CA2133097A1 - Immunoassays employing generic anti-hapten antibodies and materials for use therein - Google Patents

Abstract

The present invention relates to immunoassay methods for detecting and measuring the amount of an analyte in a sample by means of generic anti-hapten antibodies. Also disclosed are multi-analyte immunoassay methods. Reagents, devices, and kits using the anti-hapten antibodies are also disclosed. The present invention also relates to dyed erythrocytes, preferably fixed, which are coated with antibodies. Also disclosed is the use of these dyed erythrocytes in agglutination assays to detect and measure the presence of an analyte in a sample. The analyte can be a hapten, an antigen, or an antibody. Also included are agglutination assays, compositions and kits using these dyed and coated erythrocytes.

Description

WO 93/20446 PCr/US93/~2920 2 1 ~?~
IMMUNOASSAYS FMPLOYING GENERIC ANrl-HAPTEN ANTIBODIES
AND MATERIALS FOR USE THEREIN

Field ~f Inv~n~.ion S This invention relat~s to the field of immunoassays.

Back~ro~nd Qf_~hQ Inv~nti~n In-vitro diagnostic tes~s via immunoassays typically involve antibodies with specific binding affini~y toward the analytes of interest. In such test configurations, antigenic analytes either bind directiy with the antibody or compete with a hapten-label conjugate (competition assay~.
In the ~ormer case, the antibody-antigen complex may be allowed to grow as in agglutination assays. Alternativçly, in sandwich assays, another antibody with affinity to the antigen can be conjugated to a signal label and allowed to bind the;
primary an~ibody-antigen cornplex. The signal r~sponse is directly propo~ional ~o the analyte concentration.
In cornpetition. assays9 competition is sstablished : ~ ~ 20 betw~en the analytes, typically small molecules in nature, andthe hapten~label conjugates. The signal respons~ is inversely propottional to the analyte concentration.
Most target analytes can, in principle, be detected using either the competition assay or the sandwich assay f~rmat.
While there are numerous examples where th~ stated methods s~ved the purpose very well, the utility of ~h~se approaches however, were somewhat limited in that a sin~le assay is ~pecific for a single anaiyt~ only. A multi-analyte assay is difficult.

3 0 The following exemplifies the different available ~, ~ immunoassay ~orrnats. U.S. Pat. No. 4,185,0~4 ~o I~Aochida et al., discloses non-homogeneous assays with wash and separation steps. An insolubilized anti-analyte antibody acts as a primary ~: capture phase tor the analy~e which is conjugated to a hapten (hapten-analyte conjugat~). After washing, soluble lab011ed anti~hapten antibody is added and the complex ~f ~anti-analyte antibody/hapten-analyte conjw9ate/anti-hapter, antibody) is wo 93/20446 PCr/US93/02g20 ~ ~3 3 2 detected. U.S. Pat. No. 4,243,749, to Sadeh et al., discloses another similar sandwich assay format. Sadeh et al., is specifically oriented towards measuring low molecular weight (hapten) antigens. The unknown analyte and the hapten-analyte 5 conjugat~ are incubated together with the insoluble anti-analyte antibody in a compe~i~ive assay format. Following the wash step, soluble labeled anti-hapten antibody is added and the sys~em is washed again and the labeled complex of tanti-analy~e antibody/hapten-analyte conjugate/anti-hapten 10 antibody) detectsd.
Kang et al., ~lin ~;hem., ~(9~:1682-1686 (1 9B6) describes two assay ~ormats~ The first assay format pres~nts an enzyme immunoassay which uses an anti-hapten antibody .
~: coated microparticle (the common capture particle~, an anti-15 analyte antibody conjugated to a hapten (hapten-anti-analy~e-antibody conjugate), and a labeled anti-analyte an~ibody. If the analyte is present in a sample, a complex of (anti-hapten :~ antibody coated microparticle/hapten-anti-analyte-antibody -~ :: conjugate/analyt~/labeled anti-analyte antibody) is formed and detected. ~luorescein serves as the capture hapten. The micropar~iGle is a latex particle.
Kang et al.'s second assay format is similar to tha~ of Bunting, U.S. Pat. No. 4,271,140. The assay format consists of 1 an anti-hapten antibody bound to a solid phase, a hapten ~:: 2 5 corljugated to an anti-analyte antibody (hapten-anti-anal~te anti~ody) and labeled analy~. The complex of all thr~e components are detected.
: ~ . In agglutination assays, either the antibodies or the antigens (or hapten) may be bound to small pa~icles. The particles that have been used as agglutable carriers include latex, charcsal, kaolinite, bentonite, inorganic colloîdal particles, as well as both microbial cells and erythrocytes. See Mochida, lJ.S. Pat. No. 4,308,026. When these c~ated particles (coa~ed with either antibodies or antigens) are mixed with ~amples containing antigens or antibodies, the coated particles would form visually det~ctable agglutination. Agglutination is characterized by the ciumping of the lat~x polyrner particles WO 93/20446 PCr/US93/0292Q
3 3 ~ ~, 7 from an otherwise smooth suspension. Qualitative latex agglutination t~sts can be carried out on a simple slide without the aid of any instn3mer)tation. More than one antigen can be detected simultan~ously by means of differently colored latex particles which have each been sensitized with antibodies of different specificity and then mixed together. Hadfield, S.G., 8t al., I. Imn~3nol .~lethQds, ~7. 153-8 ~1987). U.S. Pat. No.

4,745,075 to Hadfield e~ al, May 17, 19~8. U.S. Pat. No.
4,419,453 further discloses latex particles dyed with Amocid yellow, brilliant crocein 3BA r~d dye, and Calco C)il Blue N Dye.
U.S. Pat. No. 4,745,075 discloses that there are also marketed test ki~s for the grouping of Beta Haemolytic Streptococci which in~lude reagsnts in which the solid phase is a suspension of killed red-dyed or blue-dyed St~phylQçoc~us ~ure~ cells.
tlillyard et al., U.S. paten~ number 5,086,002, discloses an erythrocyte agglutination assay in which the ~gglutination reagent comprises at least one erythrocy~e binding mol~cule coupled to at least one sp~cific analyte binding molecuie wherein the erythrocyt~ binding molecule does not caus~
~: 20 agglutination when incubated with ery~hrocytes in the absence of arlalyte ~in the case o~ a direct assay~ or analyte binding ~: reagent (in the case of an indirect assay). Th~ erythrocy~es are preferably endogenous to the blood sample to be tested.
Mixtures of conjugates and conjugates of anaiyte analogu~s wi~h ery~hrocyte binding molecules may also be used as agglu~ina~ion reagents. Chang, U.S. Pa~. No. 4,433,059, discloses an agglutination immunoassay reagent in which ~wo antibodies are covalently link~d ~tail-to-~ail~, so as not to alter their sp~cif~city. One antibody is specific for an an~igen bome by an indicator subs~ance, such as an ery~hrocytes.

