IMMUNOASSAY FOR MULTI-DETERMINANT ANTIGENS

Abstract: Simultaneous sandwich immunoassay employing high-affinity monoclonal antibodies. This simultaneous sandwich assay has surprising sensitivity compared to forward and reverse sandwich assays for the detection of multi-determinant antigens such as hepatitis B surface antigen.

Description: IMMUNOASSAY FOR ' MULTI-DETERMINANT ANTIGENS Description Technical Field This invention is in the field of immunology and particularly relates to an immunoassay for the detect¬ ion of multi-determinant antigens. Background Art The desirability of having an immunoassay capable of being employed by hospitals and clinical laborator- ies for the early detection of viral infections has been widely recognized. The need for such an assay can be illustrated, for example, by acute hepatitis B, one •form of the various hepatitis viral infections. Acute hepatitis B causes significant mortality and morbidity and chronic infection with this virus is also associ¬ ated with hepatocellular carcinoma, chronic active hepatitis and cirrhosis. In view of this need, much research has been devoted to the development of immuno- assays capable of detecting viral antigens such as hepatitis B surface antigen (HBsAg), the viral marker for hepatitis B, in the blood stream of patients. Generally, the immunoassays which have been devel¬ oped to date can be classified as radioimmunoassays (RIA) or immunoradiometric assays (IRMA). In a RIA, the amount of antigen present in a sample is measured indirectly employing a limited amount of antibody to compete for labeled antigen. In an IRMA, antigen is assayed directly by reacting the antigen with excess labeled antibody. In one class of IRMA assays, the unknown antigen is insolubilized and reacted with labeled antibody. When the antigen is insolubilized by reaction with solid-phase antibody, the assay is termed a n two-site IRMA", "junction test", or "sandwich assay". Sandwich assays are further classified according to their meth¬ odology as forward, reverse or simultaneous sandwich assays * Heretofore, the most widely used immunoassay for viral hepatitis B has been the forward sandwich assay. In this assay, a * patient sample containing HBsAg is initially incubated with a solid-phase im unoadsorbent containing immobilized antibody for HBsAg. Incubation is continued for a sufficient period of time to allow HBsAg i the patient sample to bind to immobilized an¬ tibody on the solid-phase immunoadsorbent. After this first incubation, the solid-phase immunoadsorbent is separated from the incubation mixture and washed to re¬ move excess, antigen , and other interfering substances, such as non-specific binding proteins, which also may be present in the patient sample. The solid-phase im¬ munoadsorbent containing HBsAg bound to immobilized an¬ tibody is subsequently incubated for a second time with labeled antibody capable of binding to HBsAg, which is multivalent. After the second incubation, another wash is performed to remove unbound labeled antibody from the solid-phase immunoadsorbent thereby removing non- specifically bound labeled antibody. Labeled antibody bound to the solid phase immunoadsorbent is then de- tected and the amount of labeled antibody detected serves as a direct measure of the amount of HBsAg pres¬ ent in the original patient sample. Such forward sand¬ wich assays are described in the patent literature, and in particular, in ϋ. S. Patent Nos. 3,867,517 and 4,012,294, issued to Chung-Mei Ling. Despite their widespread use, forward sandwich assays do have certain inherent disadvantages. For example, the requirement to perform two separate incu¬ bations as well as two separate washings means that the 5 assay is relatively time-consuming and requires a num¬ ber of manipulative operations which must be performed by skilled technicians thereby making the forward sand¬ wich assay relatively costly. In addition, the kine¬ tics in the second incubation are relatively slow since 10 labeled antibody must react in this step with antigen bound to antibody immobilized on the solid-phase immu¬ noadsorbent. The reverse sandwich immunoassay was developed to overcome some of the disadvantages of the forward sand- 15 wich assay. In a reverse sandwich assay, the patient sample is initially incubated with labeled antibody af¬ ter which the solid-phase immunoadsorbent containing immobilized antibody is added and a second incubation is carried out. Thus, the initial washing- step of a 20 forward sandwich assay is not required, although a wash is performed after the second incubation period. The major advantages to the reverse sandwich assay