CA1230552A - Device and method for performing qualitative enzyme immunoassays - Google Patents
Device and method for performing qualitative enzyme immunoassaysInfo
- Publication number
- CA1230552A CA1230552A CA000440575A CA440575A CA1230552A CA 1230552 A CA1230552 A CA 1230552A CA 000440575 A CA000440575 A CA 000440575A CA 440575 A CA440575 A CA 440575A CA 1230552 A CA1230552 A CA 1230552A
- Authority
- CA
- Canada
- Prior art keywords
- syringe
- piston
- tube
- enzyme
- outlet
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/81—Packaged device or kit
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
- Y10S436/808—Automated or kit
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/815—Test for named compound or class of compounds
- Y10S436/817—Steroids or hormones
- Y10S436/818—Human chorionic gonadotropin
Abstract
Abstract The invention relates to a device and method for performing qualitative enzyme immunoassays. The device comprises at least one tube having antibody, antigen or hapten attached to an internal surface thereof. A first syringe is connectable to a first end of the tube or tubes and supplies a flow of test liquid therethrough. A second syringe having a two piston arrangement is connected to the tube so as to allow a first wash solution followed by an enzyme conjugate solution to be flowed there-through. Finally a third syringe, also having a two piston configuration, is connected to the tube so as to allow a second wash solution followed by an enzyme substrate and metabolite indicator solution to be flowed therethrough.
Description
The present invention relates to a clevice and method for yerEorm-ing qualitative enzyme immunoassays. The invention provides a portable compact device which may be used by an untrained persotl or which is suitable for use by medical personnel in the Eield or elsewhere when a quick determination is required for diagnostic purposes. The device is particularly suitable Eor testing urine samples but also may be used for performing enzyme immunoassays using a wide variety of test solutions.
In its preferred form, the invention may be used to detect the presence of an anti~en or hapten in urine or serum. This assay is accomplished by the known method of contacting the sample with an antibody specific to the antigen or hapten being assayed for, which antibody is attached to a solid support such as the wall of a tube. Antigen which is captured by the fixed antibody is then detected by contacting the solid phase with an antibody/enzyme conjugate followed by treatment with an enzyme substrate and suitable indicator.
The present device is capable of detecting the substance being assayed for in lower concentration and with much greater speed than has heretofore been possible with prior art devices. Prior devices for the detection of an antigen or hapten employ an antibody attached to a solid support which is contacted sequentially with static volumes of test liquid, conjugate and substrate/indicator solutions. A period of incubation must be observed for each of these contacting steps as the reactions involved are diffusion controlled. That is, the reactions take place only at the surface of the solid support and sufficient time must elapse to enable enough antigen to migrate to the reaction vessel wall to give a positive test result. Increasing the surface area to volume ratio by carrying out these reactions in a tube having a small bore reduces incubation periods to about ten minutes each.
The present device employs a method whereby a continuous flow of the various reactive solutions are passed through a tube having the antibody affixed thereto so that the principles of affinity ~3C~S~
capture and concentration are utilized. That is, the reaction at t~1e tube wall is forced to completion ~uickly by continuously bathing the solid reactant with a solution having a constant con-centration of the co-reactant. This also means that the sensi-tivity of the assay is increased since a much lower concentration of co-reactant in the test sample can be detected by this flow through method. Thus, the present invention provides significant advantages over the devices and methods previously known. In the case of a pregnancy test, discussed in detail belowr the assay for the presence of human chorionic gonadotropin (HCG) in the urine may be carried out as quickly as about three minutes using the device of the invention as compared to prior art pregnancy test devices which require 20 to 120 minutes to perform the assay. Also, the present device is much simpler to use than prior devices and many fewer operations need be performed when using the device in order to carry out tne assay than is required by prior devices.
It should also be clear that the present device may be used generally for all enzyme immunoassays and should not be construed as being restricted to the detection of antigens or haptens in the manner just summarized. Without limiting the possible applications of the present invention, the device may be used as suggested to detect antigen or hapten in an antibody sandwich-type assay, or a double antibody sandwich antigen assay may be used, or a competitive antigen assay can be performed.
When detection of antibody is desired, the device can be used to do a sandwich-type assay or a double antibody sandwich antibody assay. For example, in an antigen assay for ~ hepatitis subunit or virus using the double antibody sandwich antigen assay, the solid phase comprises anti-hepatitis Ab type 1 (e.g. sheep), the second antibody used is anti-hepatitis Ab type 2 (e.g. rabbit), and the conjugate is anti-type 2 Ab/enzyme.
The present device is especially suitable if a large volume of test sample is available, such as urine. A particularly suitable assay using the present device is a test for the presence of HCG
(human chorionic gonadotropin) in urine to determine pregnancy.
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Because of the aforementioned features, pregnancy can be quickly detected at an early stage by the woman herself using the present device.
Accordingly, the invention provides a device for performing an enzyme immunoassay, comprising at least one tube having an anti-body, antigen or hapten attached to an internal surface thereof.
A first syringe is connectable to a first end of tube or tubes for supplying a flow of test liquid therethrough, and a second syringe is connected to the first end of the tube or tubes for supplying a flow of a first wash solution followed immediately by a flow of enzyme conjugate containing solution therethrough, said second syringe having a first unattached piston and a second piston attached to a plunger, the first piston being positioned to separate the first wash solution from the conjugate solution, and said syringe also having a wall with a groove therein connecting the syringe outlet with the conjugate solution only when the first piston is fully depressed. A third syringe is connected to the first end of the tube or tubes for supplying a flow of a second wash solution followed immediately by a flow of solution containing enzyme substrate and enzyme metabolite indicator therethrough, said third syringe having a structure like that of the second syringe.
The invention also provides a method for performing an enzyme immunoassay, comprising the steps of:
attaching an antibody antigen or hapten to an internal surface of a tube;
flowing a test liquid through the tube for about one minute;
flowing a first wash solution through the tube to flush out the test liquid;
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flowing an enzyme conjugate solution through the tube ~Eollowed by an incubation period of at least one minute;
flowing a second wash solution through the tube at the end of the incubation period to flush out the enzyme conjugate solution; and flowing a solution containing enzyme substrate and enzyme metabolite indicator through the tube followed by an incubation period of up to about 10 minutes.
A preferred embodiment of the invention will be described with reference to the drawings in which:
Figure 1 is a perspective view partially cut away of the testing device;
Figure 2 is a perspective view from below the device with the bottom plate removed; and Figure 3 is a cut away view of an alternate double piston syringe which may be used in the present device.
While the present device may be used for a number of different assay applications, and indeed may be used for performing multiple assays on a single test sample, the preferred embodiment hereinafter described relates to the use of the invention for testing for pregnancy by assaying for the presence of HCG (human chorionic gonadotropin~ in urine.
The preferred testing device of the invention comprises a body 10 in which is centrally disposed a tube 11 having applied to the inner wall thereof a coating of anti-HCG, i.e. HCG antibody. The anti-HCG coating is preferably covalently bonded to the tube wall/ but it may be affixed by adsorption. For the purpose of providing a control, the tube 11 is connected in series to a plain uncoated tube 12. The control tube 12 is in turn connected to a waste reservoir 15 as described below. In order to make good use of the advantayes of the flow through technique, it is ~30~5~
desirable to have a surface area to volume ratio in the tube 11 as large as is practical given the various parameters surrounding the manufacture and use of the preferred device. Thus, a tube 11 on the order of about 25 mm in length having a bore of about 1 mm in diameter is quite suitable in the present context.
The various solutions required to perform the assay are passed sequentially through the tubes 11 and 12 by means of syringes 17, 18 and 19. A test sample syringe 17 is removable from a receptacle 21 therefor which is integral with the body 10. The test sample syringe 17 comprises a body 23 having an outlet 24 at one end and a plunger 25 having a piston 26 attached at an end thereof which may move slidably within the syringe body 23 to fill and empty the syringe 17 with a liquid test sample. A urine sample of about 5 ml is reasonable for use in the particular pregnancy test device described herein.
The body 10 preferably has two additional syringes 18 and 19 integral therewith. However, the preferred device may comprise removable syringes 18 and 19, and indeed this may be desirable in certain circumstances. The syringe 18 comprises a receptacle 30 having an outlet 31 at its lower end and a groove 32 communicat-ing with the outlet 31 in the wall thereof. The syringe receptacle 30 is provided with an unattached piston 33 and a plunger 34 having a piston 35 attached at one end thereof. The free piston 33 is used to separate two solutions contained within the syringe receptacle 30, one solution contained in an upper portion 36 of the syringe 18 between the free piston 33 and the plunger 34, and the other solution contained in a lower portion 37 between the free piston 33 and the outlet 31. The final syringe 19 is of a structure identical to that of syringe 18.