One aspect of ~he invention presen~s an immunoassay for ~: an analyte ~A~. The immunoassay uses an anti-hapten antibody : 35 (aH), a hapten conjugated to an analyte (H-A), and an an~i-analyte antibody (aA). The aA can be labell~d to allow ~or det~ction. If the analyte is not present in the sample, a WO 93/20446 PCI`/US93/02920 3~
complex o~ {(atl)(H-A~(aA)} is forrned. If the analyte is present in the sannple, the analyte will compete with ~ A) for (aA~, to form the complex of ~(aA)(A~}. After an appropriate incubation period, the presence of ~(aH)(H-A)(aA)3 or {~aA~(A)} is detected S or measured. T~e amount of ~(aH)(H-A~(aA~} is inverse~y proportionai to the amoun~ of analyte in the sarnple, whereas the amount of {(aA)(A)} is directly proportionai to the presence of the analyte in th~ sample.
The above assay can be presented in an agglutination assay format wherein the anti-hapten antibody is coated onto a particle, preferably a microparticle. In this case, a separation step is not required. The formation of the complex of {(aH)(H-A)(aA)} can be visually detected in the agglutination o~ the coa~ed par~icles.
Another aspect of the invention presents an immunoassay ~or an antibody (aA) in a sample, by exposing the sample to anti-hapten antibodies (aH~, and hapten conjugated to antigen of the antibody to be assayed ~H-Ag), and labeled antibody (aA~.
~: Th~ resulting complex of {(aH)(H-Ag)~aA*)}, if any, is inversely pr~portional to the amount of the antibody (aA) in the sample, :; and the amount of remaining (aA~) is directly proportional to the amount ~ (aA) in the sample Another aspect of the invention presents a direct -agg3utinati~n assay for antibodies (aA) in a sample, which 25~ exposes the sample to particles coated wlth anti hapten antibodies ~P-aH), and conjugates comprising haptens and antigens to the antibodies (H~Ag~. Agglutination of the coated particles is directly proportional to the amount of an~ibodies in :~ the sample.
Ano~her asp~ct of the invention presen~s the preceding agglutination assays with the addition of parlticles not coated with the an~i-hapten antibodies, the color of the coated and uncoated particles are such that they enhance visualization of the agg!utination or lack thereof.
3 5 Ano~her aspect of the invention presents multi-analyte assays which employ anti-hap~en antibodies attached to a solid phase, and the solid phase is preferably a particle. The multi-W093/20446 ` 2J ~. X~ 7 PCI`/US93/02920 analyte assays can be conducted in competitive agglutination assay formats.
Another aspect of the invention presents reagents ~nd kits for corsducting the above assays.
S Another aspect of the invention presents a multi-analyte assay device with different chambers, wherein each chamber contains a reagent for a specific analyte, and the reagent is a hapten conjugated to the specific analyte to be assayed.
Another aspect of the invention pr~sents dyed and preferably fixed erythrocy~es which have been coated with anti-hapten antibodies, and can be used in agglutination assays.
Also presented are: agglutination assay kits containing the dyed and coated erythrocytes, and compositions c~mprising dyed and :; coated erythrocytes for use in agglutination assays.

rief Q~s~ri~tiQn Qf Drawing~
:~: Figure 1 iliustrates the agalutination of anti-hapten micropartiGles in the absence of analytes.
Figure 2 illustrates the inhibition of agglutination in the presence of analytes.
Figures 3A and 3B illustrate an ~xample of a multi-analyte assay device.
~: Figure 4 illustrates a sandwich assay format for antibody test.
Figure 5 presen~s phQtographs of the negative, threshold, ., ~
positive reactions: in drug assays.

The invention presents an immunoassay for an analy~e (A~.
3 0 The immunoassay uses an anti-hapten an~ibody (aH), a hap~en ' conjugated to an analyte ~H-A), and an anti-analyte antibody :~: (aA). The aA can be labelled to allow for detection. If the analyte is not present in the sample, a complex of {(aH~(H-A)(aA)} is formed. If the analyte is present in the sample, the analyte will compete with (H-A) for (~A), to fom the csmplex :~ of ~(aA)(A)}. After an appropriate period of incubation, the complex of {(aH)(H-A)(aA)} is then separated ~rom the :

wo ~3/20446 P~r/l S93/02g20 3~9 ~

uncomplexed tl-A, aA, or the complex of {~aA)(A)}, if any. The presence of {(aH)(H-A)~aA)} or {(aA)(A)} is then detected or measuFed. The amount of ~(aH~(H-A)(aA)} is inYersely propor~ional to the amount of analyte in the sample, whereas S the amount of {~aA)(A)~ is directely proportional to the presence of the analyte in the sample.
The above assay can be conducted in a competitive assay forrnat, whereby the anti-hapten antibody can be attached to a solid phase.
The above assay can also be presented in an agglutination assay format wherein the anti-hapten antibody is coated onto a particie, preferabiy a microparticle. In agglutination assays, no ~eparation steps are required. The formation of the complex of {(aH)(H-A)(aA)} can be visually detected in the agglutination of the coated particies.
~:: Also disclosed is a direct agglutination assay for the analyte with multiple epitopes using particles coated with ~: anti-hapten antibodies (P-aH~, conjugates of hapten and antibodies against the analyte (H-aA), the degree of 2 0 a~glu~ination of ~he coated particles is directly proportional with the presence of the analyte in the sample.
Also disclosed herein is an immunoassay method for detehing and measuring an antibody (aA) in a sample1 by : ~ exposing the sample to anti-hapten antibodies (aH), and ~apten 25 ~conJugated to antlgen to which the antibody binds (H-Ag), and labeled antibody (aA~). The resulting complex of {(aH3~H-: Ag)(aA~}, if any, is: inversely proportional ~o th~ amount of the antibody (aA) in th~ sample, and the amount oî remainin~ (aA~) -~ is directly proportional to the amount of (aA) in the sample.
301 Also disclosed herein i8 a direct agg!utination assay for antibodies (aA) in a sample, which exposes the sarnple to particles coated with anti-hapten antibodies ~ aH)1 and conjuga~es comprising haptens and antigens to the antibodies ~H-Ag). Agylutination of the coated particles is directly ~-: 3 5 prsportional to the amount of antibodies in th~ sample.
Also disclosed herein are multi-anaiyte assays and assay devices which ernploy anti-hapten antibodies. Reagents and W~ ~?3/204~16 PCI /US93/û~920 kits for conducting all the above assays, for example dyed fixed ~rythrocyt~s coa~ed with anti-hapten antibodies, are also discJosed herein.

s ~m~
The invention can b~ practiced in formats that include:
homogenous, sandwich, competitive, and ag~lutination assay ~urmats. In the sandwich or competitive assay formats, the an~i-hapten antibodies can be attach~d to a solid phase, thus 10 rendering the solid phase into a generic solid phase that can be used to assay different analytes. Materials for solid phase can be any ~f those used for immunoassays. Natural, synthe~ic or naturally occurring materials that are synthetically modified can bs used. They include: polysaccharides, e.y., cellulose 15 materiais including pap~r, cellulose and c~llulose deriv~tives such as cellulose acetate and nitrocellulose; silica; ~iberglass;
inorganic n aterials such as deactivated alumin, diatomaceous arth or other inorganic finely divided material uni~ormly ispers~d in a porous polymer matrix made of polymers such as 2 0 vinyl chloride, vinyl chloride-propylene copolymer, and vinyl chlorid~-vinyl acetate copolym~r; cloth, both naturally occwrring ~e.g., co~ton) and synthetic (e.g., nylon); porous gels such as silica gel, agarose, deadran and gelatîn; polyme~ic films ; ~ such as polyacrylamide; magnetic particl~s; microtitre p!ates;
25 polystyrene tubes; protein binding membranes; agaro~e;
Sephadex (Pharrnacia Fine Chemicals, Inc., Piscataway, N.J.);
Trisac~ Poin~et-Girard, France~; silicon particies; porous fibrous matrixes stc.
One embodiment of the invention presents a eomp~itive 30 1 assay ~orma~ wherein the ~ample ~or which the analy~ss are to be assayed is mixed with hapten-analyte conjugate (I~-A) and antibodies to the specific analytes to be assayed ~anti-analyts antibodies, aA~. The anti-analyte antibodies are labeled for det~ctiont e.g. with enzyme, radioactive, ~luorescent, or 35 chemical labels. The mix~ure is ~hen passed over ~he solid phase, ~o which anti-hapten antibodies have been attached, and incubated for a suffici~nt time to allow the complex o~ ~(aH)(H-WO 93/20446 PC~/US93~0292û

