US 3896218 A
Antibodies to serum hepatitis are coupled to carbohydrate macro particles which are then contacted with blood fluids, suitably plasma, which are believed to contain serum hepatitis antigen. The serum hepatitis antigen is thus complexed with the bound antibody and the particles bearing both the antibody and the antigen are removed from the blood fluid. The antigen is then cleaved from the antibody by alkaline treatment, and may, if desired, be itself isolated, suitably by dialysis. The antibody/agarose complex is then available for recycling in a further batch of contaminated blood fluids. There is also disclosed a novel method of highly sensitive measurement of the extent of contamination of blood fluids with serum hepatitis antigen.
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United States Patent 11 1 Charm et al.
1 1 RADIOMMUNOASSAY DETERMINING THE HEPATITIS ASSOCIATED ANTIGEN CONTENT OF BLOOD  inventors: Stanley E. Charm. Newton; Bing Lou Wong, Somerville, both of Mass.
 Assignee: Research Corporation, New York,
 Filed: July 13, 1972 21 App1.No.:271,452
 US. Cl. 424/]; 23/230 B  Int. Cl A6lk 27/04  Field of Search 424/1. [2; 23/230 B; 250/106 T  References Cited UNITED STATES PATENTS 3,555,143 1/1971 Axcn ct al. .1 424/1 3,645,852 2/1972 Axen ct al. t. 424/1 OTHER PUBLICATIONS Aach et al., Proc. Nat, Acad, Sci USA, Vol. 68, No. 5, pp. 1056-1060, May 1971,
[ 1 July 22, 1975 Wide, Radioimmunoassay Methods, Editors Kirkham and Hunter, (1970).
Salmon et al., The Journal of Immunology, Vol. 104, No. 3, March 1970, pp. 665672,
Primary Examiner-Benjamin R. Padgett Attorney, Agent, or FirmOmri M, Behr  ABSTRACT Antibodies to serum hepatitis are coupled to carbohydrate macro particles which are then contacted with blood fluids, suitably plasma, which are believed to contain serum hepatitis antigen. The serum hepatitis antigen is thus complexed with the bound antibody and the particles bearing both the antibody and the antigen are removed from the blood fluid. The antigen is then cleaved from the antibody by alkaline treatment, and may, if desired, be itself isolated, suitably by dialysis. The antibody/agarose complex is then available for recycling in a further batch of contaminated blood fluids. There is also disclosed a novel method of highly sensitive measurement of the extent of contamination of blood fluids with serum hepatitis antigen.
4 Claims, 4 Drawing Figures SHEET PATENTEDJUL 2 2 ms 9 E o. 9 o. m .21
n n u 4 M M m h Haifa JOKFZOU mum. WELPdEUI m0 PATENTEDJUL 22 ms BHAB (lml) 20mg ANTI BODY SALINE SALINE pH ll SALINE pH 7.2
PLASMA WITH HAA MIXING 8, 3OHRS- FILTER NO. I
WHATMAN PLASMA HAA FREE BHAB- HAA (lml) WASH AND FILTER SALINE WITH TRACES OF PLASMA BHAB-HAA DISSOCIATION OF BHAB-HAA SALINE WITH HAA 1(5Om1) Pmmmwm I915 3 8 96,218
TEST CONTROL BHAB PLASMA BHAB SALINE INCUBATE INCUBATE CENTRIFUGE CENTRIFUGE AND WASH AND WASH ADD ADD HAA 1 HAA WASH WASH CENTRIFUGE CENTRIFUGE 0R FILTER 0R FILTER COUNT OF RADIO coum' OF RADIO ACTIVITY B ACTIVITY B RADIOMMUNOASSAY DETERMINING THE I'IEPATITIS ASSOCIATED ANTIGEN CONTENT OF BLOOD The invention described herein was made in the course of work under a grant or award from the Department of Health, Education and Welfare.
FIELD OF THE INVENTION Purification of serum hepatitis contaminated blood.