The outlets of the syringes 17, 18 and 19 are connected to the lower end of the tube 11 by means of channels 40, 41 and 42 formed in the base 43 of the body 10 (Fig. 2). The channel 40 connects an opening 45 for the outlet 24 of the sample syringe 17 to an opening 46 for the tube 11. Likewise channels 41 and 42 connect the outlets 31 of syringes 18 and 19 with the opening 5;~
46. An opening 48 is provided in the base 43 for the outlet end of the control tube 12 which is in turn connected to the waste reservoir 15 by means of a channel 49~ Finally, a vent shaft 51 is provided in the body 10, the lower end of which is connected to the waste reservoir 15 by means of a slot 52 in the body 10.
The base 43 comprises a flat surface which fits flush against an unbroken flat surface 55 of a bottom plate 56 for the device.
For the purpose of providing a pregnancy testing device, the syringe 18 is filled with a solution containing an enzyme/anti-HCG conjugate in the upper portion 36. A urease/anti-HCG
conjugate is preferred in this regard. The lower portion 37 of the syringe 18 is filled with a wash solution compatible with the subsequently added conjugate solution.
The upper portion 36 of the syringe 19 is filled with a solution containing substrate for the enzyme of the conjugate and an indi-cator to detect the conversion of substrate by the enzyme in the tube 11. Preferably, the substrate is urea which is metabolized by the urease of the conjugate to give ammonia and carbon di-oxide. The pH rise caused by the release of ammonia can then be detected by a pH indicator such as bromthymol blue. The lower portion 37 of the syringe 19 is filled with a suitable wash solution, which in particular device described herein is the same as in the syringe 18.
To operate the present device to determine a suspected pregnancy, the syringe 17 is filled with a urine sample and inserted into the receptacle 21. With the syringe 17 in place in the device, the plunger 25 is slowly depressed so that the urine sample flows through the tubes 11 and 12 over a period of about one minute.
Next, the plunger 34 of the syringe 18 is fully depressed causing a first wash solution to flow through the tubes 11 and 12 thereby washing out retained urine sample, and then causing a flow of enzyme/antibody conjugate solution to pass through the tubes 11 and 12. An incubation period of about two minutes should be observed at this point to allow the conjugate time to react with any HCG captured by the anti-HCG held at the tube 11 wall. If required, a longer incubation period may be used to increase the sensitivity of an assay.
After the incubation period has elapsed, the plunger 34 of the syringe 1~ is fully depressed causing a second wash solution to flush the conjugate from the tubes 11 and 12, and then causing a flow of substrate/indicator solution to pass through the tubes 11 and 12. If the woman is pregnant and HCG is therefore present, the captured enzyme in the tube 11 will metabolize the substrate and cause the indicator to be activated. Use of a color change indicator such as bromthymol blue in a urea/urease system will give a change in the color of the liquid in the tube 11 from yellow to blue. This color change can be compared with the color in the control tube 12 which should remain yellow. A relatively high concentration of HCG in the test sample will give a color indication almost immediately, however, a low concentration of HCG will require up to 10 minutes to give a positive indicator response.
Because of the aforementioned features of the present device, low levels of HCG on the order of 50 mIU/ml can be detected. This is important so that pregnancy can be diagnosed at an early stage and so that a quick test can be carried out to reliably indicate an ectopic pregnancy.
The preferred enzyme for use in the pregnancy test device of the invention is urease because it is easily detected by the pH rise caused by metabolism of its substrate urea. Also, urea is stable in aqueous solution for at least six months at room temperature.
This is in contrast to other substrates such as hydrogen per-oxide, used with horseradish peroxidase, which breaks down rather quickly. However, without refrigeration, urea in water will eventually break down thereby making the device unusable. To provide a device having a long shelf life, i.e. two years, at room temperature, the syringe 19 may be modified as shown in Figure 3.
3~52 The modified syringe 19 has a plunger 60 equipped with a rod 61 slidably disposed longitudinally within it. The rod 61 has a piston 62 affixed at its lower end, and the piston 62 has a piercing member 63 protruding from the lower surface thereof.
The plunger 60 has a rupture membrane 65 covering the opening at its lower end, and the chamber 66 created by the membrane 65, the interior of the plunger 60 and the lower end of the rod 61 provides a space for storing powdered urea or other unstable component which may be used in another application. When the pregnancy test is to be performed, the rod 61 is depressed causing the piercing member 63 to rupture the membrane 65 thereby dispensing the powdered urea into the aqueous medium in the upper portion 36 of the syringe 19. The urea will quickly dissolve and the test can be immediately conducted in the manner described above. The piercing member 63 may also assist the dissolution of the powdered urea by causing a portion of it to be retained about the teeth 67 thereof. The retention aids in evenly distributing the powdered urea through the aqueous medium for rapid dissolution.
As mentioned above, syringes 18 and 19 may also be removable, and as such may have a body and outlet as shown in Figure 1 for the syringe 17. This arrangement would be applicable when different wash solutions are required in the lower portions 37 of the syringes 18 and 19 since in the preferred device just described, such wash solutions are in communication with one another.
Finally, in its commercial form, the present device will be sold with a protective hard cover member 70 as shown in Fig. 1. The cover member 70 will fit snugly about a peripheral shoulder 71 about the bottom portion of the body 10 thereby protecting all of the components of the device prior to use. A projection (not shown) within the cover 70 may be desirable to plug the vent 51 while in storage thus preventing evaporation of wash solutions in the lower portions 37 of the syringes 18 and 19. Removal of the cover 70 unplugs the vent 51 in readiness for performance of the test.
~2;~52 Supplementary Disclosure ~ . _ A second preferred embodiment of the present invention will be descri~ed with reference being made to the following figures in which:
Figure 4 is a perspective view of a second preferred embodiment of the device;
Figure 5 is a sectional view of a modified plunger for the sample syringe;
Figure 6 shown on the same sheet as Fig. 8, is a plan view of the base of the second preEerred device showing a more preferred circuitry for the liquid flows;
Figure 7 shown on the same sheet as Fig. 5, is a sectional detail view showing a modified upper piston for the second or conjugate containing syringe; and Figure 8 is a sectional view of the details pertaining to an alternate third or substrate containing syringe.
While the embodiment of the invention described above and shown in Figs. 1-3 provides a basic device for performing qualitative enzyme immunoassays cheaply and quickly, it has been found that occasionally false positive results may be obtained. The source of this problem has been found to reside in the failure of the wash solution in the lower portion 37 of the syringe 19 (Fig. 1) to completely remove all traces of conjugate solution from the channel 41 and the outlet 31 of the syringe 18 IFig. 2). This residual conjugate solution may subsequently contact the substrate solution as it is ejected from the third syringe 19.
The resulting mixture seeps into the tube 11 where it causes a false positive readinq.
Likewise, it has been found that contamination can occur from residual sample liquid persisting in the channel 40 and the t'~
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opening 45 for the outlet 24 of the sample syringe 17. While this is probably of less seriousness to the effective operation of the device, it is conceivable that an erroneous result may be obtained if residual sample liquid can seep into contact with subsequently used reagents.
The use of built-in plungers 34 for the syringes 18 and 1g has been found to be disadvantageous from a practical point of view.
The plungers 34 must remain withdrawn from the syringes 18 and 19 until such time as the assay is to be conducted. Premature insertion of either plunger 34 will render the device useless.
Also, packaging and transport of the device having extended plungers 34 requires the use of an oversized cover 70 (Fig. 1) which adds to the size of the device.
Finally, a further modification of the substrate syrinqe 19 for the situation where the substrate is a solid has been made to the second preferred embodiment. This modified syringe 19 is contained within the body 10 of the device and is felt to have certain advantages over the structure shown in Fig. 3.
As with the previous embodiment, the pregnancy assay for human chorionic gonadotropin (HCG) in urine will be used for illus-trative purposes in describing the operation of this embodiment.
Fig. 4 shows the second embodiment of the invention having a body 100 and a centrally disposed tube 101 having applied to the inner wall thereof a coating of antibody suitable for the particular assay to be conducted. As with the previous embodiment, the tube 101 is connected in series to a plain uncoated tube 102 which acts as a control.
The device shown in Fig. 4 has a detachable sample syringe 110, a second syringe 111 containing enzyme/antibody conjugate solution, and a third syringe 112 containing enzyme substrate. The sample syringe 110 is insertable in the cylindrical receptacle 113 provided in the body 100. The syringe 110~has a plunger 116 (Fig. 5) having a piston 117 equipped with a central frusto-conically shaped pluq 118 which is snugly insertable into the frustoconically shaped hollow tip 119 of the syringe 110 so that , i~
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the syringe opening 120 may be sealed to prevent seepage of any residual sample contained within the syringe 110.