A)(o~A)} to form. Next, the unbound reagents are separated, e.g.the unbound reagents are dissolved in an aqueous medium and wash~d away from the solid phase and the ~onnation of the complex of {~atl)(H-A)(aA)} on the solid phase is detected by S detecting the labeled aA. If--the anaiyte is n~ present, the complex will be present.: If the sampl~ contains the analytes, the analytes will bind the labeled anti-analyte antibodies, and no complex will be present, or the amount of complexes will be reduced. Thus, the presence of the complex is inversely 10 proportional to the analyte concentration in the sample.
Altematively, one can assay for the presence ot the remaining unbound la~elled aA in the aqueous rnedium. Methods for conducting a competitive assay, including the wash step, are weli known in the art, see e.g. Mochida et al., U.S. Pat. No.
15 4,185,084. An exarnple of the competitive assay format is shown below in Example 13.
~: The above approaches can also be applied to agglutination :~ assays. Currently, to test different analytes, especially in agglutination assay format, a specific set of reagents have to 20 be made for each of the analytes. This not only increases the cost of manufacturing, but also makes it impossible to perform a single multi-analyte test. The present invention presents the following advantages:
The anti-hapten coa~ed particle (P~aH) is a generic 2 5 r~ag~nt, which can be used in a variety of tes~s.
2)The as~ay ~ormat makes it possible to run multi-~: analyte tes~s, i.e. to perform multiple tests simult~neously with a single specimen.
Compared to the prior art agglutination immunoassays 301 discussed in the a~k~cQund of the Inv~ntiQn, the present ! ~' invention offers ~he advantages ot simplicityl rapidity, clari~y, economy, sensitivity and specificity. The aggiutination assays presentsd here allow tor visual detection o~ the result and do - not involve wash and separation steps. Whereas the prior art 3 5 agglutination assays require particles that are sensitized to the specific analyte to be detected and do not prwide for multi-analyte tests; the present invention allows for the ~0 93/20446 PCI`/US93/02920 'J~ 7 performance of multi-analyte test with a single specimen, and the use of a generic reagen~ for diff~rent tests.
Th~ pref~rred configuration is as follows:
There are four major components in the system, the microparticle coated with an anti-hapten antibody (P-aH), the hapteri-analyte conjugate (H-A), the anti~analyte antibody (aA
and the specimen containing the analyte (A). In the absence of the analyte ~A~, agglutinates of complexes of {(ocH)~H-A)~aA~}
are ~ormed. This results in agglu~ination tsee Fig. 1~. In the presenGe of the analyte ~A~ however, all the antibodies (aA) are bound to the analyte (A), leaving no ~ree antibody (aA) to bridge ~he complex fonnation (see Fig. 2). Thus no agglu~ination occurs. Therefore, a negative sample results in agglutination, a positive sample gives no or reduced agglutination. Figure 5 shows the agglutinations in the cases of: negative, threshold, and positive rsactions.
One skilled in the art would also realize tha~ the hapten-anti-hapten pairing can be replaced with ligand-recep~or ~; :: pairings; biotin-avidin pairings; pairings of eomplementary nucleic acids; and any pairing that would allow for : agglutination.

One aspect of the invention can be conducted on a rnulti-~:~ 25 chamber aggl~ina~ion device. Each chamber is specific for d0tecting a speoi~ic analyte, and contains a reagen~ specific for tha~ particular analyte. The deviGe p~eferabiy allows the sample/reaction mixture to simultan~ously ~low into each ~hamber and to r.~act within the chamber, but does not allow 30 ! the reverse flow or intermixing of the reaction mixture in one chamber with that of ~he other chambers.
~: The test ~or a given analyte ~Ax~ requires speeific :: eonjugate (H-AX) and anti-Ax antibody (aAx). The eoated partiele (P-aH3 howeYer, is eommon for all analytes in this test 3 5 format, and beeomes a generie reagent in the system.
~or multi-anaiyte assay, one or both of the analyte-speeifie eomponents (H-AX) or (aAx)can be pre-packaged in WO 93/2~446 ~ P~/US93/02920 respective chambers for each analyte in a manifold-chamber device. The preferred devices are disclosed in U.S. Patent Applications Serial No. 138,253, ~iled on December 23t 1987, entitled UAgglutination Reaction Device~ to Parsons, R.G., et al.;
Serial No. 614,762, filed November 16, 1990, entitled ~Improved Agglutination Reaction Device Utilizing Selectively Impregnated Material~, to Forney, R.~l., et al.; Serial No. 614,895, filed November 16, 1990, entitled ~Improved Agglu~ination Reaction Device Utilizing Porous Absorbent Materi~lU to Ropella, P.J., et al; and Serial No. 614,817, ~iled November 16, 1990,~1mproved Agglutination Reaction Device Having Geometrically Modified Chambers~, to Parsons, R. G. et al. These applications ar0 herein incorporated by reference.
One example of these multi-chamber devices is shown in Figures 3A and 3B, which show two views o~ a multi-charlnel device. Preferably, ~different hapten-analyte conjugate (H-A) is contained within each channel. In each chamber, the conjugates (H-A) bear the specific analytes to be det~c~ed in that particular chamber. In a competitive assay, the base of the chamber can be made of the any of the soiid phase materials described above. The device contains means for introducing a portion o~ the sample into each chamber and mixing it with the Gonjugates to ~orm a mixture and yet preventing ~he misnure from entering another chamber. Similarly, the device wo~ld also contains means for allowing the unbound reagents to be separated from the complex bound to the solid phase. For example, if washing is used as a separation step, the device similarly allows the wash solution to enter and exit each chamber carrying with it the unbound reagents, and yet does not ; 30 1 allow the wash solution with the unbound reagents to enter another chaml~er. To achieve this latter end, the same means that separate the sample mixture in one chamber from the other may be used.
Alternatively, a simple microtiter well plate with anti-hapten antibodies bound to its welis could be used to oarry out the competitiYe assay format, as exemplified in Example 13 below.

t,~ ",~,~",",,,"";~ ~",,, ~,~"~

~WO 93/20446 ~ ,~ 3 o 9 ~ PCI`/US93/02920 In the most preferred embodiment, an agglutination format is utilized for the multi-analyte assay. The H-A
conjugates can be dissolved in an aqueous solution, and spotted onto the base of each of the channels and allowed to dry before S the sample nixture is introduced, see e.g. Examp~e 10 below.
The mixture containing the specimen, the coated particle (P-aH) and the (aAx 's) are introduced to the sample loading ~one. As a portion of the mixture flows into the individual channels, the specific conjugates b~come mixed to form complete reaction 10 mi~nures in each channel. The agglutination reaction does not start until the reaction mixture is completed by the combination of microparticles coated with antihapten antibodies, hapten-analyte conjugate, and anti-analyte antibody. By placing different hapten-analyte conjugates (H-15 Ax, H-Ay, tl-Az...) in each channel, and using a cocktail of anti-:~ analyte antibodies (aAx, o~Ay, ocAz, .. ) mixed with the microparticles, dis~inct, simultaneous assays ~or analytes x, y, z, ~tc. will occur in each respective channel. Test results for ; ~; different analytes will be manifested in the individual channels 2 O which contain the specific hapten-analyte conjugates.
Alternative v~rsions o~ test configurations can be realized. All versions can be implemen~ed as the panel test ormatl as well as the singie test forma~.
Examples of the components of the assays ar~ as f~llows:

~ab~
The analyte in~ludes low molecular weight substances~
e.g., steroids such as testosterone, steriol, progesterone, corticosterone, a3dosterone; thyroid horrnones such as thyroxine , ~ i 3 O , and triiodothyronine; physio~ogically active pep~ides e.g. bradykinin, angiotensin, thyroid hormone-releasing hormone, and luteinizing hormone-releasing hormone;
physiologically ac~ive amines such as epinephrine, norepinephrine, histamine, and serotonin; prostaglandin;
35 relatively low molecular weight substances, e.9., insulin, giucagon, adrenocorticotropic hormonel and gastrin; and high molecular weight substancesl e.g.l human chorionic WO 93/20446 PCl`JUS~3/02~2û

~;3~3 1 12 gonadotropirl, grouth hormone, human placental lactogen, immunoglobulin E, alpha-fetoprotein, hepatitis B antig~n. The analyt~ can be a hapten, an antigen, or an antibody. Examples of antigens include antigens of micro-organisms such as human 5 immunodeficiency virus (HIV~ antig~ns, tumor-specific antigens, cell or tissue antigens, and serum antigens. The analyte is preferably small molecules such as ~herapeutic drugs, drugs of abuse, and toxins.

B. ThQ~icles and ~thQds for G~a~. Them Th~ particles are preferably microparticles that are visually ~etectab~e, colore~ microparticles which enabl~ a dir~ct visual readout o~ ~he presence or concen~ration of the analyte in the test sample without the need ~or using additional 15 signal producing reagents. Materials ~or use as such particles include colloidal metals, such as gold and dyed particles as disclosed in U.S. Patent Nurrbers 4,313,734 and 4,373,932. The preparation and use ~ non-metallic colloids, such as colloidal selenium particles, are disclosed in co-owned and copending 20 U.S. Patent Application Serial No. 072,084, filed July 9, 19~7, which is incorporated by r~ference herein. C)rganic polymer lat~x particles can also be used. They are disclosed in co-owned and copending lJ.S. Pat~nt Application Serial No. 248,858, ~: filed Septembsr 23, 1988, which is incorpora~ed by re~erence 25 herein. Other particles of natural or organic polymers can also be used. Othsr preferred particles are cells which can agglutinate, e.g., ~rythrocytes, preferably fixed erythrocyt~s such as Duracyte~M c~lls (Abbot~ Laboratories, North Chicago, :~ Illinois). An example ~ how the erythrocytes can be ~ixed (i.e.
30 stabilized) is shown in J~lmmunQlo~y, 10Q (3):641 (~988). The selection of a particular particle is not critical, so long as the particle is capable o~ agglutination and such agglutination can be visually detected.
Anti-hapten antibody is attached to the particl~ via 35 covalent binding and/or adsorption using known rnethods. For example, particles such as latex particles can be passively coated with ~he antibodies (Hadfield, et al., J. Imm. I\/lethQ~ls, WO 9~/20446 PCI`/US93/02~20 t~ à~

supr~ The method for coating antibodies onto selenium particles disclosed in the Examples below can also be used to eoat other metal ,oarticies, such as gold particl~s. For an ~Iternative method, see e.g. the method disclosed in U.S. Patent 5,07~,100 to Seno, ~or preparing iron colloid-labeled antibodies.

~. Th& lla~tçns and Anti-H~t~n An~i~di~
Th~ hapten can be any small molecule capable of eiiciting immune responses in laboratory animals, usually when conjugated to a protein. Preferably, the hapten only has one antig~nic site. Examples of these haptens are fluoresoein, rhodamine, bio~in, and dinitrophenyl groups. Anti-hapten antibodies can be produced with methods known in the-art, and ~he antibodies can be polyclonal or monoclonal antibodies.
Poiyclorlal antibodies can be produced ~or example, by injecting ~:; a host animal such a~ rabbit, r~t, go~t, mouse etc. with the hapten. Before injection, the hapten can be first conj~Jgated with carriers such as keyhole limpet hemocyanin or bovine serum albumin. Monoclonal antibodies can be p~duced, e.g.
according to the method disclosed in Kohler Muls~ein, Nature, 495-497 (1975) . The antibodies can also be reoombinant monoclonai antibodies, for examplel produced according to the methods disclosed in Reading U.S. Pa~en~ 4,474,893, or C:,abilly et al., U.S. Patent 4,816,567. Within the scope o~ antibodies are :~ also antibody fragments such as Fab, F(ab')2, and Fv fragmen~s.
Such ~ragments can b~ produced by known tschniques.

D. The H~p~çn-Analyte (::or~uga~
30 I The hapten~analyte conjugates, such as fiuorescein-analyte conjugates are commercially available for the commonly assayed analytes, for example they are used as the tracers in Abbott Laboratories' TDX's FPIA ~comrnercially available from Abbott Laboratories, Abbott Park, IL). The hapten-analyte conjugates can also be produced according to methods known in the art, such as disclosed in U.S. Pat. No.
4,668,640 to Wang et al.

WO 93/2û446 PCI/U~93~02920 9 ~ f~

The following are examples of assay formats that can be used for any analyte:
a. (:~ompetitive Assay ~ . Members o~ the immunoreaction consist of the micropartiole coated with anti-hapten antibody, the hapten-analyte conjugate, the anti-analyte antibody and specimen.
In the test, the analyt~ in the specimen competss with the hapten~analyte conjugate for the anti-analyte antibody. The more anaiytes are pr~sent in the specimen, the less the anti-analyte antibodies will be available for agglutination; and vice versa~ Thus the observed agglutination is inversely proportional to the analyte concentration in ~he specimen.
b. P~: L~I~G~fQr ~nti~Qd~
Similarly, the configuration can also be arranged to test ~or antibodies (Ab). Thus, the antibodies of interest becom~ the analytes. The sample is rnixed with the anti-hapten antibody coat~d microparticles ~P-vH), and conjugates of the t-aptens with an~igens for which the antibodies of interest are specific for (H-Ag). Ths concentration of antibodies in the sample is 0 direc~ly proportional to the arnount of aggllltir)ation caused by he formation ~ complexes of {(P-aH)(H-Ag)(Ab~} (as shown in Figure 4).

