ANTI-IDIOTOPIC IMMUNOMETRIC ASSAY

Abstract: An immunometric assay is disclosed for preselected monoclonal antibody in a biological sample comprising, forming a complex of a first labeled anti-idiotypic monoclonal antibody, the preselected monoclonal antibody, and a second anti-idiotypic monoclonal antibody which can be bound to an insoluble substrate and detecting the amount of labeled antibody associated with the complex. The assay is characterized by employing first and second monoclonal antibodies which react with an idiotypic site on the preselected monoclonal antibody.

Description: ΪL I2I0 0PJC_ MMUN0METRI C_AS^AY Il_-_-_-Α∑_i}_-i--_.-iϊ.--_- _i—Σ_-∑_i_±_ι_--iϊ- The recent development of monoclonal antibody technology has made it possible to produce unlimited amounts of homogeneous antibody preparations with single specificity. A variety of human monoclonal antibodies have been developed that are specific to antigens present in Rh blood groups [Evans, e t a_1. , J_. J_πιmuno_l->gy_, 1^0:941 (1988)] , human immuno- deficiency virus [Thompson e t al . , Iminuno ]__ _gy_ , 5J3.157 (1986)] , malaria parasites [Udomsangpetch , e t __λ - > i£ilS£ - 2:231 (1986)] , endotoxin [Teng e t a I . , PNAS, 8.0:7308 (1985)] , human platelets [Nugent e t _-_- ■ < Α1.2.°._\ < 19. : -*-6 (1987)] , and human tumor cells including melanoma [Yamaguchi e t ______ . , PNAj3 , J34.2416 (1987)] , breast [Schlom et a1. , PNAS, 77:6841 (1980)] and colon cancer [Haspel- et _____ • > _ __l _-___l Re_search , 45_:3951 (1985)] . Monoclonal antibodies such as these may be useful as therapeutic reagents. In addition, they can have both safety and product standardization advantages as compared to the use of human pooled hyperimmune sera. Furthermore, their use would have presumably fewer problems with immunogenicity than with use of murine monoclonal antibodies . Clinical studies involving human monoclonal antibody products will require analysis of the pharmacokinetics of such reagents. These analyses will enable a rational design of treatment doses and administration schedules. Radiolabeled antibodies have been used to determine the pharmacokinetics of monoclonal antibody products in man [Sears, et al. , J. Biol. Resgonse ^ Kod.. , : 138 (1984) and Meeker, et £_λ - > Blood, 6j5:1349 (1985)] , but this method has the disadvantage of requiring use of a radiolabeling procedure that might alter the molecule, thus resulting in alteration in pharmacokinetics . The pharmacokinetics of an injected dose can also be measured by quantitation of the circulating antibody at various times following administration [Khazaeli et a.1. , Clin^. Res^, 35.:615A (1988)] . This approach -would require a specific and sensitive assay capable of detecting the monoclonal antibody in the presence of a vast excess of normal human immunoglobulins in serum. S^mmarγ_o_f__the__I ; n enti.£tι The present invention provides an immunometric assay for a preselected antibody in a biological fluid such as a blood sample. The assay comprises forming a complex of a first monoclonal antibody that is labeled, the preselected antibody, and a second monoclonal antibody and detecting the amount of label associated with the complex as indicative of the presence or the amount of preselected anti¬ body in the biological fluid. The first and second monoclonal antibodies are specific for idiotopes of the preselected antibody. The preferred assay is a solid phase assay where at least one of the antibody constituents of the complex is bound to a solid phase, either before or after formation of the complex. The first antibody can be labeled before or after formation of the complex. _Ϊ _i_l-. ^^^ R_lk_i_l—_i_l_._l-l_i_.