DESCRIPTION OF THE PRIOR ART It is well known that a small, but. from a clinical point of view. statistically excessive proportion of blood available for transfusion purposes is contaminated by serum hepatitis. This proportion rises steeply in the case of blood from commercial rather than volunteer sources. Heretofore. the vast majority of efforts to isolate serum hepatitis contamination from the patient who will receive the blood or portions thereof have been directed to detection of the presence of serum hepatitis antigen therein. Even if these tests were substantially more sensitive than any available at this time. a positive reading showing contamination would cause the abandonment. for transfusion purposes. of the batch of blood tested. In view of the extreme shortage of blood this is of its very nature an unfortunately wasteful proceeding. On the other hand. a negative reading is no insurance that the blood is totally uncontaminated. In the utilization of red blood cells it is known to wash these cells to remove the serum hepatitis antigen therefrom. this procedure has been found satisfactory. however, it is extremely expensive. This method cannot. of course. be utilized for the purification of plasma which is one of the principal fluids utilized in blood transfusions. While whole blood has a very limited shelf life even under optimum conditions, plasma has a fairly substantial shelf life, thus, it tends to be in great demand when fresh whole blood is not available.
The coupling of an antibody with an antigen is a well known immunological procedure which has found in its various modifications. considerable use as a basis of diagnostic tests wherein the contact of the two particles, one of which may be attached to a carrier, gives rise to agglutination in the test device. The basic problem with extending this method to removal of particles from, say. plasma. is that antibody per so could not be added to the plasma since this would leave antibody contamination in the blood.
A second problem lies in the very costly nature of serum hepatitis antibody. It appears that serum hepatitis is a disease which is substantially confined to man although there are reports that the disease may be caused in certain primates such as chimpanzees. Thus, any antibody which is produced must originate from serum hepatitis antigen of human origin. Hence. from a commercial point of view. a purification method utilizing antigen would only be practical where the nature of the method permitted very substantial re-use of the antibody after the antigen extracted by it from the blood fluid had been removed. In order to be viable such a procedure would have to operate with a regeneration loss of less than 1%. preferably of the order of U.l7 regeneration loss per cycle.
The coupling of an antibody to a suitable carrier such as agarose is known. The most recent work is reported by Cuatrecasas in Bio. Chem. Bio. Phys. Res. Com.. 38, 947 (i970) which cites other work in this area. Cuatrecasas work reports the coupling of insulin antibodies to a particulate form of agarose known Sepharose 2B utilizing the complex to remove small amounts of insulin from body fluids and regenerating the sepharose/antibody complex by removal of the insulin. This work. however, shows a regeneration loss of l()% per cycle which is vastly in excess ofa commercially viable procedure. Thus, the discovery of a suitable mode of cleaving the antigen/antibody complex in an efficient manner is of prime importance.
The amount of serum hepatitis antigen particles present in most blood fluids such as plasma varies widely. however, such fluids generally contain between It) and I0 particles per ml. Many methods of detecting serum hepatitis are known. however, in view of the practical problems caused by the presence of small amounts of serum hepatitis antigen particles much research effort has been directed to detecting trace amounts of antigen. Heretofore, it has been possible to use the technique of irnmunoelectro-osmophoresis (IEOP) to bring the level of sensitivity to about l0 to l0 particles per ml. It is generally estimated that this level of detection would only give a positive reading for from about 25 to about 50% of blood contaminated with serum hepatitis. It should be noted moreover. that the IEOP method requires sophisticated equipment and sophisticated operation. Certain radioimmune assays have been developed Hollinger. et al (J. Immunol. 107. I099) disclose a technique wherein a test sample was incubated for 6 hours with serum hepatitis antibody to which was added serum hepatitis antigen of known ""I radio activity. Incubation was continued for a further 18 hours. and immuno precipatating antibody was then added to the mixture and the precipitate separated out 24 hours later. This method permits the detection of particles down to the level of about If) through 10'" particles per ml. However, it will be seen that the time factor. 2 days, renders the test somewhat questionable for routine testing of blood samples prior to use. And. furthermore, an increase in its sensitivity would be highly desirable.
A further problem associated with the techniques of study of serum hepatitis is the difficulty of obtaining pure serum hepatitis antibody. The techniques of antibody production are well known, however, if the antibody producing system is infused with proteinaceous material of different immunological characteristics it may produce antibodies corresponding to each of those immunological characteristics. Thus, a mode of isolation of high purity antigen is clearly to be considered a highly desirable goal.