E. _Dy~ içlç~andlh~ D~ng ~ho~s The pr~sent invention also presents dyed erythrocytes, preferably dyed ~ixed e~ythrocy~es, ~o enhance visualization of the agglutination process. U.S. Pat. No. 4,745,075, to Hadfield et al., in column 3, has sugges~ed dyeing ery~hrocytes that are used agglutination assays. The patent indicated that the 30: 1 eeythrocytes may be prepared or dyed according to standard methods, see e.g. U.S. Pa~. No. 4,419,453 and German P~tent Applica~ion DT~3000-483, and that particularly suitable colors include red, yellow, blue, green, biacki cyan, magenta, and white.
3 5 The current invention presents erythrocytes which can be dyed, ~or example, red, green or blue The dyed erythrocy~es, preferably ~ixed e~throcytes such as the commercially 6 PCI`/US93/02920 2 ~ f ? ~

available Duracy~eTM cells, can be coated with antibodies and the antib~di~s still maintain their abilities to bind their antigens and cause agglutination of the dyed erythrocytes. Dyed particles can be used at lower concentrations than their non-S dyed counterparts with comparable performance. Preferably, the dyes firmly adhere to the cells and do not leach into the surrounding assay solution. The preferred dyes for cells such as fixed erythroeytes are: Cibachrome Blue 3GA (Sigma Chemical Co., St. Louis, M0); the Reactive Color Seri~s ~rom Sigma Chemical Co., such as Reactive Red, Reactive Green, Reactive Yellow, etc.; diazonium dyes (such as Fast Black K, Fast Blue 8 ~rom Sigma Chemical Co., St. I ouis, M0); and organic dyes with lodoacetamide or maleimide coupling chemistry (e.g. Rhodamine iodoacetamide, Rhodamine maleimide, Eosine iodoacetamide, Eosine maleimide, Tetrame~hylrhodamine maleimide, and.
Tetramethylrhodamine iodoaf~etamide). In the case of fixed erythrocytes, an important ~eature of the above listed dyes is :~ that they couple to the erythrocytes via functional groups other ~: than amino groups, since the erythrocytes have no or very ~ew :: 20 free amino groups available tor binding dye. The most preferred dyes are those which covalently bind to the erythrscytes, preterably fixed erythrocytes. The intensities of the colors of the different particles are preferably balanced to achieve good di~ferentiation between agglutination and lack thereof.
The dyed particles can be used in th~ above agglutination reactions wherR ~wo or more populations of particles (of different colors) can be mixed to perform m~sltiple assays. The ~:~ different colored parlicles are preferably present in about :: equal amounts. Reactions with either of the popula~ions of 30 I particles cause an overal3 change in the color of the solution which can be easily visualized. In the multi-analyte assay format using generic anti-hapten par~icles, o~her colored particles can be used, for example, differently colored latex and plastic particles. This greatly enhances one's ability to read 3 5 these tests and allows for multiple tes~s to be run simultaneously. Thus, ~he invention poses an advantage over WO 93J204~16 P~/US~3/02920 J~ 16 prior art where each individual assay and control are run separately, incurring extra ~ime and labor.

EXAMPLES

~inin~ of Fixed Human Erythro~vtes with Co~rn~ssie Briliiant Fixed human erythrocytes (Duracyte~', Abbott 10 Laboratories, ~) were suspended in 0.1 M citrate buffer, pH
3.0 ~t a final concentration of 5% (v/v). Coomassie Brillian~
Blue R-250 dye (Biorad Labs., Richmond, CA) was added to a final ~onGentration of .5% (wlv) and the cells were allowed ~o incubate at room temperature for 1.5 hours. Finally the cells 15 wer~ alternately centrifuged (1 000 xG, 1 min) and washed with phosphat~ buffered saline (PBS, 10 mM sodium phosphate and .15 M NaCI, pH 7.4) until the supernatailt had very littie residual color. The resul~ant susp~nsion of cells had a dark purple coior.

Staininq of Fixed Hum~n Er~hro~vt~jl~
A 10% suspension of DuracyteTM cells was prepared in 50 mM NaOH. Cibacron Blue 3GA (Sigma Chemieal Co., St. Louis, MO) was added to th~e suspcnsion to a finai eoncentration ~ 50 mg/ml. The susperlsion was allowed to mix at room :~ tempe~ature for 1.5 hours. The cells were similariy centrifugedand washed ~s in Example 1. The resultan~ celi suspension had a dark blue color.

3 0 E~CAMPLE 3 S~ai s with Gi~c~n R~d 2 A 10% suspension of DuracyteTM cells was stained red using the procedure described in Example 2 and Reactive Red 2 (Sigma Chemical Co., St. Louis, MO). The resultant cell suspension had a red color.

WO 93/~044i6 PCI/US~3/02920 $

~oa~ing of Fix~ Human Erythro~y~ uQres~
A 10% (v/v) suspension of Duracyte~ cells were coated with affinity-purified rabbit anti-fluorescein at a S concentra~ion of 100 ~lg/mL in the presence o~ .05% (w/v) chromic ehlorid~ in .1 M sodium acetate buffer at pH 4Ø The suspension was incubated at 30C for 1 hour with occasional mixing via inverting the reaction test tube. After centrifugation ~1000 xG, 1 min), the cells were washed two times with 8X Volume of PBS and then incubated with 1% (w/v~
human serum albumin (Sigma C~hemical Co., St. Louis1 MO) in 25 mM Tris/HCI buffer (pH 8.0) at room ~emperature for 30 minutes. The cells were finally resuspended in PBS (see Example 1) to a final ceill concentration of ~0% (v/v).

Çoatinq ~f C::~llQLdal S~l~nium with Anti ~ ein :: About 57 mL of stock selenium colloid (ODsso 12.3 (prepared according to the method disclosed in U.S. paten~
:~ 20 application Serial No. 072,084, $upra) was centrifLIged at 750 x G ~or 25 min. The soft pellet was suspended in 20 mL of Milli-(;2 water (Millipore Corp., Bedford, MA). The centrifugation and r~suspension was repisated twice. The pH of 400 ml Milii-Q
water was carefully adjusted with .2% sodium carbonate to 7.8.
About 8.7 mL ((:)D 550 = 688) of the selenium was added to the pH 7.8 solution. Then .5 mg of protein-A purified rabbit-anti-:~ fluorescein JgG was added to the selenium suspension. This ;:~ was gently stirr~d at 2-8C overnight. Bovine s-orum a~bun in (BSA, Sigma Chemical Co., St. Louis, MO) was added to the 30 j suspension to a concentration of .5%. Stirring was continued for anothér 2 hours. The mixture was washed as described above via cen~rifugation. After the last centrifugation, the pelle~ was suspended in .1% BSA, 5mM HEPES (N-[2-hydroxyethylJpiperazine-N'-[2-ethanesulfonic acid~, Sigma 3 5 Chemicai Co" St. Louis, MO), pH 7.8 to give an OD550 of 2.0 when 15 ~lL w~s diluted into 1 mL of water. This was stored at 2-8C.

WO g3/2~)~46 PCl /US93/û~92~) ~ s 3 3 ~ ~ 18 ~atLn~ of CollQidal P~lypyrrole with AntifluQre~in Stock polypyrrole ~PP) at t mL, ~350 OD800/ml was S washed 3 tirnes witl~ water (1 mL) via centrifugation at 1000 x G, 5 min each (Intema~ional Equipment Co., Needham Height, MA~. About 200-300 ~LI of the washed PP was mixed with .5 ml of 250 mM MES (2-~N-morpholino~ethan~sulfonic acid, Sigma Chemical Co., St. Louis, MO~ pH 7.0 and 4.5 mL water and 10-2~0 ~9 of protein-A purified rabbit anti-fluorescein IgG for 1 0 min.
BSA was added to ~he mixture to a concentration of .05%. The suspension was mixed for another 10 min. The preparatlon was then washed 3 times with (1 mL) .5% BSA, 35 mM MES at pH 7Ø
The mixture was finally suspended in 50 1ll of the same buffer.