Σ_zK-l _i_l Figure 1 shows a HPLC profile of purified anti-idiotypic murine monoclonal antibody, 15B2.2. (A) Analysis using a Bio-Rad Quick Check Analyzer with a Bio-Sil TSK 250 Column (300 x 7.5 mm) . The sample was eluted with 10 mM phosphate, pH 6.8 containing 0.3 M NaCl, 10% dimethyl- sulfoxide (v/v) buffer at 1.0 ml/min. (B) Analysis of 15B2.2 post radiolabeling using HPLC as described above, with the addition of a radioisotope monitor to detect radioactivit . Figure 2 shows cross reactivity of normal human immunoglobulins . Using a solid phase radiometric assay, increasing concentrations of HA-lA normal human IgG, IgM, IgA, IgE and IgD were incubated in the assay as described in materials and methods. (°) HA-lA, (o) other normal human immunoglobulins. -The cross reactivity was calculated as less than * 0.1%. Figure 3 shows a dose response curve of HA-lA in solid phase radiometric assay. Increasing concentrations of HA-lA were incubated with 9B5.5- coated beads for 2 hours , washed and incubation was 125 continued for 1 hour with I-15B2.2. Bead associ- ated radioactivity was determined and a standard curve was constructed using logit-log transformation regression analysis. Figure 4 shows the serum concentrations of human IgM monoclonal antibody HA-lA in a patient who received 100 mg of the antibody. Serum samples obtained from the patient were assayed at appro¬ priate dilution in the solid phase radiometric assay. The values are the mean of triplicate determinations ±IS.D. Detaj.led Description of the Invention The present invention provides a novel immuno- assay for a preselected antibody in liquid sample. In the immunoassay of the invention, a complex is formed comprising a first labelled monoclonal antibody, the antibody to be measured (the "antigen") , and a second monoclonal antibody. The first and second monoclonal antibodies react with an idiotopic site on the antibody to be measured. Preferably, they are derived from the same hybridoma cell line and react with the same idiotopic site on the antigen. The immunoassay can be conducted in a reverse, simultaneous, or forward format. Following formation of the complex, the amount of preselected monoclonal antibody is quantified by detecting the amount of label associated with the complex. Preferred assays are those where the complex is immobilized on the solid phase. This invention is based on the recognition that antibodies are themselves antigenic. It is poss¬ ible, therefore, to induce antibodies that will recognize antigenic determinants on both the con¬ stant and the variable regions of immunoglobulin chains . Antigenic determinants on the variable regions of L and H chains that are associated with the antigen-binding site of an antibody are called idiotopes . The set of idiotopes on an individual antibody molecule defines the idiotype of that antibod . The first and second monoclonal antibodies of this invention are anti-idiotypic. That is, they will react solely with antigenic determinants (idiotopes) associated with the antigen-binding site of a pre-selected antibody molecule. Preferably, though not necessarily, these monoclonal antibodies are specific for a "private idiotope". The term "private idiotope" is based on the fact that several idiotopes may make up an immunoglobulin idiotype. Therefore, if two or more antibodies bind different antigens and are derived from independent V and V genes, each individual idiotope is considered "private" to one of the several antibodies. How¬ ever, a portion of the V„ or region away from the antigen binding site may have an idiotope in common between several antibodies. This particular anti- body has a "public" idiotope. Since the "private" character of the antibodies of this invention may not be a necessary requirement, the more common anti- idiotyp-ic "public" antibodies (Kiyotaki, M. et: *λ-- > 138 :4150-4158 (1987)) can also be effective, although the likelihood of their cross- reacting with normal immunoglobulin would be higher than "private" anti- idiotypes . Antibodies useful in the invention can be "obtained by immunizing an animal, preferably a mouse, with a preselected antibody. Antibody produced cells are formed by fusing antibody produc¬ ing cells from the immunized animal and an im¬ mortalizing cell such as myeloma. Particularly preferred monoclonal anti- idiotypic antibodies of this invention are "private" anti-idiotypic antibodies, and are produced as described herein. A particular example of such a monoclonal anti-idio ypic antibody is one that specifically reacts with idiotopes of human mono¬ clonal antibody HA-lA. In preferred embodiments, anti-idiotypic monoclonal antibodies of this invention are produced by hybridomas formed by fusion of: a) a mouse myeloma which does not secrete antibody with b) human lymph node spleen cells which secrete anti- idiotypic antibodies obtained from mice immunized against a preselected human