MONOCLONAL ANTIBODY DETECTION SYSTEM

Abstract: Use of monoclonal Rheumatoid antibody for assaying biological fluids.

Description: Monoclonal Antibody Detection System FIELD OF THE INVENTION The invention is directed to compositions and methods of detecting monoclonal antibodies and to assay systems based on such, methods. More particularly, the invention can be applied in analysis of biological fluids, such as serum or urine, to determine the anti¬ body, antigen and/or antigen-antibody complex content of such fluids. BACKGROUND OF THE INVENTION Analysis of biological fluids for antibody, antigen and antigen-antibody complex content is a useful tool for the diagnosis of disease. That is, many diseases are characterized by the presence in the circulation of anti- gen-antibody complexes. Antigens are proteins or other structures which may be present due to bacteria or virus or due to their release from human tissue or cancer cells. Antibodies are predominantly immunoglobulins of the IgM or IgG class, synthesized by the lymphoid system. An antibody is said to be specific to a particular antigen, as the antibody will complex with its specific antigen. That complex formation between the antigen and its specific antibody is the basis of present analysis of biological fluids for detection of antigens, antibodies and complexes thereof. Various techniques for detecting antibody, antigen and antibody-antigen complex content in biological fluids are known and are referred to as assays. The quantification techniques are referred as "immuno-assay" procedures. One of such assays depends on the reaction of an antigen-antibody complex, with OMH Rheumatoid factor (RF) or with components of complement (C); such an assay was apparently first described by Agnello et al, J. Exp. Med. , 134, 228, 1971 and later developed in U.S..Patent Nos. 4,062,935 and 4,143,124. Present assay and immunoassay techniques, based on antiglobulin reagents, involve collecting the reagents or their precursors from human serum or immunizing animals with, for example, purified imraunogloulin to produce anti-immunoglobulin antibodies, although widely used in various assay procedures, possess various disadvan¬ tages. Pools of such anti-immunoglobulin antibodies, produced by animals, are not identical, since each animal may respond to immunization in a slightly different way. Accordingly, use of pools of systems based on such anti-immunoglobulin antibodies involves constant recali- bration. Moreover, activity and specificity of such anti-immunoglobulin antibodies vary widely. A new art is developing based on the discovery that antibody forming cells, plasma cells, can be immortalized and can then produce unlimited amounts of the antibody, specifically the monoclonal antibody. Particularly, fusion of antibody forming cells from mammals, such as mice and rates, leads to the immortalization of the anti¬ body forming cell, a plasma cell, and the ability to obtain unlimited amounts of its antibody, otherwise referred as" monoclonal antibody. This development has greatly altered the way in which antigens-will be assayed in the future. Research units are now producing mono¬ clonal antibodies to a variety of antigens. Furthermore, at least two techniques have been developed to detect the binding of conventional or mono¬ clonal antibodies to the desired antigen. In one tech¬ nique, which is probably the most popular technique, the conventional or monoclonal antibody is reacted with bound antigen, antigen bound, for example, to plastic dishes or beads; then the mixture is washed to remove unbound anti¬ body; the anti-immunoglobulin reagent is added, and then the mixture is washed again to remove unbound anti- immunoglobulin reagent. The anti-immunoglobulin reagent can be iodinated (I 125 ) or coupled with an alkaline phos- phatase or peroxidase for detection in an Elisa System, described in the literature by Engvall, E. and Perlmann, Journal of Immunology, Vol. 109, pg. 129 (1972). Detec- tion in an Elisa System involves having an enzyme coupled to the conventional anti-immunoglobulin reagent. The addition and degradation of the enzyme substrate can be measured photometrically. The color developed is propor¬ tional to the amount of unknown antibody present in the test solution. A second technique involves labeling a monoclonal anti-immunoglobulin antibody, either by internal labeling (C 14 ) or external labeling (I , alkaline phosphatase, etc.) and using it to detect the first antibody. Both techniques for detecting the unknown conventional or monoclonal antibody involve cumbersome wash procedures which decrease efficiency and potentially decrease accuracy of the technique. Moreover, the latter tech¬ nique requires the use of a scintillation counter for all isotope tagged samples and requires tagging or labeling an enormous number of reagents. SUMMARY OF THE INVENTION The invention is directed to compositions and methods for detecting either conventional or monoclonal antibodies bound to antigens. In accordance with the invention, the reagent used for detection includes mono¬ clonal Rheumatoid antibodies. The methods of the invention involving the use of monoclonal Rheumatoid antibodies obviate multiple wash M stages in the assay. Moreover, the methods of the inven¬ tion are highly specific and