:: Fix~d ~lum~n ~hrocyl~n~i-fl~;uçs~çin A$say f~r_ÇQ~aine :: ~etab~lite~ ~BenzQ~ognine) One mL of anti-fluorescein-coated DuracyteTM cells (see Example 4) was resluspended in 3 ml Duracyte buffer (0.û67 M
~: sodium phosphate a~ pH B.0, .75 M NaCI, 20 mM EDTA
(ethylenediamine tetraacetic acid, Sigma Chemical Co., St.
Louis, MO~, 1.5% fetal ~alf s~rum, 6% Amphol~rte (Phannacia LKE3 Biotech., Piscataway, NJ) and .1% sodium azide). For this assay, ~ 25 25 1ll of the above anti-fluorescein DuracyteTM cells, 5 ~11 of : ~: sheep anti-cocaine antisera, 10 ~11 of the urine specimen and 5 111 of a diluted fluorescein-cocaine conjugat~s ~TDx cocaine tracer diluted at two-fold with 0.15 M NaCI; The tracer was :~ ob~ained from TDx Cocaine Metabolites Kit, commercially available from Abbott Laboratories, North Chicago, IL) were mixed, via repeated aspirating and dispensing, and then added ~o a larninated test card (similar to that disclosed in Example 1 of pending U.S. application Serial No. 07/6~4,B17, ~pra, except that each card used herein contained ten reaction channels).
Within 5-10 min, the viewing areas of the test cards were visually examined for agglutination patterns indicative of positive or negative results. A granular agglutination pattern rWO 93/20446 PCI`~US93/02920 in the viewing zone indicated the absence of cocaine (Ben~oylecognine) in the samples (neg~tive result). A smooth pattern without any agglutination was found when samples with Ben~oy~ecognine were used (positive result). Twenty known S benzoylecognine-positive samples and 18 negative samples were tested. All positive samples gave positive results and all negative samples gave negatiYe results.
Tests for opiates, cannabinoids, amphetamine and ph~ncyclidine were analogously configured. Known positive and 10 negative samples for eaoh of the analytes all give the corresponding positive and negative results respectively.

Coll~i~l Selen~m ~ fluorQscein Assay ~or Phencyçlidine Phencyclidine-fluorescein conjugate ~PCP tracer from TDX
PCP Reagenl Ki~, Abbot~ Laboratories, supra) was mixed with a ~; ~ stook preparation of anti-fluorescein-coated selenium (see ~: Example 5) at a concentration of 0.04% (%). Thirty ~l of the mixture was aliquoted into each of 4 test tubes. Five ~11 of 20 urine samples containing 0, 25, 60, and 120 ng/mL
phencyclidine were added to each of the test tubes~ Then 5 lli of anti-phencyclidine antibody (TDX PCP antisera from TD"PCP
Reagent Kit, Abbott Laboratories, supra) was added. After ~ 8 ~: min at room temperature, 1 ml of water was added to each of 25 : the test tubes. The mixtures were vortexed and measured for optical d~nsity at 550 nm (nanometer). The results were:
Phencyclidine Concentration ODss~
(nglrD~
3 0 ! 0 . 2 0 7 .51 6 0 .758 1 2~ .823 Tests for opiates, cannabinoids, cocaine and Thyroxine (T4) were analogously configured except that serum instead of ,.~f.. ~ ,.. fi " " .~ " " ~ " ~

WO 93/20446 PCI`/U~;~3/02920 urine samples were used in the case of T4. The results all showed increased OD ~50 ~Nith increasing concen~ration of analytes.

S E)(AMPLE 9 PolypyrrQI~-anti~ Qre~ein As~ay for Thyr~xinç
In each o~ 2 test tubes, 7 1ll of anti-fluorescein-coated polypyrrole ~se~ Exarnple 6) and 2 lli of thyroxine standards containing 0 ,ug/mL and 0.24 ~g/ml in serum, resp~ctively, and 1.5 ul of the TDx fluorescein-thyrsxine tracer and lQ 1ll of anti-thyroa~ine antisera from T[:)x Thyroxine Kit, Abbott Laboratories, ~, were mixed. After 10 min incubation at room tempe~atwre, the reaction mixtures were quenched with 1 ml of water. Optical density at 800 nm were measured for both mixtures. The results were:

Thyroxine Concentration C)800 g/ml ~;~ 0 ~.~2 0.25 3.73 : ~ ~ EXAMPLE 10 0 :~ 25 ~
: H3Jnan Ery~hrocyt~s 1: : An aliquot t1 IlL) of the TDx Fluorescein-drug tracer solution from each of the TDx assay kits ~or Cocaine Metabolites, Opiates, and PCP (Abbo~t I abora~ories, ~) was 30 , placed and dried in a differ~nt channel of a laminated reaction card (The reaction card is described in Example 7~. These ~: reagent spots wers positioned in the narrow, straight portions he reaction channels. An antisera cocktail was made by rnixing 20 ~11 from each of the three antisera solutions 35 (Antisera solutions trom TDx reagent kits) trom each of the same three assay kits along wi~h 16 ~l of a 10% suspension ot anti-fluorescein coated Blue DuracyteTM cells (Examples 2 and ~,t~

WO 93/20446 PCT/US93/~2920 X ~, J ~

4), and 64 ,ul Duracy~e Buffer (Example 7). This cocktail (140 ul) was mixed with 40 lli of a normal (drug free) urin~ sample and 45 ,ul aliquots of the resultan~ solution were added to the laminated reaction cards and from there the solution ~lowed 5 (through capillary action) into the channe~s having each of ~he three respective tracers. Within 5 minutes strong agglutination pattems were visible in each of the three channels. This experiment was repeated using samples which contained various concentrations of either Benzoylecognine, morphin~, or 10 PCP and the results are presented in the Table below:

REACTION NUMBER

~ ~RUG IN SAMPLE (ng/mL~

: ~ Benzoyl 0 1670 0 0 1670 1670 0 1670 Morph 0 û 330 0 33û 0 330 330 PCP 0 û 0 167 0 167 167 167 :
Tl:l~EB AGGLOTINATION IN C:HP.NNELS

Cocaine + - +
2 5 Opiate ~ ~ - + - ~ - -PCP + + ~ +
:

In every case when a given drug (either Benzoylecognine, 3 0 , Morphine, or PCP) was pres~nt in ~he sample, ~he agglutination reaction was inhibited in the reac~ion card channel that had the Tracer corr~sponding to that drug. The pr~sence of a given drug did not have any effect on the aggiutina~ion reac~ions in the channels that contained tracers for the other drugs.

WO ~3J2044~ PC~/U~i~3/0292~

c~3 ~ 22 (~o~ting Qf. Fixed H~man Erythro~ s wi~ atiti~ B ~iurfa~e Anti~en ~i IBsAg~
Cibacron Blue 3GA-stained fixed human erythrocytes (see 5 Example 2) were coated with monoclonal anti-HBsAg at a final conc~ntration of 120 ~giml using the procedure described in Example 4. The coated cells were suspended in Duracyte buffer (Example 7) at a ~inal concentration of 10% (v/v). These cells (25 ~lL) were mixed with 25 ~lL of either serum containing 12 10 ng/ml HBsAg or serum without any HBsAg (25 IlL) and added to the channels of laminated reaction cards (Example 7). The cells mixed with serum containing HBsAg formed strong agglutinates ;; whereas the cells mixed with serum without HBsAg did not agglutinate.