The plunger 116 is hollowed out longitudinally and open at the top thereof so that a second plunger 125 may be stored therein during shipment and storage of the device. The plunger 125 is used to depress the pistons in the syringes 111 and 112 as will be described below.
As shown in Fig. 6, this embodiment incorporates a modified cir-cuitry for the flow of fluids from the syringes 110, 111 and 112 to the tube 101. The base 130 of the body 100 is provided with an opening 131 for receiving the tip 119 of the sample syringe 110, openings 132 and 133 for the tubes 101 and 102, and openings 134 and 135 for the second and third syringes 111 and 112. The openings 131 and 132 are connected by means of a channel 140 formed in the base 130, and openings 132 and 135 are connected by means of a channel 141. The opening 134 is indirectly connected to the opening 132 through a channel 142 running between openings 131 and 134. Finally opening 133 for the tube 102 is connected to a waste reservoir 147 by a channel 148.
This circuitry is used in conjunction with an improved upper piston 150 for the second syringe 111 as shown in Fig. 7, to solve the problem of false positive results obtained by contamination from residual conjugate solution remaining in the circuitry. As shown in Fig. 7 the syringe 111 has an upper piston 150 which comprises a structure having an upright wall 151, a flanged bottom member 152, and a flanged top member 153.
The circular circumferential edges of the flanged members 152 and 153 engage the cylindrical wall of the syringe 111. The wall 151 is provided with a plurality of small apertures 155 spaced about the circumference thereof at the juncture of the wall 151 with the bottom flanged member 152. These apertures 155 allow communication into the hollow interior of the piston 150. A
central top vent structure 156 is provided in the top flanged member 153 and preferably comprises a frustoconial member extending inwardly as shown in Fig. 7. It may also be desirable ~,'~ - 11 -~Z30S5i~
to provide one or more small vent apertures 158 through the wall 151 near the top member 153 to provide for overflow of back flush liquid entering the hollow interior of the piston 150.
The piston 150 operates in conjunction with the modified circuitry in the following fashion. After the sample liquid has passed through the tube 101 via the channel 140, the plunger 125 is applied to the upper piston 150 located near the top of the syringe 111. As in the previously described device, the syringe 111 is provided with a volume of wash solution in a lower portion 160 and an enzyme/antibody conjugate solution in an upper portion 161. The upper and lower portions 160 and 161 are defined by a piston 162. Pressure exerted by the plunger 125 on the upper piston 150 forces the wash solution from the lower portion 160 out the opening 134 and through the channels 142 and 140 to the opening 132. The channel 141 will already contain sample liquid a portion of which will be washed through the tube 101 along with the wash solution. Continued pressure on the piston 150 by means of the plunger 125 will cause the conjugate solution to flow from the upper portion 161 through the groove 165 in the wall of the syringe 111 as described above. In the present instance however, full travel of the upper piston 150 causes the edge of the lower flange member 152 to extend below the top of the groove 165 so that the interior of the piston 150 is in communication with the circuitry in the base 130 via the apertures 155 in the wall 151 of the piston 150.
The circuitry in the base 130 may at this point be purged of any extraneous sample of conjugate liquids by activation of the third syringe 112 with the plunger 125 forcing a second wash solution from a lower portion 167 thereof (Fig. 8). The wash solution from the syringe 112 proceeds through the opening 135 and the channel 141 to the opening 132 where it proceeds through the tube 101. At the same time wash solution also proceeds along the channels 140 and 142 and throuqh the opening 134, into the groove 165, through the apertures 155 in the piston wall 151, and final-ly into the interior of the piston 150. This back flush into the interior of the piston continues so long as the vent structure ~X;~)55~
156 remains open to the atmosphere. When the liquid level in the piston 150 closes the vent structure 156, the pressure buildup within the piston 150 retards the inward flow of back flush liquid thus ensuring that the principal liquid flow from the third syringe 112 proceeds through the tube 101. Thus, this arrangement of circuitry in the base 130 and the modified upper piston 150 in the syringe 111 eliminate the possibility of contamination of the substrate solution delivered from the third syringe 112 through the tube 101, thereby ensuring that false positive results cannot be obtained.
Finally, modifications to the construction of the third syringe 112 may be made as shown in Fig. 8. The cylindrical body 170 of the third syringe 112 comprises a lower portion 167 for the second wash solution and an upper syringe insert 171 containing substrate solvent and indicator. The syringe insert 171 has an outside diameter just less than the inside diameter of the bore of the syringe 112, and is provided with an upper rim 172 which engages the inner wall of the syringe 112 to make an interference fit therewith. That is, the insert 171 by virtue of the upper rim 172 is held in place within the cylindrical body 170 until it is used, at which time it can be forced down the bore of the syringe 112 by means of the plunger 125.
The insert 171 has a lower portion 173 which tapers frusto-conically from the diameter of the body thereof to a lesser diameter cylindrical tip 175. The tip 175 has a castellated rim 176 and is inserted sealingly into a piston 177 provided with a cylindrical cavity 178 formed into the upper portion thereof for the purpose. The piston 177 forms the barrier between the wash solution in the lower portion 167 and the substrate and substrate solvent in the upper part of the syringe 112. The lower portion 173 of the insert 171 is provided with a circular flange 179 for engaging the inner wall of the syringe body 170. The space 180 defined between the top of the piston 177 and the flange 179 and the lower portion 173 of the insert 171 is used for storing substrate powder. The assembly of the syringe 112 is completed by capping the insert with a piston 182.
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123055;~:
The operation of the syringe 112 is as follows. Pres~sure is exerted on the piston 182 of the insert 171 by means of the plunger 125 which, as mentioned above, was stored within the hollow sample syringe plunger 116 (Fig. 4). Hydraulic pressure from the piston 182 forces the lower piston 177 to pop off the tip 175 of the insert 171. Liquid comes out of the tip 175 forcing the piston 177 downward and causing the powdered substrate in the space 180 to dissolve therein. Upon full travel of the upper piston 1~2 to the bottom of the insert 171, continued pressure thereon by means of the plunger 125 causes the whole insert 171 to slide down the bore of the syringe 112. The substrate solution now in the lower portion 167 of the syringe 112 passes out through the opening 135 via a groove (not shown) in the wall of the syringe as previously described.
The modified syringe 112 is designed to operate satisfactorily even when the sequence of events does not occur precisely as just described. Thus, initial pressure by the plunger 125 on the upper piston 182 may cause the insert to commence sliding down the bore of the syringe body 170. ~owever, at the same time liquid from the insert 171 will be forced out through the castellated rim 176 of the tip 175, and this liquid will lubricate the surfaces of the tip 175 and piston 177 engaging one another thereby promoting disengagement of the piston 177 from the tip 175. Should the insert 171 travel the full length of the syringe 112 without disengagement of the piston 177 therefrom, continued pressure applied by the plunger 125 will force such disengagement and allow the final step of the assay to proceed.
In its preferred form, the invention may be used to detect the presence of an anti~en or hapten in urine or serum. This assay is accomplished by the known method of contacting the sample with an antibody specific to the antigen or hapten being assayed for, which antibody is attached to a solid support such as the wall of a tube. Antigen which is captured by the fixed antibody is then detected by contacting the solid phase with an antibody/enzyme conjugate followed by treatment with an enzyme substrate and suitable indicator.
The present device is capable of detecting the substance being assayed for in lower concentration and with much greater speed than has heretofore been possible with prior art devices. Prior devices for the detection of an antigen or hapten employ an antibody attached to a solid support which is contacted sequentially with static volumes of test liquid, conjugate and substrate/indicator solutions. A period of incubation must be observed for each of these contacting steps as the reactions involved are diffusion controlled. That is, the reactions take place only at the surface of the solid support and sufficient time must elapse to enable enough antigen to migrate to the reaction vessel wall to give a positive test result. Increasing the surface area to volume ratio by carrying out these reactions in a tube having a small bore reduces incubation periods to about ten minutes each.