SUMMARY OF THE INVENTION In the process of the present invention serum hepatitis antibody (goat) is coupled to agarose. suitably Sepharose 28. by the cyanogen bromide method (See Borath. ct al. Nature, 2l5. l49l (1967)). The thus bound antibody. (hereinafter BHAB) is then mixed with a blood fluid. suitably plasma. and agitated for up to 30 hours. The plasma is separated from the BHABI HAA complex by filtration. The complex is dissociated by washing with saline at pH 9-12, suitably 10.5-1 l .5 and the saline wash containing the HAA concentrated by dialysis. The BHAB is then washed again and utilized for the removal of HAA from a further batch of plasma. In a modification of this procedure, the BHAB may be utilized to test the presence of HAA down to a level of IO to 10 particles per ml. In this procedure BHAB is incubated with the test plasma at about 37 for about 3 hours and a similar sample similarly incu bated in saline to serve as control. The BHAB is separated from the test plasma suitably by filtration or centrifugation and incubated for 1 hour with "I-labeled hepatitis antigen. The control BHAB is of course similarly treated. The radio activity associated with the test and control samples of BHAB is then measured and the ratio compared to a plot of a similar ratio against the number of antigen particles in a test sample set forth on a standard curve produced by serial dilution methods. In place of agarose, filter paper may be used as a support for the test procedure.
The method of HAA removal as set forth hereinabove, may be extended to the preparation of hepatitis free fibrinogen, a clotting factor of great utility in the treatment of the hemophilia and similar conditions. In this procedure, plasma is treated with BHAB as hereinabove, and further treated with agarose to which antifibrinogen has been coupled by similar means. The fibrinogen is then cleaved from the agarose/antifibrinogen complex and is available for use as a hepatitis-free product.
DESCRIPTION OF THE DRAWINGS FIG. I is a graphic plot of HAA particles per ml. of standardized fluid on the linear X axis plotted against the ratio of radio activity of a BHAB/HAA/labeled HAA sample to the radio activity of a BHAB/labeled HAA sample plotted on a logarithmic Y axis.
FIG. 2 is a multiple plot of the number of HAA particles shown to remain in a fluid containing said particles after treatment by the removal method of the present invention and recovered, by standard radio tracer methods, IEOP and the radio immuno assay method of the present invention plotted on a logarithmic Y axis against contact time of the removal agent with the HAA contaminated fluid, plotted on a linear X axis.
FIG. 3 is a flow diagram of the HAA removal method of the present invention.
FIG. 4 is a flow diagram of the HAA detection method of the present method.
DESCRIPTION OF THE PREFERRED EMBODIMENTS In the process of the present invention serum hepati tis antibody (suitably but not critically goat antibody) is coupled to agarose particles, preferably Sepharose 28 by a modification of the cyanogen bromide method. While agarose has been found particularly suitable as a support phase (hereinafter BHAB-A) the invention is not limited thereto. Physically stable supports containing carbohydrate groups may be employed. For example, cellusosic materials such as cotton cloth have been found operative, and, indeed high grade filter paper is the carrier of choice in the testing modification of the invention. In this procedure cyanogen bromide is agitated with the carrier, suitably agarose and the pH increases to between 10 to 12, suitably to about pH II, and maintained at this level for from about 5 to about 20 minutes, suitably for from about 8 to about l0 min utes. The aqueous supernate is removed, suitably by filtration and the suspension washed at reduced temperatures suitably between about 0C and 10C, preferably at about 4C at pH from about 7 to about 8.5, suitably about 7.8. To this suspension of activated agarose is added antibody. The amount of antibody utilized will of course depend upon the titer of the antibody used. However, it has been found suitable to utilize approximately I50 milligrams of antibody to l0 ml. of say sepharose where the hemagglutination titer of the antibody is l:l28,()00. Contact between the antibody and the activated carrier is maintained for about 12 hours with very gentle stirring in which time more than of the antibody is bound to the sepharose. The antibody/carrier complex suitably antibody/sepharose complex (hereinafter BHAB) is then thoroughly washed with a slightly alkaline wash, suitably 0.1 M sodium bicarbonate at pH 8, therafter with an acetate buffer at about pH 4 and finally with a tris buffer at pH 7.4. Suitably this last wash contains a trace suitably about 0.2% of bovine serum albumin. All steps are carried out in the temperarange of 0 to 10C, suitably at about 8C.
In the preferred modification of the method for ex ample. in the removal of serum hepatitis antigen (hereinafter HAA) the BHAB, suitably BHAB-A, in con tacted with the plasma and the mixture agitated. The agitation is suitably carried out by means of gentle shaking. The contacting between the BHABA and the plasma containing the HAA may take place at any temperature between about 0C and 45C. The tempera ture decided upon will be as a result of a compromise between two competing factors. Clearly, the higher the temperature the more rapid the reaction between the BHAB-A and the HAA. On the other hand, side reac tions and deterioration of the plasma is less likely to occur ar lower temperatures. Generally speaking, it has been found most satisfactory to operate in the temperature range of between 4C and 10C, preferably around 8C. At this temperature it has been found by the method discussed hereinbelow that the number of HAA particles are reduced by approximately every 2 hours in a substantially straightline logarithm against time relationship. Thus, over a period of about 24 to 30 hours, presuming an average number of about lO particles per ml, under the general conditions of the process 99999999999999? of the particles of HAA will have been removed. (See FIG. 2) The procedure is operated utilizing approximately I ml. of BHABA, the BHAB-A having 20 mg/ml of antibody protein as sociated with it, per about 8 ml. of plasma.