compared to the forward sandwich assay are the elimination of the first wash step and the improvement in kinetics of 25 reaction since the labeled antibody and antigen in the patient serum are initially incubated together. Thus, for a total incubation period less than that required , to reach equilibrium, it would be expected that the sensitivity of the reverse assay would be higher than i30 that of the forward assay. A reverse sandwich assay has been described in the patent literature in U. S. Patent No. 4,098,876, issued to Roger N. Piasio et al. Although reverse sandwich assays have potential advantages over forward sandwich assays, they still re¬ quire two separate incubation periods and one washing. Simultaneous sandwich assays, although known, have 55 not found widespread use. In a simultaneous sandwich assay, the patient sample, immunoadsorbent having immo¬ bilized antibody thereon, and labeled antibody are in¬ cubated simultaneously in one incubation step. Al¬ though offering certain potential advantages due to the UX single incubation and lack of washing steps required, simultaneous sandwich assays have previously been found to be less sensitive and reliable than forward and re¬ verse sandwich assays. This would be expected, of course, since the patient sample often contains many S non-specific binding molecules, such as proteins, which are not washed from the assay as they are in the for¬ ward sandwich assay. Disclosure of the Invention This invention relates to a simultaneous sandwich GD assay for detecting the presence of multi-determinant antigens in liquid samples, such as the blood of a pa¬ tient. As pointed out above, in a simultaneous sand¬ wich assay, the patient sample is incubated simultane¬ ously with both an immunoadsorbent containing immobi- S lized antibody for the antigen and labeled antibody for the antigen. Thereafter, labeled antibody bound to the immunoadsorbent is detected as an indication of the amount of antigen present in the patient sample. In a broad sense, this invention comprises the i - 03 provement of employing high-affinity monoclonal anti¬ bodies for both immobilized antibody and labeled anti¬ body in a simultaneous sandwich assay. These high- affinity, antibodies can comprise either monoclonal IgG L-ϋS.-c*i, or monoclonal IgM antibodies. One preferred embodiment of this simultaneous sandwich assay employs high-affinity monoclonal IgM antibody for HBsAg for both immobilized and labeled antibody in a particularly sensitive assay * 5 for viral hepatitis B. The simultaneous sandwich assay described herein allows the realization of the previously recognized * potential advantages of a simultaneous sandwich assay. For example, the assay is simpler and requires less 10 steps since there is only one incubation and since no washing steps are required. This reduces the amount of skilled technician time required which, in turn, re¬ duces the cost of performing the assay. More importantly, however, is the surprising 5 sensitivity which can be obtained when monoclonal anti¬ bodies are employed in a simultaneous sandwich assay. For example, for total. incubation times which are comparable, it has been found that the simultaneous sandwich assay described herein can be more sensitive 20 than either a forward or . reverse assay for- he detec¬ tion of HBsAg. Another unexpected advantage of the assay de¬ scribed herein is the wide range of antigen concen¬ tration which can be detected, despite the fact that 25 only one incubation is employed and no washing steps are employed. For example, it has been found that this assay is capable of detecting HBsAg over a greater than ten million-fold dilution range. • '. Best Mode for Carrying Out the Invention • 0 The simultaneous sandwich assay described herein is useful for the detection of multi-determinant anti¬ gens. By "multi-determinant," it is meant that the antigen contains a plurality of the same antigenic deter- minant (epitope) . Sometimes, such antigens have also been referred to in the literature as "polydeterminant" antigens, but the terminology "multi-determinant" will be used herein to describe such antigens. B Another term used to describe antigenic determi¬ nants is the term " ultivalent." The term "multivalent"- is used herein to mean that the antigen has a plurality of different antigenic determinants or epitopes thereon. Many viral antigens are both multi-determinant and multivalent. For example, the hepatitis B virus is known to be a complex 42 nanometer (nm) particle having at least three . well defined antigens. These include the hepatitis B core antigen (HBcAg) , the hepatitis B e antigen (HBeAg) , and the hepatitis B surface antigen (HBsAg) . The HBsAg antigen is a high molecular weight complex protein which occurs on the surface of the. intact hepatitis B virus. However, HBsAg is also present in the blood of patients infected with hepatitis B as a circulating 22 nm ' particle or filamentous .for . Because of its presence in the bloodstream of infected patients, HBsAg has become the most widely employed specific viral marker for acute and chronic hepatitis B infection. HBsAg has been found in patient serum samples at concentrations of up to about 800 micrograms/ml. Data indicate, however, that serum concentrations as low as about 50 pg/ml can be infectious. This represents an extraordinarily wide range for an assay to cover. Although much of the description of the simultane¬ ous sandwich assay decribed herein is presented in terms of the detection of HBsAg, it should be recog¬ nized that the assay itself is suitable for the detec- 5 tion of any multi-determinant antigen. This includes other viral antigens which are multi-determinant, such as Herpes Simplex viruses I and II, Herpes Virus Zoster, Cytomegalovirus, Bpstein-Barr virus, Papova vi¬ ruses such as BK or JC virus, measles virus, rubella virus, influenza or parainfluenza viruses, etc. Viral subunits, such as virus capsids, can also be detected as long a such subunits are multi-determinant. Addi¬ tionally, antigens need not be viral in nature to be multi-determinant and the simultaneous sandwich assay decribed herein is suitable for detecting such non- viral multi-determinant antigens. Such non-viral multi-determinant antigens might include, for example, cells, immune complexes, membrane preparations, bac¬ teria, etc. Such multi-determinant antigens are assayed in a liquid sample. Often, the liquid sample is a sample of a patient's blood or a blood component such as plasma or serum. -Other liquid samples, such as urine, could of course be assayed by the simultaneous sandwich assay decribed herein. • In this assay, high-af inity monoclonal antibodies are employed for both immobilized antibody bound to an immunoadsorbent as well as for labeled antibody. The term "high-affinity" is employed herein to mean that the antibody has an affinity binding constant (K) of greater than 10 9n—1, and preferably- greater than 10 11n-1. Binding constants are determined from Scatchard plots employing known techniques, and are, of course, determined for the whole antigen and antibody molecules rather than on a per site basis. ^uE:. The term "monoclonal" means that the antibody has been produced by a. cell line cloned from a single anti¬ body producing cell. Monoclonal antibodies are extra¬ ordinarily pure, uniform and reproducable since each S antibody is effective against a single antigenic deter¬ minant. Monoclonal antibodies can be obtained in signifi¬ cant quantities from hybridoma cells. Hybridoma cells are fused cells resulting from the fusion of antibody UX producing cells with tumor cells. The initial work re¬ lating to the production of such hybridoma cells was done by Cesar Milstein and George Kδhler employing mouse myeloma cells with spleen cells taken from mice immunized with sheep red blood cells. See, * Kohler et I al. , Sur. J. Immunol. , 6_, 511-19 (1976); Kohler et al.. Nature, 256, 495-7 (1975); and Milstein, Scientific American, 243 ( ) , 66-74 (1980). More recently, Hilary Koprowski and collegues have extended;the original hybridoma work by developing hybridoma ZQD cell lines capable of- producing monoclonal antibodies against specific viruses and tumors. See, ϋ. S. Patent Nos. 4,196,265 and 4,172,124, respectively. Even more recently, the hybridoma technology has been further ex- " tended to produce hybridoma cell lines capable of producing 255 monoclonal antibodies to hepatitus virus. See U. S. application Serial No. 86,947, filed October 22, 1979 in the names of Jack R. Wands and Vincent R. Zurawski. It has also been recently discovered that monoclonal IgM antibodies produced from hybridoma cell lines can be 33T 5 employed in certain i munoassays to improve the sensi¬ tivity and specificity thereof. See, U. S. application Serial No. 188,735, filed September 19, 1980, in the names of Jack R. Wands, Vincent R. Zurawski, and Hubert J. P. Schoemaker. 