The present device employs a method whereby a continuous flow of the various reactive solutions are passed through a tube having the antibody affixed thereto so that the principles of affinity ~3C~S~
capture and concentration are utilized. That is, the reaction at t~1e tube wall is forced to completion ~uickly by continuously bathing the solid reactant with a solution having a constant con-centration of the co-reactant. This also means that the sensi-tivity of the assay is increased since a much lower concentration of co-reactant in the test sample can be detected by this flow through method. Thus, the present invention provides significant advantages over the devices and methods previously known. In the case of a pregnancy test, discussed in detail belowr the assay for the presence of human chorionic gonadotropin (HCG) in the urine may be carried out as quickly as about three minutes using the device of the invention as compared to prior art pregnancy test devices which require 20 to 120 minutes to perform the assay. Also, the present device is much simpler to use than prior devices and many fewer operations need be performed when using the device in order to carry out tne assay than is required by prior devices.
It should also be clear that the present device may be used generally for all enzyme immunoassays and should not be construed as being restricted to the detection of antigens or haptens in the manner just summarized. Without limiting the possible applications of the present invention, the device may be used as suggested to detect antigen or hapten in an antibody sandwich-type assay, or a double antibody sandwich antigen assay may be used, or a competitive antigen assay can be performed.
When detection of antibody is desired, the device can be used to do a sandwich-type assay or a double antibody sandwich antibody assay. For example, in an antigen assay for ~ hepatitis subunit or virus using the double antibody sandwich antigen assay, the solid phase comprises anti-hepatitis Ab type 1 (e.g. sheep), the second antibody used is anti-hepatitis Ab type 2 (e.g. rabbit), and the conjugate is anti-type 2 Ab/enzyme.
The present device is especially suitable if a large volume of test sample is available, such as urine. A particularly suitable assay using the present device is a test for the presence of HCG
(human chorionic gonadotropin) in urine to determine pregnancy.
~230~S;~
Because of the aforementioned features, pregnancy can be quickly detected at an early stage by the woman herself using the present device.
Accordingly, the invention provides a device for performing an enzyme immunoassay, comprising at least one tube having an anti-body, antigen or hapten attached to an internal surface thereof.
A first syringe is connectable to a first end of tube or tubes for supplying a flow of test liquid therethrough, and a second syringe is connected to the first end of the tube or tubes for supplying a flow of a first wash solution followed immediately by a flow of enzyme conjugate containing solution therethrough, said second syringe having a first unattached piston and a second piston attached to a plunger, the first piston being positioned to separate the first wash solution from the conjugate solution, and said syringe also having a wall with a groove therein connecting the syringe outlet with the conjugate solution only when the first piston is fully depressed. A third syringe is connected to the first end of the tube or tubes for supplying a flow of a second wash solution followed immediately by a flow of solution containing enzyme substrate and enzyme metabolite indicator therethrough, said third syringe having a structure like that of the second syringe.
The invention also provides a method for performing an enzyme immunoassay, comprising the steps of:
attaching an antibody antigen or hapten to an internal surface of a tube;
flowing a test liquid through the tube for about one minute;
flowing a first wash solution through the tube to flush out the test liquid;
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flowing an enzyme conjugate solution through the tube ~Eollowed by an incubation period of at least one minute;
flowing a second wash solution through the tube at the end of the incubation period to flush out the enzyme conjugate solution; and flowing a solution containing enzyme substrate and enzyme metabolite indicator through the tube followed by an incubation period of up to about 10 minutes.
A preferred embodiment of the invention will be described with reference to the drawings in which:
Figure 1 is a perspective view partially cut away of the testing device;
Figure 2 is a perspective view from below the device with the bottom plate removed; and Figure 3 is a cut away view of an alternate double piston syringe which may be used in the present device.
While the present device may be used for a number of different assay applications, and indeed may be used for performing multiple assays on a single test sample, the preferred embodiment hereinafter described relates to the use of the invention for testing for pregnancy by assaying for the presence of HCG (human chorionic gonadotropin~ in urine.
The preferred testing device of the invention comprises a body 10 in which is centrally disposed a tube 11 having applied to the inner wall thereof a coating of anti-HCG, i.e. HCG antibody. The anti-HCG coating is preferably covalently bonded to the tube wall/ but it may be affixed by adsorption. For the purpose of providing a control, the tube 11 is connected in series to a plain uncoated tube 12. The control tube 12 is in turn connected to a waste reservoir 15 as described below. In order to make good use of the advantayes of the flow through technique, it is ~30~5~
desirable to have a surface area to volume ratio in the tube 11 as large as is practical given the various parameters surrounding the manufacture and use of the preferred device. Thus, a tube 11 on the order of about 25 mm in length having a bore of about 1 mm in diameter is quite suitable in the present context.
The various solutions required to perform the assay are passed sequentially through the tubes 11 and 12 by means of syringes 17, 18 and 19. A test sample syringe 17 is removable from a receptacle 21 therefor which is integral with the body 10. The test sample syringe 17 comprises a body 23 having an outlet 24 at one end and a plunger 25 having a piston 26 attached at an end thereof which may move slidably within the syringe body 23 to fill and empty the syringe 17 with a liquid test sample. A urine sample of about 5 ml is reasonable for use in the particular pregnancy test device described herein.
The body 10 preferably has two additional syringes 18 and 19 integral therewith. However, the preferred device may comprise removable syringes 18 and 19, and indeed this may be desirable in certain circumstances. The syringe 18 comprises a receptacle 30 having an outlet 31 at its lower end and a groove 32 communicat-ing with the outlet 31 in the wall thereof. The syringe receptacle 30 is provided with an unattached piston 33 and a plunger 34 having a piston 35 attached at one end thereof. The free piston 33 is used to separate two solutions contained within the syringe receptacle 30, one solution contained in an upper portion 36 of the syringe 18 between the free piston 33 and the plunger 34, and the other solution contained in a lower portion 37 between the free piston 33 and the outlet 31. The final syringe 19 is of a structure identical to that of syringe 18.
The outlets of the syringes 17, 18 and 19 are connected to the lower end of the tube 11 by means of channels 40, 41 and 42 formed in the base 43 of the body 10 (Fig. 2). The channel 40 connects an opening 45 for the outlet 24 of the sample syringe 17 to an opening 46 for the tube 11. Likewise channels 41 and 42 connect the outlets 31 of syringes 18 and 19 with the opening 5;~
46. An opening 48 is provided in the base 43 for the outlet end of the control tube 12 which is in turn connected to the waste reservoir 15 by means of a channel 49~ Finally, a vent shaft 51 is provided in the body 10, the lower end of which is connected to the waste reservoir 15 by means of a slot 52 in the body 10.
The base 43 comprises a flat surface which fits flush against an unbroken flat surface 55 of a bottom plate 56 for the device.
For the purpose of providing a pregnancy testing device, the syringe 18 is filled with a solution containing an enzyme/anti-HCG conjugate in the upper portion 36. A urease/anti-HCG
conjugate is preferred in this regard. The lower portion 37 of the syringe 18 is filled with a wash solution compatible with the subsequently added conjugate solution.
The upper portion 36 of the syringe 19 is filled with a solution containing substrate for the enzyme of the conjugate and an indi-cator to detect the conversion of substrate by the enzyme in the tube 11. Preferably, the substrate is urea which is metabolized by the urease of the conjugate to give ammonia and carbon di-oxide. The pH rise caused by the release of ammonia can then be detected by a pH indicator such as bromthymol blue. The lower portion 37 of the syringe 19 is filled with a suitable wash solution, which in particular device described herein is the same as in the syringe 18.
To operate the present device to determine a suspected pregnancy, the syringe 17 is filled with a urine sample and inserted into the receptacle 21. With the syringe 17 in place in the device, the plunger 25 is slowly depressed so that the urine sample flows through the tubes 11 and 12 over a period of about one minute.
Next, the plunger 34 of the syringe 18 is fully depressed causing a first wash solution to flow through the tubes 11 and 12 thereby washing out retained urine sample, and then causing a flow of enzyme/antibody conjugate solution to pass through the tubes 11 and 12. An incubation period of about two minutes should be observed at this point to allow the conjugate time to react with any HCG captured by the anti-HCG held at the tube 11 wall. If required, a longer incubation period may be used to increase the sensitivity of an assay.
After the incubation period has elapsed, the plunger 34 of the syringe 1~ is fully depressed causing a second wash solution to flush the conjugate from the tubes 11 and 12, and then causing a flow of substrate/indicator solution to pass through the tubes 11 and 12. If the woman is pregnant and HCG is therefore present, the captured enzyme in the tube 11 will metabolize the substrate and cause the indicator to be activated. Use of a color change indicator such as bromthymol blue in a urea/urease system will give a change in the color of the liquid in the tube 11 from yellow to blue. This color change can be compared with the color in the control tube 12 which should remain yellow. A relatively high concentration of HCG in the test sample will give a color indication almost immediately, however, a low concentration of HCG will require up to 10 minutes to give a positive indicator response.