monoclonal antibody (e.g. , HA-lA) . The mice are immunized with a primary injection of monoclonal antibody followed by a number of boosting injections of the same antibody. During or after the immunization procedure, sera of the mice may be screened to identify those mice in which a substantial immune response to the antibody has been evoked. From selected mice, the spleen cells are obtained and fusions are performed. Suitable fusion techniques are the Sendai virus technique. ohler, G. and Milstein, C , Nature 25_ :495 (1975) , or the polyethylene glycol method, Kennet, R.H. in "Mono¬ clonal Antibodies, Hybridomas -- A New Dimension in -Biological Analyses", ed. R.H. Kennet, T. J. McKearn and K.B. Bechtol, Plenum Press, N.Y. , 1980. Also, electrofusing techniques may be employed. Zimmer¬ man, U. and Vienken, J. , J .___Membrane Bio . 6.7.: 165 (1982) . The hybridomas are then screened for production of anti-idiotypic antibody. A suitable preliminary screening technique is an enzyme-linked im uno- sorbent assay (ELISA) using plates precoated with the monoclonal antibody of interest and control immunoglobulins. That is, solid phase immuno- adsorbent is prepared by coupling, for example HA-lA, to an insoluble matrix. The immunoadsorbent is brought into contact with culture supernatants of hybridomas. Hybridomas secreting antibodies re¬ active with the preselected monoclonal antibody but not with other immunoglobulins are selected and subcloned. Anti-idiotypic antibodies can then be tested for "public" and "private" specificity using immunofluorescence analysis. Kiyotaki, M. ______ ___!___ , I _._∑ _l_l-l°λ- ill:^150 ( 1987 ) . Monoclonal anti-idiotypic antibodies for use in the assays can be produced in large quantities by injecting anti- idiotype-producing hybridoma cells into the peritoneal cavity of mice and, after an appropriate time, harvesting ascites fluid from the mice which yields a very high titer of homogenous anti-idiotypic antibody. The monoclonal antibodies are isolated therefrom. Alternatively, the anti¬ bodies can be produced by culturing anti- idiotype producing cells ______ vitro and isolating secreted monoclonal anti- idiotype antibodies from the cell culture medium directly. Because the anti-idiotypic antibodies of this invention discriminate between the preselected antibody and the normal suite of human immunoglobulins, they permit a sensitive immunochemical assay of the preselected monoclonal antibody. A particularly preferred type of immunochemical assay is a sandwich immunometric assay in which antigen (i.e. , natural or recombinant the monoclonal antibody) is measured directly by re¬ acting it with murine anti-idiotypic antibody that is labeled, or capable of being labeled. In sandwich assays of this invention, a complex is formed comprising: 1) a first antibody specific for an idiotope on a preselected monoclonal anti¬ body, 2) the preselected antibody (the "antigen") , and 3) a second antibody specific for an idiotope of the preselected antibody. This first antibody is labeled either before or after formation of complex. The label can be attached directly or indirectly to antibody. For example, the antibody can be com- plexed with biotin, to which a label can be attached via avidin linkage. The complex can be formed before it is im¬ mobilized onto a solid phase. In other embodiments, the complex can be immobilized on the solid phase at - the same time that it is formed. In preferred assays, the antigen is immobilized on an immuno- adsorbent which specifically "captures" or binds the preselected antibody ("antigen"). This immuno- adsorbent is formed by affixing to it an antibody specific for an idiotope- of the preselected mono- clonal antibody. In preferred sandwich assays of- this invention, two anti-idiotypic antibodies which recognize identical idiotopes on the antigen can be used. Thus, for most purposes, the same anti- idiotypic antibody used to form the immunoadsorbent can be used as the labeled antibody. Sandwich assays may be performed in forward, reverse or simultaneous mode. In a forward sandwich assay for an antibody, a monoclonal antibody directed against an idiotope of the antibody is affixed to a solid phase. A liquid sample to be tested is incubated with the immunoadsorbent . Incubation is maintained