selective with respect to the antigen to be detected. Recalibration of the rea¬ gent, in accordance with the invention, is substantially, if not completely, eliminated, and the invention allows quantitative antigen assays. DETAILED DESCRIPTION OF THE INVENTION Complex formation between an antigen and its speci¬ fic conventional or monoclonal antibody results in con- formational changes with the heavy chain portion of the antibody molecule and thus in a number of sites which can bind other substances. At least one of those sites can bind monoclonal Rheumatoid antibodies; at least one of those sites can bind components of complement (C) . Neither monoclonal Rheumatoid antibodies nor components of complement can bind the antigen, the conventional or the monoclonal antibody in the absence of those sites. Rheumatoid antibodies are circulating antibodies that react with sites on activated immunoglobulin (immunoglobulin bound to its antigen) . Parenthetically, they are called Rheumatoid factors (RF) because they were originally discovered in patients with Rheumatoid Arthritis. The methods " of detecting conventional or monoclonal. antibodies,- in accordance with the invention, involve inducing conformational changes in the respective anti¬ bodies to produce sites which will bind Rheumatoid anti¬ bodies and providing monoclonal Rheumatoid antibodies which will bind at such sites. The methods of the inven- tion differ from each other in the step of detecting the binding of monoclonal Rheumatoid antibodies to those sites. Those sites can be produced by combining the conventional or monoclonal antibody with its specific antigen to form an activated antigen antibody complex, O P which expressed the site that allows the monoclonal Rheumatoid antibody to bind. Thus, the invention provides an assay, a method of analyzing a biological fluid sample for conventional or monoclonal antibody, for antigen or for monoclonal anti¬ body-antigen complex content thereof which includes the step of adding to the sample, before or after the addi¬ tion of other reagents, a solution containing monoclonal Rheumatoid antibody to bind with the antigen: ntibody complex. Thus the assay method includes providing a source of antigen, such as a sample of biological fluid, for detecting the presence of a specific antigen therein; adding to the antigen source an amount of conventional or monoclonal anti- body specific to the antigen to be detected to allow the formation of the complex between said monoclonal antibody and its specific antigen; and adding a known amount of monoclonal Rheumatoid antibody to bind with a complex of said monoclonal antibody and its specific antigen. Lastly, the method includes detecting the binding of the monoclonal Rheumatoid antibody to said complex. The methods of the invention differ with respect to the means of detecting the binding of the monoclonal Rheumatoid antibody to said complex, as will be seen in the follow¬ ing discussion. Many cultures of fused cells secreting monoclonal antibody have been produced. The methods of producing the monoclonal antibody generally involve fusion of anti- body-forming cells which are plasma cells to hybridoma tumors, leading to the immortalization of the plasma cell and the ability to obtain unlimited amounts of its anti¬ body, i.e. the monoclonal antibody. By way of illustra¬ tion, it is noted that tissue culture cell lines which secrete monoclonal anti-sheep red blood cell antibodies were made by the fusion of a mouse myeloma and mouse spleen cells from an immunized donor and described in NATURE, Vol. 256, pp. 495-497 (August 7, 1975) by Kohler et al. Since the Kohler et al description, monoclonal antibodies have been made by the spleen cell fusion tech¬ nique of Kohler et al against a variety of antigens, as reviewed in Curr. Topics Microbiol. Immun., 81 (1979), and adopted by Secher et al, NATURE, Vol. 285, pp. 446- 450 (1980) . The preparation of a monoclonal Rheumatoid antibody involves immunizing the donor by injecting the donor with aggregated immunoglobulins, and then isolating the spleen cells of the donor and fusing them to a tumor; these aggregated immunoglobulins are heat aggregated purified gamma globulin molecules from any species that are to be utilized in the test system. In general these would be human, mouse, rabbit, etc. The method involves adding an amount of the mono¬ clonal Rheumatoid antibody to the antigen source, prior to, during or after addition of the conventional or mono- clonal antibody specific to the antigen to be detected, to allow binding of the monoclonal Rheumatoid antibody to the complexed antigen. A known amount of monoclonal Rheumatoid antibody is generally used, and preferably that amount is in excess of that amount needed to bind to all antigen antibody complexes. Preferably, the mono¬ clonal Rheumatoid antibody will be used in solution form, although the invention embraces its use in insolubilized form. For practical reasons, preferably the amount of antibody specific to the antigen to be detected is used in an amount in excess of that amount necessary to complex all of that antigen. Although the amounts of specific conventional or monoclonal antibody and of monoclonal Rheumatoid antibody are preferably in excess of those amounts necessary to complex all antigen to be detected