Because of the aforementioned features of the present device, low levels of HCG on the order of 50 mIU/ml can be detected. This is important so that pregnancy can be diagnosed at an early stage and so that a quick test can be carried out to reliably indicate an ectopic pregnancy.
The preferred enzyme for use in the pregnancy test device of the invention is urease because it is easily detected by the pH rise caused by metabolism of its substrate urea. Also, urea is stable in aqueous solution for at least six months at room temperature.
This is in contrast to other substrates such as hydrogen per-oxide, used with horseradish peroxidase, which breaks down rather quickly. However, without refrigeration, urea in water will eventually break down thereby making the device unusable. To provide a device having a long shelf life, i.e. two years, at room temperature, the syringe 19 may be modified as shown in Figure 3.
3~52 The modified syringe 19 has a plunger 60 equipped with a rod 61 slidably disposed longitudinally within it. The rod 61 has a piston 62 affixed at its lower end, and the piston 62 has a piercing member 63 protruding from the lower surface thereof.
The plunger 60 has a rupture membrane 65 covering the opening at its lower end, and the chamber 66 created by the membrane 65, the interior of the plunger 60 and the lower end of the rod 61 provides a space for storing powdered urea or other unstable component which may be used in another application. When the pregnancy test is to be performed, the rod 61 is depressed causing the piercing member 63 to rupture the membrane 65 thereby dispensing the powdered urea into the aqueous medium in the upper portion 36 of the syringe 19. The urea will quickly dissolve and the test can be immediately conducted in the manner described above. The piercing member 63 may also assist the dissolution of the powdered urea by causing a portion of it to be retained about the teeth 67 thereof. The retention aids in evenly distributing the powdered urea through the aqueous medium for rapid dissolution.
As mentioned above, syringes 18 and 19 may also be removable, and as such may have a body and outlet as shown in Figure 1 for the syringe 17. This arrangement would be applicable when different wash solutions are required in the lower portions 37 of the syringes 18 and 19 since in the preferred device just described, such wash solutions are in communication with one another.
Finally, in its commercial form, the present device will be sold with a protective hard cover member 70 as shown in Fig. 1. The cover member 70 will fit snugly about a peripheral shoulder 71 about the bottom portion of the body 10 thereby protecting all of the components of the device prior to use. A projection (not shown) within the cover 70 may be desirable to plug the vent 51 while in storage thus preventing evaporation of wash solutions in the lower portions 37 of the syringes 18 and 19. Removal of the cover 70 unplugs the vent 51 in readiness for performance of the test.
~2;~52 Supplementary Disclosure ~ . _ A second preferred embodiment of the present invention will be descri~ed with reference being made to the following figures in which:
Figure 4 is a perspective view of a second preferred embodiment of the device;
Figure 5 is a sectional view of a modified plunger for the sample syringe;
Figure 6 shown on the same sheet as Fig. 8, is a plan view of the base of the second preEerred device showing a more preferred circuitry for the liquid flows;
Figure 7 shown on the same sheet as Fig. 5, is a sectional detail view showing a modified upper piston for the second or conjugate containing syringe; and Figure 8 is a sectional view of the details pertaining to an alternate third or substrate containing syringe.
While the embodiment of the invention described above and shown in Figs. 1-3 provides a basic device for performing qualitative enzyme immunoassays cheaply and quickly, it has been found that occasionally false positive results may be obtained. The source of this problem has been found to reside in the failure of the wash solution in the lower portion 37 of the syringe 19 (Fig. 1) to completely remove all traces of conjugate solution from the channel 41 and the outlet 31 of the syringe 18 IFig. 2). This residual conjugate solution may subsequently contact the substrate solution as it is ejected from the third syringe 19.
The resulting mixture seeps into the tube 11 where it causes a false positive readinq.
Likewise, it has been found that contamination can occur from residual sample liquid persisting in the channel 40 and the t'~
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opening 45 for the outlet 24 of the sample syringe 17. While this is probably of less seriousness to the effective operation of the device, it is conceivable that an erroneous result may be obtained if residual sample liquid can seep into contact with subsequently used reagents.
The use of built-in plungers 34 for the syringes 18 and 1g has been found to be disadvantageous from a practical point of view.
The plungers 34 must remain withdrawn from the syringes 18 and 19 until such time as the assay is to be conducted. Premature insertion of either plunger 34 will render the device useless.
Also, packaging and transport of the device having extended plungers 34 requires the use of an oversized cover 70 (Fig. 1) which adds to the size of the device.
Finally, a further modification of the substrate syrinqe 19 for the situation where the substrate is a solid has been made to the second preferred embodiment. This modified syringe 19 is contained within the body 10 of the device and is felt to have certain advantages over the structure shown in Fig. 3.
As with the previous embodiment, the pregnancy assay for human chorionic gonadotropin (HCG) in urine will be used for illus-trative purposes in describing the operation of this embodiment.
Fig. 4 shows the second embodiment of the invention having a body 100 and a centrally disposed tube 101 having applied to the inner wall thereof a coating of antibody suitable for the particular assay to be conducted. As with the previous embodiment, the tube 101 is connected in series to a plain uncoated tube 102 which acts as a control.
The device shown in Fig. 4 has a detachable sample syringe 110, a second syringe 111 containing enzyme/antibody conjugate solution, and a third syringe 112 containing enzyme substrate. The sample syringe 110 is insertable in the cylindrical receptacle 113 provided in the body 100. The syringe 110~has a plunger 116 (Fig. 5) having a piston 117 equipped with a central frusto-conically shaped pluq 118 which is snugly insertable into the frustoconically shaped hollow tip 119 of the syringe 110 so that , i~
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the syringe opening 120 may be sealed to prevent seepage of any residual sample contained within the syringe 110.
The plunger 116 is hollowed out longitudinally and open at the top thereof so that a second plunger 125 may be stored therein during shipment and storage of the device. The plunger 125 is used to depress the pistons in the syringes 111 and 112 as will be described below.
As shown in Fig. 6, this embodiment incorporates a modified cir-cuitry for the flow of fluids from the syringes 110, 111 and 112 to the tube 101. The base 130 of the body 100 is provided with an opening 131 for receiving the tip 119 of the sample syringe 110, openings 132 and 133 for the tubes 101 and 102, and openings 134 and 135 for the second and third syringes 111 and 112. The openings 131 and 132 are connected by means of a channel 140 formed in the base 130, and openings 132 and 135 are connected by means of a channel 141. The opening 134 is indirectly connected to the opening 132 through a channel 142 running between openings 131 and 134. Finally opening 133 for the tube 102 is connected to a waste reservoir 147 by a channel 148.
This circuitry is used in conjunction with an improved upper piston 150 for the second syringe 111 as shown in Fig. 7, to solve the problem of false positive results obtained by contamination from residual conjugate solution remaining in the circuitry. As shown in Fig. 7 the syringe 111 has an upper piston 150 which comprises a structure having an upright wall 151, a flanged bottom member 152, and a flanged top member 153.
The circular circumferential edges of the flanged members 152 and 153 engage the cylindrical wall of the syringe 111. The wall 151 is provided with a plurality of small apertures 155 spaced about the circumference thereof at the juncture of the wall 151 with the bottom flanged member 152. These apertures 155 allow communication into the hollow interior of the piston 150. A
central top vent structure 156 is provided in the top flanged member 153 and preferably comprises a frustoconial member extending inwardly as shown in Fig. 7. It may also be desirable ~,'~ - 11 -~Z30S5i~
to provide one or more small vent apertures 158 through the wall 151 near the top member 153 to provide for overflow of back flush liquid entering the hollow interior of the piston 150.
The piston 150 operates in conjunction with the modified circuitry in the following fashion. After the sample liquid has passed through the tube 101 via the channel 140, the plunger 125 is applied to the upper piston 150 located near the top of the syringe 111. As in the previously described device, the syringe 111 is provided with a volume of wash solution in a lower portion 160 and an enzyme/antibody conjugate solution in an upper portion 161. The upper and lower portions 160 and 161 are defined by a piston 162. Pressure exerted by the plunger 125 on the upper piston 150 forces the wash solution from the lower portion 160 out the opening 134 and through the channels 142 and 140 to the opening 132. The channel 141 will already contain sample liquid a portion of which will be washed through the tube 101 along with the wash solution. Continued pressure on the piston 150 by means of the plunger 125 will cause the conjugate solution to flow from the upper portion 161 through the groove 165 in the wall of the syringe 111 as described above. In the present instance however, full travel of the upper piston 150 causes the edge of the lower flange member 152 to extend below the top of the groove 165 so that the interior of the piston 150 is in communication with the circuitry in the base 130 via the apertures 155 in the wall 151 of the piston 150.