for a sufficient period of time to allow the antibody in the liquid sample to bind to its immobilized anti-idiotypic antibody on the immunoadsorbent. After this first incubation, the solid phase immunoadsorbent is separated from the sample. The immunoadsorbent is washed to remove unbound monoclonal antibody and interfering sub¬ stances, such as non-specific binding proteins, which may also be present in the liquid sample. The immunoadsorbent containing antibody bound to im¬ mobilized antibody is subsequently incubated with labeled anti-idiotypic antibody, specific for idiotope(s) on the monoclonal antibody. The incuba- tion is carried out for a period of time and under conditions sufficient to ensure binding of the labeled anti-idiotypic antibody to the antibody. After the second incubation, another wash may be performed to remove unbound label from the solid phase immunoadsorbent. The labeled anti-idiotypic antibody bound to the solid phase immunoadsorbent is then measured, and the amount of label detected serves as a direct measure of the amount of antibody present in the liquid sample. Anti-idiotypic monoclonal antibodies can provide the basis for an extremely sensitive forward sandwich immunoassay for an antigen such as HA-lA, a human IgM monoclonal antibody to endotoxin. In one configuration, murine anti-idiotypic monoclonal antibody is used to form the immunoadsorbent and also serves as the labeled antibody. The assay is performed as outlined above. With these two anti¬ bodies, the assay is specific for HA-lA and highly sensitive. Levels of HA-lA in serum or tissue culture fluid at limiting concentrations of about 25 ng/ml can be detected. The sandwich immunoassays may also be performed in reverse and simultaneous modes. In reverse modes, an incubation mixture is formed of the liquid sample to be tested and a soluble labeled anti- idiotypic antibody directed against an idiotope of a preselected antibody such as HA-lA. The mixture is incubated, then contacted with a solid phase immuno¬ adsorbent containing an anti-idiotypic monoclonal antibody directed against the same or different idiotope of the preselected antibody. After another incubation, the immunoadsorbent is separated from the mixture and the label bound to the immuno¬ adsorbent is taken as an indication of the amount of preselected monoclonal antibody in the liquid samp1e . In the simultaneous mode, an incubation mixture is formed of the liquid sample containing monoclonal antibody to be measured (e.g. , HA-lA) , labeled anti-idiotypic antibody and the solid phase immuno- adsorbent. After appropriate incubation, the solid phase immunoadsorbent is separated from the mixture and the label associated with the immunoadsorbent is measured to give an indication of the amount of monoclonal antibody in the liquid sample. For each incubation step in the various assay formats , the time and incubation conditions are selected to ensure optimal binding of antigen (i.e. , monoclonal antibody) to the immobilized anti- idiotypic antibody and to labeled anti-idiotypic antibody. In the forward sandwich immunoassay, where two incubation steps are required, the solid phase immunoadsorbent containing immobilized anti- idiotypic antibody is incubated with the liquid sample for several hours at room temperature to obtain optimal binding. The parameters which yield optimal binding of monoclonal antibody reagent may be established for other formats of the immunoassay by no more than routine experimentation. The immunoassays of this invention are used to detect and quantify monoclonal antibody in a liquid sample. Liquid samples include essentially all biological fluids such as blood, or blood-derived fluids such as plasma or serum, as well as urine, lymph, etc. Also, the liquid sample may be a sample of a liquid medium in which lymphocytes or other mammalian cells have been cultured. They may also be extracts or supernatants of microbial cultures. The assays of this invention can be used to detect any monoclonal antibody capable of forming a suitable complex with anti-idiotypic monoclonal antibodies (i.e. , labeled anti-idiotypic antibody preselected monoclonal antibody: unlabeled anti- idiotypic antibody) , mammalian, preferably human monoclonal antibodies. These monoclonal antibodies are generally useful