3 ' Thus, the general scientific and patent literature include many descriptions of hybridoma cell lines and - 9 - monoclonal antibodies produced from such cell lines. These teachings are relied on in general herein, and specifically the teachings of each of the heretofore mentioned articles and patents relating to hybridoma 5 cell lines and/or monoclonal antibodies are hereby incorporated by reference. Additionally, a clone of cell line H25D3, identified in these prior applications, has been deposited at the American Type Culture Collec¬ tion (ATCC) , Rockville, Md., and is identified by ATCC Accession No. HB8058. As mentioned above, high-affinity monoclonal anti¬ bodies for the multi-determinant antigen being assayed are immobilized to a solid-phase immunoadsorbent in the simultaneous sandwich assay of this invention. There are many such solid-phase immunoadsorbents which have been employed. Well-known immunoadsorbents include beads formed from glass polystyrene, polypropylene, dextran, and other materials; tubes formed from or coated with such materials; etc. The antibody can be either covalently or physically bound to the solid-phase immunoadsorbent by techniques such as covalent bonding via an amide or ester linkage or adsorption. Those skilled in the art will know many other suitable solid- phase immunoadsorbents and methods for immobilized anti- bodies thereon, or will be able to ascertain such using no more than routine experimentation. High-affinity monoclonal antibody is also used as the labeled antibody in the simultaneous sandwich assay described herein. Such antibodies can be labeled with a radioactive material, such as I; labeled with an optical label, such as a fluorescent material; labeled with an enzyme; or labeled by some other known technique. The liquid sample, solid-phase immunoadsorbent with immobilized monoclonal antibody, and labeled solu- ble monoclonal antibody are " incubated under conditions and for a period of time sufficient to allow antigen to antibody. In general, it is usually desirable to provide incubation conditions sufficient to bind as much antigen as possible because this maximizes the binding of labeled antibody to the solid phase thereby increasing 5 the signal. Typical conditions of time and temperature are two hours at 45° C or twelve hours at 37° C. Labeled antibody typically binds to antigen more rapidly than immobilized antibody since the former is in solution whereas the latter is bound to a solid- 0 phase support. Because of this, labeled antibody should be employed in a lower concentration than immo¬ bilized antibody and it is also preferable to employ a high specific activity for labeled antibody. For example, labeled antibody might be employed at a concen- 5 tration of about 1-50 ng/assay whereas immobilized antibody might have a concentration of 100-500 ng/assay. The labeled antibody might have a specific activity with, for instance, one iodine per IgM or as high as two labeled iodines per molecule of IgM angibody. 0 Of course, the specific concentrations of labeled and immobilized antibody, the temperature and time of incubation as well as other such assay conditions will depend upon many factors, including the specific anti¬ gen being assayed, the concentration of the antigen, 5 and the monoclonal antibody or antibodies employed. Those skilled in the art will be able to determine op¬ erative and optimal assay conditions for each determi¬ nation desired by employing routine experimentation. After the single incubation period, the solid— Q phase immunoadsorbent is removed from the incubation mixture. This can be accomplished by any of the known separation techniques, such as sedimentation or centri- fugation. A washing step is not required prior to de¬ tection of bound labeled antibody. Detection can be performed by a scintillation counter, for example, if the label is a radioactive gamma emitter, or by a fluorometer, for example, if the label is a fluores¬ cent,material. In the case of an enzyme label, the detection can be done by colorimetric methods employ¬ ing a competing substrate for the enzyme. As noted above, results which have been achieved employing a simultaneous sandwich assay as described herein have demonstrated that such an assay can be more sensitive than forward and reverse sandwich assays wherein the total incubation time for each assay is the same. This is surprising, and the reason for such sensitivity is not well understood. While not wishing to be bound by this theory, one possible explanation is that the monoclonal IgM antibodies employed tend to form a crosslinked structure in the incubation mixture. Because of this, greater -amounts of antigen and labeled antibody can be bound to the solid- phase immunoadsorbent. Data indicate, indirectly, for example, that more antigen molecules can bind to the N - solid phase than would be expected from the number- - l of antibody molecules thus supporting the concept of crosslinking or layering. Although data presented in the examples