and to bind the said complex, it is understood that determination of the presence or the absence of the antigen to be detected is not critically dependent on those amounts; for non-quantitative determination, those amounts are only dependent on the sensitivity of the equipment used in detection. When the monoclonal Rheumatoid antibody binds with the complex, discernible agglomeration may occur. The invention includes other additional ways of detecting the binding of monoclonal Rheumatoid antibody to the com- plex. Accordingly, those agglomerates can be centrifuged and washed and the monoclonal Rheumatoid antibody content of the agglomerate measured. In accordance with the invention, detection and/or measurement of the monoclonal Rheumatoid antibody bound to the complex can be undertaken in the following ways. The monoclonal Rheumatoid antibody may be measured directly if it is labeled, tagged and/or contains a developing marker, all of which can be detected by con¬ ventional means. Thus, the monoclonal Rheumatoid anti- body may contain internal C or 125 labeling or a fluorescent or a co-enzyme label. Preparation of mono¬ clonal antibodies containing radioactive isotopes is described in Hybridoma Technology with Special Reference to Parasitic Diseases, pg. 73 (for 3 H ) and 74 (for C 14 ). Undr/World Bank/Who World Health Organization Publication:, (1980) , Switzerland 80/4675 - Reggiani - 1000. Preparation of monoclonal antibodies containing I 12 labeling is described by Yallow, R. S. and Berson, S. A. , 1960, J. of Clinical Investigation, Vol. 13, pg. 1157 and Marchalonis, J. J. , 1969, Biochemistry Journal, Vol. 113, pg. 299. Preparation of monoclonal antibodies containing a fluorescent is described in the Handbook of Experiment of Immunology, Vol. 1, Immunochemistry Section 18.9, edited by D. M. Weir, Backwell Scientific Publica- tions, London, second edition. Preparation of monoclonal antibodies containing co-enzyme labeling is described by Nakane, P. K. , Annals of New York Academy of Sciences, Vol. 254, pg. 203, 1975 and Engvall, E. and Perlmann, P., 1971, Immunochemistry, Vol. 8, pg. 871. Thus, the mono- clonal Rheumatoid antibody bound to the antigen-antibody complex may be detected by a scintillation counter or a spectrophotometer. The agglomerate may be treated with a substrate for detection in the Elisa System. The binding of the monoclonal Rheumatoid antibody to complexed antibody may be detected by complement (C) or complement component activation. The "complement" system is the primary humoral mediator of antigen-antibody reac¬ tions and consists of at least 15 chemically and immuno- logically distinct serum proteins which may interact with each other, with antibody and with cell membrane. In accordance with this aspect of the detection method of the invention use of both synthetic and naturally occurring complement and complement components are con¬ templated. Activation of the complement system is well document, e.g. , "Basic and Clinical Immunology," second edition, Lange Medical Publications, Los Altos, California, pp. 66-77 (1978). Thus, detection can be based on the capacity of certain monoclonal Rheumatoid antibodies (1) to undergo conformational changes on binding and (2) to induce, in the presence of complement, activation of complement components. Such activation of the complement can be measured by providing a substrate which undergoes changes as a direct result of activation and .measurable by con- ventional means. The changes may be enzymatic and/or visible color or fluorescent changes which can be measured by spectrophotometers. For example, certain peptides generate a fluorescent compound, amino methyl coumarin, such as the 7-amino-4-methylcoumarin, upon cleavage by enzymes generated during complement activa- -g3P.£A " OMPI tion; cleavage of the substrate can be followed by measuring absorbance- of the coumarin derivative at 360 nM in a Cary 219 spectrophotometer, J. Immun. , Vol. 126, No. 5, pp. 1963-1965 (1981). In this alternative procedure, the monoclonal Rheumatoid antibody bound to the complexed monoclonal antibody need not be isolated for treatment with complement; but rather the complement may be added directly to it as it forms, prior to, during or after addition of said substrate. Complement and complement component are produced from serum components. Fresh animal serum contains the essential complement proteins necessary for such assays. Alternatively, such com¬ ponents are available for purchase. Buxted Rabbit Com¬ plement Co., East Grinstead, England is such a commercial supplier. The monoclonal Rheumatoid antibody which undergoes conformational changes which induce complement activation are, Human IgM, IgG- j _, IgG2 / IgG β ; Mouse I 9 M / IgG2 a , IgG2- j -, Rabbit; as well as antibodies from other species. The methods described herein provide quick means for antigen or monoclonal antibody detection. The method obviates recalibration of reagents used in said detec¬ tion, obviates multiple wash stages and obviates varia¬ tions in specificity of the reagent compositions. Although the invention has been explained and illustrated by reference to specific elements of the invention, it is intended that equivalents and obvious modifications of those specific elements are embraced by the appended claims.