as therapeutic reagents. Assay methods of this invention can be used to detect monoclonal antibodies raised against carcinoma cells, lymphoma cells, fibrin (e.g. , T2G1, Kudryk, B. e_t al_ , Mol^Immunol^ li- 89 (1984)) , platelet (e.g. , 7E3 , European Patent Application No. 205,207) endotoxin (e.g. , HA-lA) and many others. Monoclonal antibodies that are chimeric (i.e. , those in which variable regions of antibodies from one mammal are joined to constant regions of antibodies from a different mammal) , can also be detected in assays of this invention. In selected solid phase immunometric assays of this invention, anti-idiotypic monoclonal antibodies reactive with preselected monoclonal antibodies can first be immobilized by affixing them to a solid phase to create an "immunoadsorbent". The anti- idiotypic antibody is therefore affixed to the solid phase before the three-part complex (i.e. , labeled anti-idiotypic antibody: preselected monoclonal antibody: unlabeled anti-idiotypic antibody) is created. This tripartate or ternary complex can also be attached to a solid phase after the complex is formed. This can be accomplished, for example, by affixing avidin to the solid phase and allowing the ternary complex to form in solution, one anti¬ body of this complex being labeled with biotin. Many types of solid-phases may be employed. Well-known solid phases include beads formed from glass, polystyrene, polyprop lene, dextran, and other materials; tubes formed from or coated with such materials, etc. The anti-idiotypic antibody can be either covalently or noncovalently bound to the solid-phase 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-phases and methods for immobilizing antibodies thereon, or will be able to * ascertain such using no more than routine experimentation. In the various solid phase assays of this invention, the immunoadsorbent can be separated from incubation mixtures containing the liquid sample, the labeled antibody or both. Separation can be accomplished by any conventional separation, filtra¬ tion, or centrifugation step. Preferably, the immunoadsorbent is washed prior to contacting it with a second incubation medium (e.g. , a solution of labeled anti-idiotypic antibody and the preselected antibody) and prior to measuring the amount of label associated with the immunoadsorbent. The washing removes nonspecific interfering substances or excess label which may affect the accuracy and sensitivity of the assay. In each of the immunoassays of this invention, monoclonal anti-idio ypic antibody directed against an idiotope of a preselected mammalian monoclonal antibody is also used as the labeled antibody (tracer) . Such antibodies can be labeled directly 125 with a radioactive material, such as I; labeled with an optical label, such as fluorescent material; labeled with an enzyme; or labeled by some other technique. These antibodies can also be labeled Indirectly (I.e. , by complexation with another labeled antibody) . To determine the amount of monoclonal antibody In the liquid sample, either the amount of label associated with the immunoadsorbent or the amount of unbound label, that is, labeled anti-idiotypic antibody which remains in soluble form, is measured. Generally, it is preferable to measure the label bound to the immunoadsorbent because at very low concentration of monoclonal in the sample, only small amounts of labeled anti-idiotypic antibody bind the immunoadsorbent. Thus, for accuracy the label associated with the immunoadsorbent should be measured directly. The label may be detected by a gamma counter, for example, if the label is a radioactive gamma emitter, of by a fluorimeter, if the label Is a fluorescent material. In the case of an enzyme label, detection may be done by * colorimetric methods employing a substrate for the enzyme . The measured amount of label detected is then compared to a quantitative relationship between the amount of preselected label and the amount of monoclonal antibody. The quantitative relationship can be determined by performing the immunoassay with standards (i.e. , liquid samples containing known amounts of monoclonal antibody) . For several samples containing different amounts of preselected monoclonal antibody, the assay is conducted and the amount of label either bound or