involves IgM antibodies, it is also believed that this simultaneous sandwich assay can employ gG antibodies as either the immobilized or labeled antibody, or both. IgG molecules have two binding sites and are capable of crosslinking antigens. The simultaneous sandwich assay of this invention will now be more specifically illustrated by way of the following examples. EXAMPLE 1 Immunoradiometric Assay for HBsAg Forward, reverse and simultaneous sandwich assays 5 were compared for detection of HBsAg. The monoclonal antibody employed in each was a monoclonal IgM antibody having high affinity for HBsAg and produced by the hybridoma cell line 5D3. The 5D3 hybridoma cell line comprises a cloned line developed 10 by fusing the myeloma cell line P3-NSl/l-Ag4-l derived from a BALB/c mouse MOPC 21 myeloma with mouse spleen cells immunized by a specific immunization procedure with HBsAg. The specific procedure employed is de¬ tailed in copending application U. S. Serial No. 1586,947, filed October 22, 1979, the teachings of which are hereby incorporated by reference. The solid-phase immunoadsorbent was provided by " 1/4 inch polystyrene beads obtained from Precision Plastic Ball Company, Chicago, Illinois. The beads 0 were coated with the monoclonal IgM antibody as follows. Ascites fluid derived from an immunized BALB/c ( mouse was diluted 1:500 to 1:5,000 in phosphate buf¬ fered saline (PBS). The beads were incubated for 16 hours at room temperature and washed three times with 5 distilled water. Monoclonal IgM antibody was labeled using the Bolten Hunter method. Specific activity ranged from about 2 to about 20 μCi/μg. The forward sandwich assay protocol involved in- 0 cubation of the beads having immobilized monoclonal IgM antibody thereon with 100 μl 1% bovine albumin in PBS and 100 μl of an HBsAg-containing patient serum for one hour at 45° C followed by washing three times with distilled water and subsequent addition of 200 μl of labeled antibody which was incubated for one hour at 45° C. In the reverse sandwich assay protocol, 100 ,ul of labeled antibody were incubated with 100 μl of HBsAg- containing patient serum in a soluble phase for 1 hour at 45° C. Subsequently, monoclonal antibody coated onto the solid beads was added for an additional 1 hour at 45° C. The beads were subsequently washed three times with distilled water and placed in a scin¬ tillation counter. The simultaneous sandwich assay protocol involved simultaneously incubating coated beads and 100 μl of labeled antibody and 100 μl of patient sample for two hours at 45 9 C. The beads were subsequently washed hree times', with distilled water and placed in a scintillation counter. Additionally, patient samples were diluted a number of times by a factor of 10 and reassayed. by each protocol to determine the limit of sensitivity for each assay protocol. A negative control consisting of human serum was employed. The results are presented in Table 1. As can be seen from the data in Table 1, the simultaneous sand¬ wich assay was more sensitive than either the forward or reverse assays where each assay employed the same monoclonal IgM antibody against HBsAg and wherein in- ' cubation conditions for each assay were the same. TABLE I COMPARISON OF REVERSE, FORWARD AND SIMULTANEOUS SANDWICH ASSAYS USING A RADIOIMMUNO ASSAY • FORWARD REVERSE SIMULTANEOUS • Negative 407.2 149.6. 63.5 -•.. Control 1 Patient 28,951 71.1 2,059 13.8 1,760 27.7 Sample 1:10 38,474 94,5 8,017 ' 54,4 • 9,246 145.1 1:100 38,100 93.6 12,673 84.5 16,650 262.2 1:1000 11,892 29.2 8,091 53.9 8,725 137.4 1:10,000 2.,921 7.2 1,840 12.3 2,311 36.4 1:100,000 799 1,9 361 2.4 1,024 16.1 1:1,000,000 546 1,3 99 0.66 582 9.2 • NOTES 1. Data is expressed in Counts Per Minute (cpm's) , 2. In all assays, identical number of total counts were added and identical reagents were used. 3. S/N in ratio of cpm in, sample divided by cpm is negative control. A S/N of greater >M than 2.0 represents a positive, cn '(1) (2) (3) (4) Patient No.Pos. No.Pos, No.Pos. No.Pos, Total No. Group SSA Comm. Anti-HBc Anti-HBs Studied Acute hepatitis B 42 42 36 42 Acute hepatitis 12 22 Post-Transfu¬ sion hepatitis 9 25 Blood donors 5 92 Alcoholic liver disease 2 0 2 0 50 Normal controls 0 0 6 7 59 Total — - 290 1. Simultaneous sandwich assay 2. Abbott Ausria II assay 3. Abbott Corab assay 4. Abbott Ausab assay 5. Anti-HBc titer> 1:1024 on 2 positives in SSA It should be noted that the SSA detected postive samples in cases of acute hepatitis and post-transfusion hepatitis whereas the commercial assay produced negative results. industrial Applicability The invention described herein is useful in the detection