It is recognized that this forward projection beyond the present sensitivity of the test method makes certain assumptions. Nevertheless, these assumptions are considered to be valid as the same approach is taken in the destruction of Clostridium botulinum in standard food processing wherein the assumption is made that the Cl.botulinum is reduced to a safe level under certain conditions of sterilization in a situation where the test method available to the art at the present time is of sufficient sensitivity to prove this postulate. [See Fundamentals of Food Engineering, 2nd Ed., p. 189, Charm, Ari Pub. Co., Westport, Connecticut (197i )1.
The BHAB-A/HAA complex is then separated from the blood fluid. such as the plasma. either by centrifugation or filtration. Either method is entirely satisfactory. Especially preferred however, is the use of a filter of polypropylene cloth.
The BHAB-A/HAA complex is then dissociated. The conditions of dissociation have been found to be critical where it is desired to recycle the BHAB-A thus produced. The BHAB-A/HAA complex is washed with alkaline saline, suitably made alkaline with aqueous ammonia. A pH range of pH 9 to pH 12 is operative. however. at pH 12 the sepharose support swells and while the antibody can be regenerated and reused at this pH such a high pH value is undesirable for purposes of multiple recycling. At pH 10.5 the dissociation rate is rather slow while the most satisfactory dissociation rate, that is to say, rapid without swelling of the se pharose occurs at pH 1 1. Hence, it is most desirable to work between pH [0.5 and pH ll.
in the dissociation step. the BHAB-A is subjected to several washes with the saline suitably about 4 washes are employed utilizing about volumes of saline for each volume of BHAB-A. the contact time in each instance being from about 15 minutes to 1 hour.
The saline containing the HAA is separated by filtration and the BHAB-A is then washed, suitably with substantially neutral saline, for example, saline at about pH 7.2, the wash separated and the regenerated BHAB-A reutilized in treating further samples of, say, plasma in accordance with the methods set forth hereinabove.
The aforementioned saline wash containing the HAA is then dialyzed, suitably in dialysis tubing surrounded by polyethylene glycol. The concentration of HAA is increased l0O-fold from the wash. The alkaline saline is removed thru the dialysis membrane. The ph of the HAA concentrate is about 7.0. The HAA tested by immunoelectrophoresis is found to be free of the plasma proteins it was originally associated with. HAA in the concentrated condition may have l0 to l0 particles/ml.
Utilizing the basic principles set forth in the present invention the detection of HAA particles down to a level of at least l0" particles per ml. may be carried out. in this procedure BHAB is incubated with test plasma, at the same time, a similar sample is incubated with neutral saline as a control.
While quantities are not critical, it has been found satisfactory to utilize BHAB-A containing about milligrams of antibody (this would correspond to approximately 1 ml. of BHAB prepared as hereinabove).
While the test is operative with BHAB-A it has been found more suitable to proceed using an antibody/filter paper complex (BHAB-P). BHAB-P is prepared in a manner similar to that utilized for BHAB-A. In the coupling step it has been found suitable to utilize filter paper of about 2,-50 sq.mm. in area. Whatman No. 3 chromatographic paper (manufactured by W&R Balston l.td., Maidenstone, England) has been found especially suitable. While the ratio of paper to antibody is not critical, it has been found satisfactory to suspend 70 mg. of antibody in a l0 ml. solution of ().l M sodium bicarbonate containing about 200 pieces of filter paper of the foregoing dimensions.
The incubation is suitably carried out at between about and suitably about 37C for from about l to about 4, preferably for about 3 hours. The BHAB is then separated by filtration or contrifugation and washed, suitably twice, with neutral saline.
Prior to the assay, HAA is labeled with or similar radioisotope compatable with and attachable to proteins by methods unknown in the art. Approximately l0 pl. of antigen (HAA), having a radio activity count of from about 2,000 to about 8,000 preferably about 5.000 counts/min. in 0.5 ml. of neutral saline are incubated with the reacted BHAB-A or BHAB-P of the pre vious step. Preferably the incubation is carried on for from about 30 to about 40, suitably about 37 for from about 30 minutes to about 2 hours, suitably for about l hour, with slow stirring.