unbound to the immunoadsorbent is determined; a curve is con- structed defining the quantitative relationship between the amount of label and the amount of monoclonal antibody. By reference to the curve, the amount of monoclonal antibody in a liquid sample containing an unknown amount of monoclonal antibody can be determined from the amount of label detected. The immunoassays described provide rapid, highly sensitive, inexpensive and reproducible methods for detection and quantification of mono- clonal antibodies. The assays provide a substitute for existing bioassays which are more time-consuming and variable and much less sensitive and specific. Thus, the assay reagents may be provided conven¬ iently in kits . The assays may be employed by hospitals or clinical laboratories to determine levels of thera¬ peutic and/or diagnostic monoclonal antibodies in * serum, plasma or other biological fluids of patients. The assay may also be used to monitor the amount of monoclonal antibody during the course of antibody therapy. This will be of predictive value in managing the course of treatment in a variety of disease .states. The reagents for performing the assays of this invention may be assembled in assay kits. For instance, a kit for performing an immunoassay for HA-lA would comprise a solid phase immunoadsorbent containing an anti-idiotypic antibody specific for one idiotope of HA-lA, a labeled anti-idiotypic antibody specific for the same idiotope of HA-lA and, optionally, an HA-lA standard. The invention is further described in the following examples wherein all parts and percentages are by weight degrees are celsius. Exam£ ______s.: Materials and Methods Gram negative lipopolysaccharide (LPS) was obtained from List Biological Laboratories, Inc. Campbell, CA. E. coli was purchased from Sigma Chemical Company, St. Louis, MO. Normal human IgG, IgM and Ig was supplied by Southern Biotechnology Associates, Inc. , Birmingham, AL and normal human IgE and IgD was supplied by Behring Diagnostics, 1 12255 LLaaJJoollllaa,, CCAA.. II wwaass <obtained from the DuPont Company, Wilmington, DE Production of HA-lA The HA-lA monoclonal antibody was produced by a hybridoma cell line generated by fusion of splenic lymphocytes with a heteromyeloma cell line (Teng, N.N.A. , K.S. Lam, F.C. Rieva and J.S. Kaplan, Proc. Natl. Acad. Sci. USA, 8j3:7308 -7312 (1983) and Bron, D. , M.B. Feinberg, N.N.H. Teng and H.S. Kaplan, Proc. Natl. Acad. Sci. USA, 1:321 (1985)) . The splenocytes were removed from a patient undergoing splenectomy during staging for Hodgkin' s disease. The patient had been immunized with the J5 mutant of Escherichia Coli (E. co_l.i) which expresses the core oligosaccharide common to the lipopolysaccharide (LPS) of all Gram-negative bacteria. The HA-lA monoclonal antibody cross -reacts with endotoxins from a wide range of unrelated species of Gram- negative bacteria and protects against lethal Gram-negative bactere ia in mice (Teng, N.N.H. , H.S. Kaplan, J.M. Herbert, C. Moore, H. Douglas, A. Wunderlich and A. Braude , Pr££_. Na ^ tl^ Acad_, £Ϊ^. U A, 8_2:1790 (1985)) . HA-lA is a human IgM k molecule. It was isolated and purified by Centocor (Malvern, PA) and provided as a solution of 5 mg/ml in 0.01 M Sodium phosphate, 0.3M NaCI, pH 7.2 buffer. ^^£du ^i 1 £n_£f_Murine__m£n £l_o_nal__an^^_ ^d^£ty2i£ anti dies_t£_HA_* : _lA Lymph node cells from BALB/c mice immunized with HA-lA were fused with non-Ig producing cell line P3-X63-Ag8.653 (Kearney, J.F. et a1. , J^ iϋSH5 ι 1_^3:1548 (1979)) . Culture supernatant from hybridoma containing' wells were tested for antibody specificity using an enzyme- linked immuno- sorbent assay (Elisa) in 96-well plastic plates pre-coated with HA-lA or control immunoglobulins. Hybridomas that secreted antibodies reactive with HA-lA, but not with other immunoglobulins including IgM or IgA paraproteins and normal IgG were selected and subcloned by limiting dilution. The anti-idio- typic reagents were subsequently tested for "public" and "private" idiotypic specificity utilizing a two-color immunofluorescence analysis as previously described (Kiyotaki, M. e t a 1 . , J_. Immuno ______ , l :4150-4158 (1987)) . The two cell lines having "private" anti- idio ypes (9B5.5 and 15B2.2) were injected intraperitoneally into pristane primed