Claims: Claims 1. A method of detecting the presence or absence of a specific antigen via antibody binding to said antigen in a fluid comprising: providing a known amount of that fluid; adding to 5 the fluid a known amount of an antibody specific to said antigens, which will thus form a complex with said antigen in the presence of said antigen, to allow formation of a complex between said antibody and said antigen; adding to the fluid a known amount 10 of monoclonal Rheumatoid antibody, wherein said monoclonal Rheumatoid antibody will bind to said comple . 2. The method of Claim 1, wherein said monoclonal Rheumatoid antibody is adapted to be detected by scintil¬ la lation counting and wherein the amount of monoclonal Rheumatoid antibody which is bound to complex is deter¬ mined by scintillation counting. 3. The method of Claim 1, wherein said monoclonal Rheumatoid antibody is adapted to be detected by U.V. 20 spectroscopy and wherein the amount of monoclonal Rheumatoid antibody which is bound to complex is deter¬ mined by U.V. spectroscopy. 4. The method of Claim 1, wherein said monoclonal Rheumatoid " antibody is adapted to exhibit a visible color 25 change when bound to said complex. 5. The method.of Claim 4, which further includes the step of adding complement component in their natural enzymatic state in combination with peptide which gener¬ ates a fluorescent compound upon cleavage by enzymes 30 generated during complement activation. 6. The method of Claims 1, 2, 3, 4 or 5 wherein said antibody is a monoclonal antibody. OMPI 7. The method of Claim 1 further includes adding complement to the fluid wherein said monoclonal Rheumatoid antibody activates the decomposition of the complement. 8. The method of Claim 7 which includes a substrate which will undergo change when said complement undergoes • activation. 9. The method of Claims 1, 2, 3 or 4 wherein the amount of said conventional or monoclonal antibody is in excess of that amount effective to form said complex and wherein the amount of monoclonal Rheumatoid antibody is in excess of that amount effective to bind all complex in said fluid. 10. A composition for the detection of a complex of a conventional or monoclonal antibody bound to its speci¬ fic antigen comprising a monoclonal Rheumatoid antibody. 11. The composition of Claim 10, which is adapted to be detected by scintillation counting. 12. The composition of Claim 10, which is adapted to be detected by U.V. spectroscopy. 13. The composition of Claim 10, which is adapted to exhibit a color change in the presence of said com¬ plex. 14. The composition of Claim 10, which includes complement, complement components or admixtures thereof. 15. The composition of Claim 14, which includes a substrate which wili undergo changes when said complement undergoes activation, said changes being detectable by conventional means. 16. A method of detecting the complex of a conven¬ tional or monoclonal antibody and its specific antigen in a sample, comprising adding to the sample an amount of OMPI the composition of Claim 10, effective to bind to said comple . 17. A method of detecting the complex of a mono¬ clonal antibody and its specific antigen in a sample, comprising adding to the sample an amount of the composi¬ tion of Claim 11, effective to bind said complex. 18. A method of detecting the complex of a mono¬ clonal antibody and its specific antigen in a sample, comprising adding to the sample an amount of the composi- tion of Claim 12, effective to bind said complex. 19. A method of detecting the complex of a mono¬ clonal antibody and its specific antigen in a sample comprising adding an amount of composition of Claim 13, effective to bind said complex. OM

Inventors: LAKE PHILIP, SELL KENNETH W, WOODY JAMES N, LAKE, PHILIP, SELL, KENNETH, W., WOODY, JAMES, N.
Applicants: UNIV GEORGETOWN, GEORGETOWN UNIVERSITY
Application year: 1982
Main classification: G01N33
IPC codes: C07K16, G01N33