The BHAB-A or BHAB-P carrying HAA from the test plasma and the "'l labeled HAA upon it, is then separated by centrifugation or, preferably, filtration and washed, suitably three times, with neutral saline until no further radio activity is noted in the wash. The radio activity of the control carrier is then read by means known to the art. This count is designated as B. The control BHAB-A (or BHAB-P) utilized in the first stage of the present process is similarly treated with labeled antigen (HAA) and washed and the radio activity associated with said control is counted and designated as B,,.
The actual number of particles of HAA per unit volume in the test plasma may be determined by, for example, by a method of direct calculation or by determination from a graph.
In both methods the experimental procedures to gen erate the required data is the same.
The number of particles of HAA per unit volume in a standard sample ("l I is determined by known methods, such as lEOP which are not as sensitive as the method of the present invention. Serial dilutions of this test sample are then prepared using bovine serum albumin in neutral saline to produce a series of test samples of known fivalue, some of these diluted standards will of course be beyond the sensitivity of the [EOF test.
In accordance with the foregoing procedures the B value is determined for each of these diluted standards and designated "B units of radio activity. The value of B is of course the same for any sample taken from a given batch of BHAB.
The radio activity of ratio of the BHAB reacted with test sample and labeled HAA and that reacted with control and labeled HAA, 8/8,, and 8/3,, is calculated for the unknown and the diluted standards respectively. in the latter case the number of HAA particles, "ficorresponding to "BIB is known. Therefore the statement may be made where k is a constant. Similarly,
where k is as above. Therefore therefore B a, a
Hence [Tmay be directly calculated for any given EXAMPLE 1 Preparation of BHAB-A Five ml. of decanted Sepharose 2B is mixed with 300 mg. of CNBr per ml. of Sepharose 2B. The pH is increased to 11 with 4N NaOH and maintained at this level by titration for 8 to 10 minutes. The suspension is washed with 0.1 M NaHCO,;. (4C) pH 7.8, and stirred gently at 4C with 150 mg. of serum hepatitis antibody (goat, Electronucleonics. 1nc., Bethesda. Maryland) in 10 ml. of mixture (hemagglutination titer l to 128,000). Based on absorbancy (280 p.) in the wash, about 77% of the antibody is bound to the sepharose. The thus formed BHAB-A was washed twice with 10 times volume of 0.1 M NaHCO;, pH 8, twice with 10 times volume 0.1 M acetate buffer, pH 4, and twice with 10 times volume of 0.1 M tris buffer, Ph 7.4, containing 0.2% of bovine sermum albumin (all steps at 4C).
EXAMPLE 11 Plasma Extraction/Purification Antibody bound to Sepharose 2B (BHAB-A), l ml. (decanted volume with mg. protein) is mixed with plasma (about 8 ml.) containing HAA (obtained from Massachusetts Red Cross) at 89C for 26 hours. Mixing is carried out with gentle stirring or shaking. Plasma is separated from the BHAB-A/HAA complex by filtration through Whatman No. 1 filter paper. Small sam ples are withdrawn at various times and tested for HAA.
FIG. I shows a plot of the ratio of HAA particles in BHAB-A treated HAA containing plasma to the number of particles in the original sample of plasma, indicating the present limits of detectability in the standard radio tracer, lEOP and modified radio immuno assay technique of the present invention.
EXAMPLE [[1 Regeneration of BHAB The BHAB-A/HAA complex is dissociated by washing with ammoniacal saline (4 X 10 ml.) at pH 11 per ml. of complex, at C for a total of 120 minutes. The BHAB-A is washed with pH 7.2 saline and finally dis tilled water. After a final vacuum filtration to remove water, the BHAB-A is ready for use again. All washes for BHAB are sterilized before use to prevent the development of microorganisms.
The regeneration efficiency of this system was mea sured by reacting about 8 ml. of HAA containing plasma with a batch of BHAB-A originally containing 20.17 mg. of serum hepatitis antibody at 37. The regenerated material was contacted with a fresh sample of plasma from the same bach, and the time required for a negative HAA reading noted using the IEOP technique. These results indicate an increase in time between the second and third regenerations, probably due to mechanical loss, but no further increase in time between the third and twentieth regenerations, indicating that after a Shakedown in the initial runs the BHAB system is stable under the operating conditions of the present invention.