syngeneic mice for ascites production. Purification of anti- idioty ^c_m£n £l nal._an^j.b di.eg The ascites fluid was purified by passage over a Bakerbond ABx HPLC column (10x250 mm) equilibrated with 25mM MES buffer pH 5.5. After the elution of unbound proteins, a gradient from 0 to 100% of IM NaOAc pH 7.0 was used over 60 min to elute the monoclonal antibody. The recovered antibody was analyzed for purity using a Quick-check analysis (BioRad, Richmond, CA) . This analytic column was a Blo-Sil TSK-250 (7.5 x 300mm) . The eluting buffer was .01M phosphate, 0.3 M NaCI , 10% dimethyl- sulfoxide (v/v) eluting at 1 ml/min. HA^lA solid phase _radiome.tric_assay Polystyrene beads, 6.4 mm diameter (Precision Plastic Ball, Inc. , Chicago, IL) , were coated with 200 μl anti-id 9B5.5 in phosphate buffered saline (PBS) at a concentration of 5 μg/ml. Beads were washed three times with PBS containing 2% bovine serum albumin (BSA) and .02% Tween 20 and allowed to stand in wash buffer for 1 hr at room temperature. The beads were air dried and stored at 4°C until used. Human monoclonal antibody HA-lA was diluted in normal human serum (NHS) at concentrations ** ranging from 12.5-6400 ng/ l. Standard, controls and patient serum at appropriate dilution were incubated (100 μl) for 2 hr in triplicate with coated beads on a laboratory rotator at room temperature. The beads were washed with 4 ml of PBS. One hundred μl of radiolabeled anti-idiotypic antibody 15B2.2 was added to each bead at a con¬ centration of 1 g/ml. The incubation was continued for an additional 1 hr . The beads were washed again with 4 ml of PBS, transferred to clean tubes and the bead associated radioactivity was determined with a Micromedic Automatic Gamma Counter (Micromedic System, Inc. , Horsham, PA) interfaced with an IBM System 2. A logit-data reduction program was used to generate the standard curve and the values of controls and patient samples. l ^ΪS^:__iϊ£B-_ _έ_£Σ £ B The purified monoclonal antibodies were labeled with 125I by a modified method of Greenwood, e t a 1 . , 1963. The radiolabeled monoclonal antibodies were analyzed using HPLC as described above, except that a radioisotope monitor was used to detect radio- activity in sequence with the U.V. monitor. The protein was measured in final preparation by the method of Lowry , O.H. e t aj.. , _J___B.i£i_. l]l 2L.. 12 . 3:265 (1951) . E am£_. _l ι D £. £S££-^_£_f—^£__ SS£££ _§.5;- ___f£_£_SΔ_. t___2^______ι__l_ιin___t_ i__.___lA Among 11 hybridomas producing antibodies which were reactive in an ELISA with HA-lA but not with other myeloma proteins or normal IgG, " only two antibodies (9B5.5 and 15.B2.2) were found by two- color immunofluorescence analysis to be non- cross -reactive with plasma cells generated from pokeweed mitogen-stimulated blood lymphocyte ( 0.1%) . The other 9 antibodies were cross - reactive to the plasma cells on the order of 0.1-2.0%. Thus, antibodies 9B5.5 and 15B2.5 were designated as "private" anti-idiotypic antibodies. The monoclonal antibodies were purified as described above. The final products were concentrated to 5.0 mg/ml on an A icon 8050 stirred cell filtration apparatus and were stored at 4 β C. Figure 1 shows the HPLC profile of the purified monoclonal anti-Id antibody 15B2.2 (A) and the radiolabeled 15B2.2 (B) . There were no aggregates or break down products seen. HA^l _ £l._-.d_£ha e_£adi : £ tri. _a a-_^ A number of studies were carried out to estab¬ lish the standard conditions of this assay. Polystyrene beads were carried with varying amounts of 9B5.5 from 0.1 μg to 1.6 μg/bead. Maximum binding occurred at 1.0 μg/bead. The amount of radiolabeled 15B2.2 used in the assay was determined by incubating the 9B5.5 coated beads with HA-lA standards followed by incubation with varying amount of 125 I - 15B2.2. The 0.1 μg of 125 I - 15B2.2 was sufficient to* have an excess of 15B2.2 available at all concentrations of standard. The two incubations were carried out, at intervals varying from 30 min to 18 hrs at both room temperature and 37° C. Greater than 90% of maximum binding occurred with 2 hour incubation with sera and one hour incubation with radioactive 15B2.2. Binding was comparable at room temperature' and 37 β C. For convenience, room temperature incubations were used. Using the HA-lA