The circuitry in the base 130 may at this point be purged of any extraneous sample of conjugate liquids by activation of the third syringe 112 with the plunger 125 forcing a second wash solution from a lower portion 167 thereof (Fig. 8). The wash solution from the syringe 112 proceeds through the opening 135 and the channel 141 to the opening 132 where it proceeds through the tube 101. At the same time wash solution also proceeds along the channels 140 and 142 and throuqh the opening 134, into the groove 165, through the apertures 155 in the piston wall 151, and final-ly into the interior of the piston 150. This back flush into the interior of the piston continues so long as the vent structure ~X;~)55~
156 remains open to the atmosphere. When the liquid level in the piston 150 closes the vent structure 156, the pressure buildup within the piston 150 retards the inward flow of back flush liquid thus ensuring that the principal liquid flow from the third syringe 112 proceeds through the tube 101. Thus, this arrangement of circuitry in the base 130 and the modified upper piston 150 in the syringe 111 eliminate the possibility of contamination of the substrate solution delivered from the third syringe 112 through the tube 101, thereby ensuring that false positive results cannot be obtained.
Finally, modifications to the construction of the third syringe 112 may be made as shown in Fig. 8. The cylindrical body 170 of the third syringe 112 comprises a lower portion 167 for the second wash solution and an upper syringe insert 171 containing substrate solvent and indicator. The syringe insert 171 has an outside diameter just less than the inside diameter of the bore of the syringe 112, and is provided with an upper rim 172 which engages the inner wall of the syringe 112 to make an interference fit therewith. That is, the insert 171 by virtue of the upper rim 172 is held in place within the cylindrical body 170 until it is used, at which time it can be forced down the bore of the syringe 112 by means of the plunger 125.
The insert 171 has a lower portion 173 which tapers frusto-conically from the diameter of the body thereof to a lesser diameter cylindrical tip 175. The tip 175 has a castellated rim 176 and is inserted sealingly into a piston 177 provided with a cylindrical cavity 178 formed into the upper portion thereof for the purpose. The piston 177 forms the barrier between the wash solution in the lower portion 167 and the substrate and substrate solvent in the upper part of the syringe 112. The lower portion 173 of the insert 171 is provided with a circular flange 179 for engaging the inner wall of the syringe body 170. The space 180 defined between the top of the piston 177 and the flange 179 and the lower portion 173 of the insert 171 is used for storing substrate powder. The assembly of the syringe 112 is completed by capping the insert with a piston 182.
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The operation of the syringe 112 is as follows. Pres~sure is exerted on the piston 182 of the insert 171 by means of the plunger 125 which, as mentioned above, was stored within the hollow sample syringe plunger 116 (Fig. 4). Hydraulic pressure from the piston 182 forces the lower piston 177 to pop off the tip 175 of the insert 171. Liquid comes out of the tip 175 forcing the piston 177 downward and causing the powdered substrate in the space 180 to dissolve therein. Upon full travel of the upper piston 1~2 to the bottom of the insert 171, continued pressure thereon by means of the plunger 125 causes the whole insert 171 to slide down the bore of the syringe 112. The substrate solution now in the lower portion 167 of the syringe 112 passes out through the opening 135 via a groove (not shown) in the wall of the syringe as previously described.
The modified syringe 112 is designed to operate satisfactorily even when the sequence of events does not occur precisely as just described. Thus, initial pressure by the plunger 125 on the upper piston 182 may cause the insert to commence sliding down the bore of the syringe body 170. ~owever, at the same time liquid from the insert 171 will be forced out through the castellated rim 176 of the tip 175, and this liquid will lubricate the surfaces of the tip 175 and piston 177 engaging one another thereby promoting disengagement of the piston 177 from the tip 175. Should the insert 171 travel the full length of the syringe 112 without disengagement of the piston 177 therefrom, continued pressure applied by the plunger 125 will force such disengagement and allow the final step of the assay to proceed.
Claims (34)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A device for performing an enzyme immunoassay, comprising:
at least one assay tube having first and second ends and having an antibody, antigen or hapten attached to an internal surface thereof;
a first syringe having an outlet connectable to the first end of the assay tube for supplying a flow of a test liquid therethrough;
a second syringe having an outlet connected to the first end of the assay tube for supplying a flow of a first wash solution followed immediately by a flow of an enzyme conjugate containing solution therethrough, said second syringe having a first piston and a second piston spaced from one another, the first piston being positioned to separate the first wash solution from the conjugate solution, the second piston having a plunger, and said syringe also having a wall with a groove therein con-necting the syringe outlet with the conjugate solution only when the first piston is fully depressed; and a third syringe having an outlet connected to the first end of the assay tube for supplying a flow of a second wash solution followed immediately by a flow of solution containing an enzyme substrate and an enzyme metabolite indicator therethrough, said third syringe having a first piston and a second piston, spaced from one another, the first piston being positioned to separate the second wash solution from the substrate, the second piston having a plunger, said third syringe also having a wall with a groove therein connecting the syringe outlet with the substrate and indicator solution only when the first piston is fully depressed.
at least one assay tube having first and second ends and having an antibody, antigen or hapten attached to an internal surface thereof;
a first syringe having an outlet connectable to the first end of the assay tube for supplying a flow of a test liquid therethrough;
a second syringe having an outlet connected to the first end of the assay tube for supplying a flow of a first wash solution followed immediately by a flow of an enzyme conjugate containing solution therethrough, said second syringe having a first piston and a second piston spaced from one another, the first piston being positioned to separate the first wash solution from the conjugate solution, the second piston having a plunger, and said syringe also having a wall with a groove therein con-necting the syringe outlet with the conjugate solution only when the first piston is fully depressed; and a third syringe having an outlet connected to the first end of the assay tube for supplying a flow of a second wash solution followed immediately by a flow of solution containing an enzyme substrate and an enzyme metabolite indicator therethrough, said third syringe having a first piston and a second piston, spaced from one another, the first piston being positioned to separate the second wash solution from the substrate, the second piston having a plunger, said third syringe also having a wall with a groove therein connecting the syringe outlet with the substrate and indicator solution only when the first piston is fully depressed.
2. A device as claimed in claim 1, wherein the antibody, antigen or hapten is covalently bonded to the internal surface of the assay tube.
3. A device as claimed in claim 1, further comprising a control tube not having any antibody, antigen or hapten attached to an internal surface thereof, the control tube being connected to one end of the assay tube.
4. A device as claimed in claim 1, further comprising means for receiving and storing liquids which have flowed through the assay tube.
5. A device as claimed in claim 4, wherein the means for receiving and storing liquids comprises a reservoir formed integrally with the device.
6. A device as claimed in claim 1, wherein the device is a pregnancy test kit having human chorionic gonadotropin antibody (anti-HCG) attached to the internal surface of the assay tube, the enzyme conjugate being urease/anti-HCG, the enzyme substrate being urea, and the enzyme metabolite indicator being bromthymol blue.
7. A device as claimed in claim 6, wherein the test liquid is urine.
8. A device as claimed in claim 6, wherein the test liquid is serum.
9. A device as claimed in claim 1, wherein the third syringe is provided with a plunger having a rod slidably disposed longitudinally within it, said rod having a piston affixed at its lower end the piston being equipped with a piercing member protruding from the lower surface thereof and the plunger having a rupture membrane covering an opening at its lower end, said membrane, the interior of the plunger and the lower end of the rod defining a chamber.
10. A device as claimed in claim 9, wherein the device is a pregnancy test kit having human chorionic gonadotropin antibody (anti-HCG) attached to the internal surface of the assay tube, the enzyme conjugate being urease/anti-HCG, the enzyme substrate being urea contained in powder form in said chamber, and the indicator being bromthymol blue.
11. A device as claimed in claim 1, wherein the second and third syringes are each provided with a rupture membrane about each outlet thereof to prevent cross contamination of solutions contained therein.
12. A device as claimed in claim 5, further comprising vent means for the reservoir and a protective hard cover for the device having means integral therewith for plugging the vent means when the cover is in place.