Time for Negative IEOP (hours) No. of Times Test No. Regcncratcd a. Standard Curve in order to determine the level of HAA contamination in a sample of plasma, the BHAB-P used in the test must be standardized. A sample of plasma containing HAA particles (H particles per unit volume) measured by known techniques (i.e. radio tracer or IEOP) is prepared and subjected to serial dilution with saline (pH 7) containing bovine serum albumin (0.2% relative to saline). "BIB is then determined by the radio immuno assay methods set forth hereinabove for a given batch of BHAB-P and B/B plotted against H 17. Preparation of BHAB for the Modified Radio Immuno Assay for HAA About a hundred pieces of Whatman No. 3 chromatography paper (6mm 6mm) are mixed with 0.6 gm. CNBr while adjusting the pH valve to 11.0 by manual titration of 4N NaOH. This level is maintained for 8-10 minutes. The papers are washed with 0.1 M NaHCO;, (4C, pH 7.8) and mixed with mg. of serum hepatitis antibody (goat, ENl Maryland) suspended in 10 ml. of mixture. The mixture is stirred very gently at 4C overnight. The thus formed BHAB-P is washed twice with ml. of 0.1 M NaHCO pH 8, twice with 100 ml. 0.1 M acetate buffer, pH 4, and twice with 10 ml. of 0.1 M tris buffer. pH 7.4. containing 0.2% of bovine sermum albumin (all steps at 4C). c. Operating Procedure 1. Each piece of BHAB-P is added to one-half ml. of test plasma; then incubated at 37C for 3 hours. At the same time another piece of BHAB-P should be mixed with one-half ml. of saline as a control. The incubation for both continues for 3 hours. 2. The BHAB moieties are separated from the plasma and saline respectively by filtration and each washed twice with saline (5 ml., 25C).
3. lOul. of a previously prepared batch of lhepatitis antigen (5,000 cpm) are incubated with each moiety of BHAB from Step 2 for 1 hour with slow stirring.
4, The BHAB-P papers are each separated and washed with saline three times (5 ml. each time, 25C).
5. The radio activity associated with each BHAB-P piece incubated with the test sample is counted. This count is designated as B.
6. Similarly the radio activity associated with the antibody incubated with the control is counted and is designated as B The value of 8/8,, is calculated and its correspondence with the number of particles of HAA per unit value (7-1 is read off from the standard curve produced in Section (a) of this Example.
I. A method of determining the HAA content of a fluid comprising an unknown quantity of HAA particles per unit value designated 77, comprising the steps of:
l. Coupling serum hepatitis antibody to a support phase comprising carbohydrate groups by the sequential steps of treating said support phase with cyanogen bromide, raising the pH of the mixture to between pH l0 and pH l2, removing the supernatant liquid, washing said support phase at from about ph 7 to about pH 8, adding said serum hepatitis antibody to the mixture, washing the mixture free of unreacted antibody, buffering the support phase/antibody complex to pH 7.8 to pH 8 to yield a complex designated BHAB,
ll. lncubating a predetermined portion of the thus produced support phase/antibody complex with the fluid containing HAA,
Ill. Washing the thus produced support phase/am tibody/HAA complex designated BHAB/HAA,
lV. incubating said BHAB/HAA with radio active HAA,
V. Washing the thus produced BHAB/HAA/radio active HAA complex,
Vl. Measuring the radio activity of said BHAB/ HAA/radio active HAA complex to obtain a value designated 8 units of radio activity,
Vll. incubating a predetermined portion of the support phase of Step (I) designated as BHAB using the same quantity thereof as utilized in Step (II) under the same physical conditions of incubation using neutral saline as the incubation medium,
Vlll. Washing the product of Step (VII),
lX. Incubating the product of Step (Vlll) with radio active HAA selected from the sample used in Step (IV) for the same time under the same conditions of temperature, pressure and pH.
X. Washing the product of Step (lX),
Xi. Measuring the radio activity of the product of Step (X) to obtain a value designated 8., units of radio activity,
X". Repeating Steps (ll) (Vl) utilizing a fluid containing a predetermined quantity of HAA particles per unit value designated as H to obtain a value designated B units of radio activity.
2. A process according to claim 1 comprising the additional step of:
determining H in accordance with the formula 3. A process according to claim 1 additionally comprising repeating the procedure of Step (Xll) thereof utilizing a plurality of solutions containing predetermined quantities of HAA particles per unit value to obtain thereby a series of values for the quantity B corresponding to predetermined values of F 4. A process according to claim 2 additionally comprising the steps of calculating an an rage value for fT/B and determining the value of H in accordance with the formula