of multi-determinant antigens, such as hepatitis B surface antigen (HBsAg). Thus,, the assay can be employed by hospitals or clinical laboratories for the early detection of viral infection or the presence of non-viral antigens. Equivalents Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims: Claims 1. In an immunoassay for detecting the presence of of multi-determinant antigen in a liquid sample wherein said sample is simultaneously incubated in the presence of an immunoadsorbent containing 5 . • immobilized antibody for said antigen and labeled antibody for said antigen, and thereafter detect¬ ing the amount of labeled antibody bound to said immunoadsorbent as an indication of the amount of antigen present in said sample: 0 The improvement wherein said immobilized antibody and said labeled antibody com¬ prise high-affinity monoclonal antibodies directed against a multiply-appearing deter¬ minant of said antigen. -5 2. The improvement of Claim 1 wherein said labeled mono ¬ clonal antibody comprises a monoclonal IgM anti¬ body. 3. The improvement of Claim 1 wherein said ϋnmobilized monoclonal antibody comprises a monoclonal IgM antibody. 4. The improvement of Claim 1 wherein each of said labeled monoclonal antibody and said immobilized monoclonal antibody comprises a monoclonal IgM antibody. 5. The improvement of Claim 1 wherein said labeled monoclonal antibody and said immobilized mono— • clonal antibody comprise the same monoclonal antibody. > 6. The improvement of Claim 5 wherein said same mono¬ clonal antibody comprises a monoclonal IgM antibody. 7. The improvement of Claims 1, 2, 3, 4, 5 or 6 wherein said antigen is a viral antigen comprising 5 a virus or antigenically-active portion thereof. 8.- The improvement of Claim 7 wherein said viral an¬ tigen comprises an hepatitis viral antigen. 9. The improvement of Claim 8 wherein said hepatitis viral antigen comprises HBsAg. 1010. An immunoassay fo ' detecting the presence of "a multi-determinant antigen in a liquid sample comprising: a. forming an incubation mixture ' . •containing: 15 i. liquid sample; ii. a solid-phase immunoadsorbent containing immobilized high- affinity monoclonal antibody directed against a multiply- 0 appearing determinant of said antige ; and, iii. labeled, soluble, high-affinity monoclonal antibody .directed against a multiply-appearing 5 determinant of said antigen? b. incubating said incubation mixture under conditions and for a period of time sufficient for antigen in the liquid sample to bind to both immobilized monoclonal anti- 0 body and labeled, soluble, monoclonal antibody; c. separatin•g•said solid-phase im uno- ' adsorbent; from the incubation mixture af er said incub ' ation; ' and, d. detecting for labeled monoclonal 5 antibody bound to said solid-phase immuno¬ adsorbent as an indication of the amount of said antigen in the liquid sample. 11. An immunoassay of Claim 10 wherein said labeled, soluble, monoclonal antibody comprises a monoclonal 0 IgM antibody. 12. ■ An immunoassay of Claim 10. wherein said immo¬ bilized monoclonal antibody comprises a monoclonal IgM antibody. 13. An immunoassay of Claim 10 wherein said labeled, 5 soluble monoclonal antibody and said ' immobilized monoclonal antibody both comprise a monoclonal IgM antibody. 14. An immunoassay of Claim 10 wherein said labeled, soluble monoclonal antibody and said immobilized 0 monoclonal antibody comprise the same monoclonal antibody. 15. An immunoassay of Claim 14 wherein said same ono- l clonal antibody comprises a monoclonal IgM antibody. 16. An immunoassay of Claims 10 , 11 ,. 12 , 13 , 14 or 15 wherein said multi-determinant antigen is a viral antigen comprising a virus or antigenically-active portion thereof. 17. . An immunoassay of Claim 16 wherein said viral antigen_comprises an hepatitis viral antigen. 18. An immunoassay of Claim 17 wherein said hepatitis viral antigen comprises HBsAg. 19. An immunoassay of Claim 18 wherein said labeled, soluble, monoclonal antibody contains a radioactive label * 20. An immunoassay of Claim 19 wherein said radioactive label comprises -'*-'*'I, 21 * An immunoassay of Claim 18 wherein said labeled,, soluble, monoclonal antibody is labeled* ' with an " . enzyme label. 22. An immunoassay of Claim 18 wherein said labeled,,, soluble, monoclonal antibody is labeled with' an optical label * - 23. An immunoassay of Claim 22 wherein said optical labevl comprises a fluorescent material.

Inventors: SCHOEMAKER HUBERT J P, WANDS JACK R, WESTRICK BARBARA L, ZURAWSKI VINCENT R JR
Applicants: CENTOCOR INC, MASSACHUSETTS GEN HOSPITAL
Application year: 1982
Main classification: A61K39
IPC codes: G01N33