13. A method for performing an enzyme immunoassay, comprising the steps of:
attaching an antibody, antigen or hapten to an internal surface of a tube;
flowing a test liquid through the tube for about one minute;
flowing a first wash solution through the tube to flush out the test liquid;
flowing an enzyme conjugate solution through the tube followed by an incubation period of at least one minute;
flowing a second wash solution through the tube at the end of the incubation period to flush out the enzyme conjugate solution; and flowing a solution containing enzyme substrate and enzyme metabolite indicator through the tube followed by an incubation period of up to about 10 minutes.
attaching an antibody, antigen or hapten to an internal surface of a tube;
flowing a test liquid through the tube for about one minute;
flowing a first wash solution through the tube to flush out the test liquid;
flowing an enzyme conjugate solution through the tube followed by an incubation period of at least one minute;
flowing a second wash solution through the tube at the end of the incubation period to flush out the enzyme conjugate solution; and flowing a solution containing enzyme substrate and enzyme metabolite indicator through the tube followed by an incubation period of up to about 10 minutes.
14. A method as claimed in claim 13, wherein the enzyme substrate is dissolved just prior to performing the assay.
15. A method as claimed in claim 13, wherein human chorionic gonadotropin antibody (anti-HCG) is attached to the internal surface of the tube, the enzyme conjugate is urease/anti-HCG, the substrate is urea, and the indicator is bromthymol blue.
16. A method as claimed in claim 15, wherein the test liquid is urine.
17. A method as claimed in claim 15, wherein the test liquid is serum.
18. A device as claimed in claim 1, wherein the device has a flat bottom surface provided with grooves therein connecting outlets for the first, second and third syringes with the first end of the assay tube, and a base plate for the device which coacts with the grooves to form channels for the flow of liquids from the syringes to the first end of the assay tube.
Claims Supported by Supplementary Disclosure
Claims Supported by Supplementary Disclosure
19. A device for performing an enzyme immunoassay, comprising:
a body defining a receptacle for receiving a first syringe containing a test liquid, and defining a second and a third syringe formed integrally in the body, the receptacle and syringes all having outlets;
at least one assay tube is provided in the body, the tube having first and second ends and having an antibody, antigen or hapten attached to an internal surface of the tube;
the body is provided with a flat bottom surface having grooves in it which connect outlets of the first, second and third syringes to the first end of the assay tube;
a channel forming means is insertable over the bottom surface of the body, the means having a flat surface which coacts with the grooves to form channels for the flow of liquids from the syringes to the first end of the assay tube; and the second and third syringes are each provided with first and second pistons spaced from one another, the second piston may be directly depressed by a plunger, and each second and third syringe having a wall with a groove in it connecting the syringe outlet with the space above the first piston when it is fully depressed.
a body defining a receptacle for receiving a first syringe containing a test liquid, and defining a second and a third syringe formed integrally in the body, the receptacle and syringes all having outlets;
at least one assay tube is provided in the body, the tube having first and second ends and having an antibody, antigen or hapten attached to an internal surface of the tube;
the body is provided with a flat bottom surface having grooves in it which connect outlets of the first, second and third syringes to the first end of the assay tube;
a channel forming means is insertable over the bottom surface of the body, the means having a flat surface which coacts with the grooves to form channels for the flow of liquids from the syringes to the first end of the assay tube; and the second and third syringes are each provided with first and second pistons spaced from one another, the second piston may be directly depressed by a plunger, and each second and third syringe having a wall with a groove in it connecting the syringe outlet with the space above the first piston when it is fully depressed.
20. A device as claimed in claim 19, wherein the second syringe is provided with a first wash solution in the space defined therein below its first piston, and an enzyme conjugate containing solution in the space defined therein above its first piston; and the third syringe is provided with a second wash solution in the space defined therein below its first piston, and a solution containing enzyme substrate and enzyme metabolite indicator in the space defined therein above its first piston.
21. A device as claimed in claim 19, further comprising a control tube not having any antibody, antigen or hapten attached to its internal surface, the control tube being connected to one end of the assay tube.
22. A device as claimed in claim 19, further comprising a waste reservoir defined in the body for receiving and storing liquids which have flowed through the assay tube.
23. A device as claimed in claim 19, wherein the third syringe is provided with a hollow plunger having a rod slidably disposed longitudinally within it, the rod having a piston affixed to its lower end, the piston being equipped with a piercing member protruding from the lower surface thereof, and the plunger having a rupture membrane covering an opening at its lower end;
the membrane, the interior of the plunger and the lower end of the rod defining a chamber.
the membrane, the interior of the plunger and the lower end of the rod defining a chamber.
24. A device as claimed in claim 23, wherein human chorionic gonadotropin antibody (anti-HCG) is attached to the internal surface of the assay tube, the enzyme conjugate is urease/
anti-HCG, the enzyme substrate is urea powder contained in the chamber, and the indicator is bromthymol blue.
anti-HCG, the enzyme substrate is urea powder contained in the chamber, and the indicator is bromthymol blue.
25. A device as claimed in claim 19, wherein the second and third syringes are each provided with a rupture membrane about each outlet thereof to prevent cross contamination of solutions contained therein.
26. A device as claimed in claim 22, further comprising vent means for the reservoir and a protective hard cover for the device having means for plugging the vent means when the cover is in place.
27. A device as claimed in claim 19, wherein the outlet of the second syringe is connected by a channel to the first end of the assay tube via the outlet of the first syringe.
28. A device as claimed in claim 19, wherein the first syringe comprises a body having a frustoconically shaped hollow tip provided with an outlet for the syringe at its end, and a plunger having a frustoconically shaped plug on its end which is insertable into the tip so that it may be sealed.
29. A device as claimed in claim 19, wherein the second syringe has a second piston which comprises a hollow body having an upright wall with a flanged bottom member attached thereto, a flanged top member having a downward depending cylindrical wall is inserted into the opening defined by said upright wall, edges of said flanged members engage the wall of the syringe, the piston wall has at least one groove about its internal surface depending from one or more corresponding notches spaced about the upper edge of the piston wall, the grooves and notches forming conduits between the opposing walls of the piston and flanged top member to the interior of the piston; and a top vent structure extending inwardly from the top flanged member into the hollow body defining an opening through the member, the groove in the syringe wall being of sufficient height to extend above the bottom flange edge when the second piston is fully depressed.
30. A device as claimed in claim 29 wherein the vent structure extends into the hollow body approximately one-half its height.
31. A device as claimed in claim 19,wherein the third syringe has a syringe insert positioned in its upper portion to contain substrate solvent, the insert having a tip insertable into a cavity formed in the top of the first piston, the end of the insert and the top of the first piston defining a space to contain powdered substrate.
32. A device as claimed in claim 31, wherein the insert has an upper rim and a lower flange which slidably engage the wall of the syringe, the structure in between the rim and flange not touching the syringe wall.
33. A device as claimed in claim 31, wherein the tip of the insert is provided with a castellated edge.
34. A device as claimed in claim 19, wherein the outlet of the second syringe is connected to the first end of the assay tube by means of a channel running to the outlet for the first syringe and then to the first end of the assay tube, the third syringe outlet being connected directly to the first end of the assay tube by means of a channel.