S~ific Çolo~-cod~d A~salL~Q~B~
An equal volume of the red stained (uncoated) DuracyteTM
~;; cells (Example 3) were mixed with the blue stained (anti-HBsAg 20 co~ted~ DuracyteTM cells (Example 11). The resulting suspension was a dark gray or black color. This mixed Duracyte~ cell suspension was mixed with serum samples containing either 25 nglml or ~ero HBsAg as desoribed in Example 11. In the sample ontaining HBsAg, the blue DuracytesT~ cells agglutinated and 25 ~ were clearly visible against a pink background tunagglutinated red DuracyteTM cells). In the sample without HBsAg, the gray-colored suspension remained as uniform color and did not show signs of agglutination.

Affinity purified anti-fluorescein is diluted with bu~fer ~: (10 ml~A Tris-HCI, pH 9.0, 150 mM NaCI) to a final concentration of 50 llg/mL. 100 ~L aliql~ots of this material is inoubated in ach of the wells of a 96 well microtiter plate for 12 hr at 37 :~ ~ 35 C. The non-bound anti-fluorescein is next aspirated from the wells, and the wells are washed five times with 100 ~L
amounts of PBS oontaining 0.1% BSA. At this point the WO 93/2044~ PCI/USg3/0~20 3~0~7 microtiter plate welis contain an adsorbed, non-soluble coating of anti-flllorescein (coated microtiter plate).
Mixtures (100 ~L) of urine samples containing known amounts of Phencyclidine ~PCP), a PCP-fluorescein conjugate, 5 and an alkaline phosphatase labeled anti-PCP antibody, are added to the wells, such that the first well contains no free PCP, the second well contains 25 ng/mL PCP and the third well contains 250 ngJmL of PCP. The microtiter plate is incubated for 1 hour at 37 C, and the contents of each well are aspirated :;: 10 and ~he wells ars washed five times with 100 ~lL of PBS
containing 0.1% BSA. Alkaline phosphatase substrate reagent (Sigrna 104 Phosphatase Substrate, Sigma Chemical Co., St.
Louis, MO) is added in 100 IlL amounts to each of the wells, and after 10 minutes incubation, a yellow color is observed to be 15 devsloping in the well which did not contain PCP, whereas the wells with in~reasing concentrations of PCP have decreasing ~: amounts of Golor~ In this assay, the amoun~ of color developedis inversely pr~porRonal to ~he amount of PCP in ~he sample.
Analogous assays for other drugs ~opiates, amphetamine, 20 etc.) are performed in other wells of the same coated rnicrotiter plate by adding samples containing mixtures of specific ~luorescein conjugates of those drugs and their respective enzyme-labeled antibody pairs, in the place of the PC~P-~luorescein and the enzyme-labeled anti-PCP used in,~he 25 example above.

,~ ~
liquots (1.5 IlL) of fluorescein-drug tracer solutions for ~: amphetamines, cannabinoids, cocaine, opiates, and PCP (from 30 the respective T~x assay kits available from Abbott Laboratories, upra) ar~ placed and dried in channels 2-6 of the laminated reaction card (Figures 3A and 3S show two views of the card). For a negative control, another 1.5 ~L of Fluoreseein labeled BSA (Sigma Chemicals, St. Louis, MO) diluted to 0.5 3 5 mg/mL in PE~S is added to char.nel 1 of each card. For a positive control, channel 7 of each of the card is left without any dried reagents. An antisera cocktail with anti-fluorescein coated WO 93/20446 P(~/US93/02920 ~,.......
3~ ' l 24 Durac~fe cells is prepared as described in Example 10, however, additional antisera directed against cannabinoids and amphetamine are also added. A 200 ~lL sample of this antibody cocktail is mixed with 20 ~lL o~ normal (drLg-free) human urine, 5 and then introduced into the center of the reaction card. The 13quid simultaneously flows into each ot the channels and mixes with the reagents dried therein. After 5 minutes, the appearance of aggregated cells is clearly visible in Channels 1-6. Channel 1 represents a negative reaction reference and it 10 will show a reaction analagous to the negative assays, independent of whether negative or positive samples are run.
Channels 2-6 provide the individual reactions for each of the 5 drugs (amphetamines, cannabinoids, cocaine, opiates, and PGP) corresponding ~o the individual drug tracers placed in the 15 channels. The Duracyte cells in channel 7 do not aggregate, since no tracer or other reagents are present in this channel, and therefore it provides a positive reaction reference.
All publications and patent applications mentioned in this Spe~ification are herein incorporated by reference to the same 20 extent as if each of them had been individually indicated to be incorpo~ated by re~erence.
Although the ~oregoing invention has been described in some detail by way cf illustration and example for purposes o~
clarity and understanding, it will be obvious that various 25 modifications and changes which are within the skill of those skilled in the art are considered to fall within the scope of the appended claims. Future technological advancements which allows for obvious changes in the basic invention h~rein are also wi~hin the claims.

':;

~' : ~

Claims (37)

We claim:

1. An immunoassay method for detecting or quantifying an analyte (A) in a sample, comprising the steps of:
(a) exposing the sample to an anti-analyte antibody (.alpha.A), anti-hapten antibody (.alpha.H), and hapten-analyte conjugate (H-A) for a sufficient time to allow the formation of a complex comprising {(.alpha.H)(H-A)(.alpha.A)} and a complex comprising {(.alpha.A)(A)};and (b) separating the complex of {(.alpha.H)(H-A)(.alpha.A)} from (.alpha.A), (H-A), and {(.alpha.A)(A)}; and (c) detecting and measuring the presence of {(.alpha.H)(H-A)(.alpha.A)} or {(.alpha.A)(A)}, wherein the amount of {(.alpha.H)(H-A)(.alpha.A)} is inversely proportional to the amount of analyte in the sample, whereas the amount of {(.alpha.A)(A)} is directly proportional to the presence of the analyte in the sample.

2. The immunoassay method of claim 1, wherein the anti-analyte antibody is labelled.

3. The immunoassay method of claim 2, wherein the anti-hapten antibody is attached to a solid phase.

4. The immunoassay method of claim 3, wherein the hapten analyte conjugate and anti-analyte antibody are in a liquid phase.

5. The immunoassay method of claim 1, wherein the analyte is selected from the group consisting of: low molecular weight substances, relatively low molecular weight substances, and high molecular weight substances.

6. An immunoassay method for detecting or quantifying an analyte (A) in a sample, comprising the steps of:
(a) exposing the sample to an anti-analyte antibody (.alpha.A), anti-hapten antibody (.alpha.H), and hapten-analyte conjugate (H-A) for a sufficient time to allow the formation of a complex comprising {(.alpha.H)(H-A)(.alpha.A)}; and (b) detecting and measuring the presence of {(.alpha.H)(H-A)(.alpha.A)}, wherein the amount of {(.alpha.H)(H-A)(.alpha.A)} is inversely proportional to the amount of analyte in the sample.

7. The immunoassay method of claim 6, wherein the anti-hapten antibodies are coated onto particles, and agglutination of the particles indicates the presence of {(.alpha.H)(H-A)(.alpha.A)}.

8. The immunoassay of claim 7, further comprising second particles uncoated with antibodies to the hapten, wherein each of the coated particles and the uncoated particles has a different color, the color of the uncoated particles is different from that of the coated particles, and the absence of agglutination is detected by the combined colors of the coated and uncoated particles, and the presence of agglutination is detected by the color of the agglutinated coated particle against the background color of the uncoated particles.