Priority Applications (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000440575A CA1230552A (en) | 1983-11-07 | 1983-11-07 | Device and method for performing qualitative enzyme immunoassays |
| US06/551,906 US4665034A (en) | 1983-11-07 | 1983-11-15 | Device for performing qualitative enzyme immunoassays |
| AU34936/84A AU579329B2 (en) | 1983-11-07 | 1984-11-02 | Device and method for performing qualitative enzime immunoassays |
| EP84307634A EP0144162A3 (en) | 1983-11-07 | 1984-11-06 | Device and method for performing qualitative enzyme immunoassays |
| JP59233396A JPS60128369A (en) | 1983-11-07 | 1984-11-07 | Device and method for executing qualitative enzyme immunity test |
| US06/933,171 US4791060A (en) | 1983-11-07 | 1986-11-21 | Device for performing qualitative enzyme immunoassays |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000440575A CA1230552A (en) | 1983-11-07 | 1983-11-07 | Device and method for performing qualitative enzyme immunoassays |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1230552A true CA1230552A (en) | 1987-12-22 |
Family
ID=4126468
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000440575A Expired CA1230552A (en) | 1983-11-07 | 1983-11-07 | Device and method for performing qualitative enzyme immunoassays |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US4665034A (en) |
| EP (1) | EP0144162A3 (en) |
| JP (1) | JPS60128369A (en) |
| AU (1) | AU579329B2 (en) |
| CA (1) | CA1230552A (en) |
Families Citing this family (35)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4791060A (en) * | 1983-11-07 | 1988-12-13 | Allelix Inc. | Device for performing qualitative enzyme immunoassays |
| JPS62257061A (en) * | 1986-04-28 | 1987-11-09 | エー・デイ・アイ ダイアグノステイクス・インコーポレーテッド | Chemical and immunochemical qualitative quantitative analyzer |
| US4729875A (en) * | 1986-06-26 | 1988-03-08 | Allelix Inc. | Device for performing immunochemical assays |
| US5714380A (en) * | 1986-10-23 | 1998-02-03 | Amoco Corporation | Closed vessel for isolating target molecules and for performing amplification |
| FR2608768A1 (en) * | 1986-12-22 | 1988-06-24 | Ecole Nat Sup Creation Ind | Manual control device for automatic colorimetric determination of hormones or hormone derivatives in plasma or urine liquids |
| US4978502A (en) * | 1987-01-05 | 1990-12-18 | Dole Associates, Inc. | Immunoassay or diagnostic device and method of manufacture |
| US4859603A (en) * | 1987-01-05 | 1989-08-22 | Dole Associates, Inc. | Personal diagnostic kit |
| US4769333A (en) * | 1987-01-05 | 1988-09-06 | Dole Associates, Inc. | Personal diagnostic kit |
| US5422270A (en) * | 1987-05-19 | 1995-06-06 | The United States Of America As Represented By The Department Of Health And Human Services | One-step tray test for release of soluble mediators and apparatus therefore |
| US5256372A (en) * | 1987-11-06 | 1993-10-26 | Idexx Corporation | Dipstick test device including a removable filter assembly |
| US4883760A (en) * | 1988-06-20 | 1989-11-28 | Adi Diagnostics Inc. | Device for performing enzyme immunoassays |
| AU2684488A (en) * | 1988-06-27 | 1990-01-04 | Carter-Wallace, Inc. | Test device and method for colored particle immunoassay |
| WO1990014163A1 (en) * | 1989-05-24 | 1990-11-29 | Ensys, Inc. | Method and apparatus for detecting environmental contaminants |
| DE3941098C2 (en) * | 1989-12-13 | 1994-02-03 | Dornier Gmbh | Device for the production of crystals |
| DE3941099A1 (en) * | 1989-12-13 | 1991-06-20 | Dornier Gmbh | HANDLING LIQUIDS UNDER WEIGHTLESSNESS |
| US5100626A (en) * | 1990-05-24 | 1992-03-31 | Levin Andrew E | Binding assay device with removable cassette and manifold |
| US5212065A (en) * | 1990-10-25 | 1993-05-18 | International Diagnostic Systems, Corp. | Rapid assay device |
| US5350693A (en) * | 1993-04-08 | 1994-09-27 | Long Island Jewish Medical Center | Multichamber syringe device for fusing cells |
| US5631734A (en) * | 1994-02-10 | 1997-05-20 | Affymetrix, Inc. | Method and apparatus for detection of fluorescently labeled materials |
| US6114122A (en) * | 1996-03-26 | 2000-09-05 | Affymetrix, Inc. | Fluidics station with a mounting system and method of using |
| EP0899572A4 (en) * | 1996-05-01 | 2005-02-02 | Sanko Junyaku Kk | Automatic immunoassay method and apparatus |
| US6511277B1 (en) | 2000-07-10 | 2003-01-28 | Affymetrix, Inc. | Cartridge loader and methods |
| US6422249B1 (en) | 2000-08-10 | 2002-07-23 | Affymetrix Inc. | Cartridge washing system and methods |
| ATE467115T1 (en) | 2002-03-15 | 2010-05-15 | Affymetrix Inc | SYSTEM AND METHOD FOR SCANNING BIOLOGICAL MATERIALS |
| US20040120861A1 (en) * | 2002-10-11 | 2004-06-24 | Affymetrix, Inc. | System and method for high-throughput processing of biological probe arrays |
| US7317415B2 (en) | 2003-08-08 | 2008-01-08 | Affymetrix, Inc. | System, method, and product for scanning of biological materials employing dual analog integrators |
| US20060246576A1 (en) * | 2005-04-06 | 2006-11-02 | Affymetrix, Inc. | Fluidic system and method for processing biological microarrays in personal instrumentation |
| DE102006002258B4 (en) * | 2006-01-17 | 2008-08-21 | Siemens Ag | Module for the preparation of a biological sample, biochip set and use of the module |
| WO2008023579A1 (en) * | 2006-08-21 | 2008-02-28 | Panasonic Corporation | Measuring device, measuring instrument and method of measuring |
| AU2008269201B2 (en) | 2007-06-21 | 2011-08-18 | Gen-Probe Incorporated | Instrument and receptacles for use in performing processes |
| US20110262895A1 (en) * | 2008-08-07 | 2011-10-27 | Harding Robert E | Methods for the diagnosis of varicella zoster virus infection |
| WO2010075025A2 (en) * | 2008-12-16 | 2010-07-01 | Shell Oil Company | Device for evaluating a heat exchange fluid |
| US9767342B2 (en) | 2009-05-22 | 2017-09-19 | Affymetrix, Inc. | Methods and devices for reading microarrays |
| DE102012109317A1 (en) * | 2012-10-01 | 2014-04-03 | Astrium Gmbh | Device for carrying out a biochemical analysis, in particular in space |
| CN117571979B (en) * | 2024-01-15 | 2024-03-19 | 佳木斯大学 | Premature rupture detection kit for fetal membranes for obstetrics and gynecology department |
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| US31006A (en) * | 1861-01-01 | Feeding mechanism for spoke-machines | ||
| CA1036935A (en) * | 1973-03-19 | 1978-08-22 | Lavell R. Johnson | Method and apparatus for radioimmunoassay with regeneration of immunadsorbent |
| US3883044A (en) * | 1973-03-30 | 1975-05-13 | Buchler Instr Division Nuclear | Micropipetter, especially for the discharge of a sample and a diluent |
| US4053284A (en) * | 1976-03-10 | 1977-10-11 | Akzona Incorporated | Continuous flow apparatus for biological testing |
| DE2719234C2 (en) * | 1977-04-29 | 1985-03-28 | Hans A. Dipl.-Chem. Dr. 8000 München Thoma | Multi-channel system for handling immobilized, biologically active substances |
| FR2396969A1 (en) * | 1977-07-06 | 1979-02-02 | Pasteur Institut | DEVICE AND METHOD FOR MULTIPLE ANALYZES |
| GB1601242A (en) * | 1978-02-06 | 1981-10-28 | Miles L E M | Multiple assay machine and method |
| JPS54111892A (en) * | 1978-02-17 | 1979-09-01 | Ii Mairusu Rooton | Method and device for inspecting numerous data |
| US4324758A (en) * | 1979-04-12 | 1982-04-13 | The United States Of America As Represented By The Secretary Of The Air Force | Analysis of lubricating oils for iron content |
| US4276048A (en) * | 1979-06-14 | 1981-06-30 | Dynatech Ag | Miniature reaction container and a method and apparatus for introducing micro volumes of liquid to such a container |
| AU553422B2 (en) * | 1980-12-22 | 1986-07-17 | Commonwealth Serum Laboratories Commission | Method and apparatus for detecting antigens and antibodies |
| NZ199286A (en) * | 1980-12-22 | 1986-05-09 | Commw Serum Lab Commission | A method of detecting antibodies or an antigenic or haptenic substance in a sample |
| US4476231A (en) * | 1981-07-22 | 1984-10-09 | International Remote Imaging Systems, Inc. | Method of analyzing the distribution of a reagent between particles and liquid in a suspension |
| AU556898B2 (en) * | 1981-09-25 | 1986-11-27 | Commonwealth Serum Laboratories Commission | Capillary based rotomat for immunoassays |
| NZ201901A (en) * | 1981-09-25 | 1986-11-12 | Commw Serum Lab Commission | An apparatus suitable for performing automated heterogeneous immunoassays in a plurality of samples |
| US4458020A (en) * | 1982-11-15 | 1984-07-03 | Quidel | Integrated single tube plunger immunoassay system having plural reagent chambers |
| US4585623A (en) * | 1984-02-27 | 1986-04-29 | Allelix Inc. | Device for performing quantitative chemical and immunochemical assays |
-
1983
- 1983-11-07 CA CA000440575A patent/CA1230552A/en not_active Expired
- 1983-11-15 US US06/551,906 patent/US4665034A/en not_active Expired - Fee Related
-
1984
- 1984-11-02 AU AU34936/84A patent/AU579329B2/en not_active Ceased
- 1984-11-06 EP EP84307634A patent/EP0144162A3/en not_active Ceased
- 1984-11-07 JP JP59233396A patent/JPS60128369A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| AU3493684A (en) | 1985-05-16 |
| JPS60128369A (en) | 1985-07-09 |
| EP0144162A3 (en) | 1986-12-30 |
| AU579329B2 (en) | 1988-11-24 |
| EP0144162A2 (en) | 1985-06-12 |
| US4665034A (en) | 1987-05-12 |
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