9. The immunoassay method of claim 8, wherein about equal numbers of the coated and uncoated particles are used.

10. The immunoassay method of claim 7, wherein the particles are microparticles.

11. The immunoassay method of claim 7, wherein the particles are selected from the group consisting of: cells, latex microparticles, plastic microparticles, selenium microparticles, iron microparticles, and gold microparticles.

12. The immunoassay method of claim 11, wherein the cells are dyed erythrocytes.

13. The immunoassay method of claim 6, wherein the analyte is selected from the group consisting of: low molecular weight substances, relatively low molecular weight substances, and high molecular weight substances.

14. The immunoassay method of claim 13, wherein the analyte is selected from the group consisting of: haptens, antigens, and antibodies, wherein said haptens are not the same as those recognized by the anti-hapten antibodies coated on the particles, or the haptens in the hapten-analyte conjugates.

15. The immunoassay method of claim 14, wherein the analyte is selected from the group consisting of: drugs, toxins, vitamins, hormones, antigens of microorganisms, cellular antigens, tissue antigens, allergens and enzymes.

16. The immunoassay of claim 15, wherein the drug is a drug of abuse or a therapeutic drug, and is selected from the group consisting of: phencyclidines, opiates, cannabinoids, amphetamines, cocaines, and steroids.

17. An immunoassay for detecting and measuring the presence and amount of an antibody (.alpha.A) in a sample, comprising the steps of:
(a) exposing the sample to anti-hapten antibodies (.alpha.H), and haptens: conjugated to antigens which the antibody binds (H-Ag), and labeled antibodies (.alpha.A*); and (b) detecting the presence of a complex of {(.alpha.H)(H-Ag)(.alpha.A*)}, or free (.alpha.A*), the amount of the complex {(.alpha.H)(H-Ag)(.alpha.A*)} being inversely proportional to the amount of the antibody (.alpha.A) in the sample, and the amount of (.alpha.A*) being directly proportional to the amount of (.alpha.A) in the sample.

18. A direct agglutination assay for detecting and assaying for the presence and amount of antibody (.alpha.A) in a sample, comprising the steps of:

(a) exposing the sample to particles coated with anti-hapten antibodies (P-.alpha.H), and conjugates comprising haptens and antigens go the antibodies (H-Ag); and (b) detecting and assaying the agglutination of the coated particles, the agglutination being directly proportional to the amount of antibodies in the sample.

19. A reagent for assaying analytes, comprising:
(a) anti-analyte antibodies; and (b) particles coated with antibodies directed against a hapten which is not the analyte.

20. An immunoassay kit comprising:
(a) a first container containing anti-analyte antibodies;
(b) a second container containing particles coated with anti-hapten antibodies (.alpha.H-P); and (c) a third container containing conjugates comprising haptens conjugated to the analytes (H-A).

21. The immunoassay kit of claim 20, wherein the anti-hapten antibodies are selected from the group consisting of:
anti-fluorescein antibodies, anti-rhodamine antibodies, anti-biotin antibodies, and antibodies directed against dinitro-phenyl groups.

22. An immunoassay kit comprising:
(a) a first container containing anti-analyte antibodies, and particles (P) coated with anti-hapten antibodies (.alpha.H-P); and (b) a second container containing conjugates comprising haptens conjugated to the analytes (H-A).

23. An immunoassay device for detecting and quantifying different analytes in a sample, comprising multiple chambers, wherein each chamber contains conjugates of a hapten and a specific analyte to be detected in the chamber, and means for introducing a portion of the sample into each chamber and mixing it with the conjugates in that chamber to form a mixture and means preventing the mixture in one chamber from entering another chamber.

24. The device of claim 23, wherein the particles are selected from the group consisting of: cells, latex microparticles, plastic microparticles, selenium microparticles, iron microparticles, and gold microparticles.

25. The device of claim 21, wherein the hapten is selected from the group consisting of: fluorescein, rhodamine, biotin, and dinitro-phenyl groups.

26. An agglutination assay method for simultaneously detecting or quantifying more than one analyte in a sample, comprising the steps of:
(a) employing a device which contains more than one chamber, wherein each chamber contains a conjugate (H-A) of a hapten specific to an analyte to be detected in said chamber, (b) mixing the sample with particles coated with anti-hapten antibodies (P-.alpha.H) and antibodies to each analyte to be detected, to form a mixture;
(c) introducing a portion of the mixture into each chamber, whereby the portion, once introduced into the chamber would not escape and enter other chambers, and the portion becomes mixed with the conjugate (H-A) in that chamber;
(d) detecting or measuring the presence of agglutination in each chamber, the amount of agglutination in each chamber being inversely proportional to the amount of analyte in the sample which is specifically being assayed for in that chamber.

27. An erythrocyte coated with antibodies and dyed a color different from the original color of the coated erythrocyte, said dyed and coated erythrocyte is capable of agglutinating in an agglutination assay.

28. The coated and dyed erythrocyte of claim 27, wherein the erythrocyte is fixed.

29. The coated and dyed fixed erythrocyte of claim 28, wherein the dye used to dye the erythrocyte is selected from the group consisting of: Coomassie Brilliant Blue R-250, Cibacrome Blue 3GA, Reactive Color Series, diazonium dyes, organic dyes with lodoacetamide or maleimide coupling chemistry.

30. The coated and dyed erythrocyte of Claim 29, wherein the organic dyes with iodoacetamide or maleimide coupling chemistry are selected from the group consisting of:
Rhodamine iodoacetamide, Rhodamine maleimide, Eosine iodoacetamide, Eosine maleimide, Tetramethylrhodamine maleimide, and Tetramethylrhodamine iodoacetamide; and the Reactive Color Series is selected from the group consisting of:
Reactive Red, Reactive Green, Reactive Yellow; and the diazonium dyes are selected from the group consisting of: Fast Black K and Fast Blue B.

31. The coated and dyed erythrocyte of claim 27, wherein the dye covalently binds to the erythrocyte.

32. An agglutination assay kit for an analyte comprising:
(a) a first container containing erythrocytes coated with anti-hapten antibodies and dyed a color different from the erythrocytes' original color, and (b) a second container containing another reagent for agglutination assay.

33. The agglutination assay kit of claim 32, wherein dye used to dye the erythrocyte is selected from the group consisting of: Coomassie Brilliant Blue R-250, Cibacrome Blue 3GA, Reactive Color Series, diazonium dyes, organic dyes with lodoacetamide or maleimide coupling chemistry.

34. The agglutination assay kit of Claim 33, wherein the organic dyes with iodoacetamide or maleimide coupling chemistry are selected from the group consisting of: Rhodamine iodoacetamide, Rhodamine maleimide, Eosine iodoacetamide, Eosine maleimide, Tetramethylrhodamine maleimide, and Tetramethylrhodamine iodoacetamide; and the Reactive Color Series is selected from the group consisting of: Reactive Red, Reactive Green, Reactive Yellow; and the diazonium dyes are selected from the group consisting of: Fast Black K and Fast Blue B.

35. The agglutination assay kit-of Claim 33, wherein the erythrocytes are fixed erythrocytes.

36. The agglutination assay kit of Claim 33, further comprising a third container containing uncoated erythrocytes of a color different from that of the coated erythrocytes.

37. An immunoassay kit for an analyte comprising dyed fixed erythrocytes of different colors coated with different antibodies.