assay as described above, increasing concentrations of HA-lA, normal human IgG, IgM, IgA, IgE and IgD . were assayed (Fig. 2) . The cross reactivity of these various immunoglobulin preparations was calculated as less than 0.1%. The sensitivity of the assay defined as 2 standard deviations above the nonspecific binding (normal human serum) was approximately 25 ng/ml . The linearity of the assay is best seen by logit-log analysis (Fig. 3) . The assay is linear between 25 and 800 ng/ml. Recovery and reproducibility studies were also carried out. Normal human serum was "seeded" with 0.07, 1.6, 8.2, 12.0 and 19.0 μg/ml of HA-lA and subsequently assayed at appropriate dilutions in three separate assays. As seen in Table 1, the average percent recovery was 116±4% and the range was 113-123% over this wide spectrum of serum concentrations . Th r V r HA lA n human um The inter-assay variance was examined by assay of three concentrations of HA-lA (200, 1000 and 2500 ng/ml in human serum) as positive controls in 10 consecutive independent assays. The means for the three HA-lA levels were 206 ± 12, 981 ± 65 and 2573 ± 161 ng/ml, respectively (a coefficient of vari¬ ation of between 5.8 and 6.6 percent) . Since this antibody (HA-lA) is likely to be used In patients with gram negative sepsis, the effects of bacteria and LPS on the assay HA-lA in human serum was studied. Human sera containing 20 μg/ml HA-lA was Incubated with varying numbers of bacteria (E. l_) or varying concentrations) LPS at 37" for 20 minutes and then assayed for HA-lA content (Table 2) . These additives had no adverse effects in detection of HA-lA. Table 2 : Eff££_t_£f_E_. coli _and .LPS o i__detecti£n of HA-lA_in human serum__ Mean HA-lA measured Serum + 201 _gg/ml HA-lA. plus: ϋ≤Zs * nothing 21.9 ± .49 <1 CFU E. coli/ml 21.4 ± 1.8 10 CFU E. coli/ml 20.2 ± 1.8 80 CFU E. coli/ml 21.02 ± 1.5 0.20 μg/LPS 22.95 ± 1.04 2.0 μg/LPS 20.5 ± 1.8 20.0 μg/ml LPS 19.9 ± 1.9 Example 2 Detectio of HA-lA from human patient A patient was given 100 g. of HA-lA and serial serum samples drawn for quantitation of HA-lA. As seen in Figure 4, mean peak serum concentration after infusion was 36.6 μg/ml, which is 101.5% of the predicted value, based on the patient's calcu¬ lated plasma volume. The data fit a one compartment plasma disappearance model having a mean plasma half-life of 24.5 hrs (Sisson, 1983) . This illustrates the ability of this assay to be used for pharmacokinetic studies. _ _l_l_---_-_-_ϊ_l_--L Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims .

Claims: CLAIMS 1. An Immunometric assay for a preselected anti¬ body in a biological fluid comprising: a. forming a ternary complex of: i. a first monoclonal antibody that is labeled, or capable of being labeled, specific for an idiotope of the pre¬ selected antibody; ii. a second monoclonal antibody specific for an idiotope of the preselected anti¬ body; and iii. the preselected antibody; b. detecting the amount of label associated with the complex formed from the com- ponents'ln step (a) , as an indication of preselected antibody in the fluid. 2. An assay of Claim 1, wherein the first labeled monoclonal antibody is labeled with Iodine-125. 3. An assay of Claim 1, wherein the second monoclonal antibody is immobilized on a solid phase before or after formation of the ternary comple . 4. An assay of Claim 1, wherein the first and second monoclonal antibodies are anti-idio ypic antibodies specific for the same private idiotope on the preselected monoclonal anti¬ body. - 25 - 5. An assay for a preselected antibody in a blood- derived sample, comprising the steps of: a. forming an incubation mixture of the sample and a solid phase immunoadsorbent 05 containing monoclonal anti-idiotypic antibody affixed to a solid phase, this antibody specific for an idiotope of the preselected antibody; b . incubating the incubation mixture under 10 conditions and for a period of time sufficient for preselected antibody in the liquid sample to bind to the immuno¬ adsorbent; c. thereafter separating the immunoadsorbent 15 from the liquid sample; d. forming an incubation mixture of the immunoadsorbent and soluble labeled monoclonal anti-idiotypic antibody, this antibody specific for an idiotope of the 20 preselected antibody; e. incubating the mixture under conditions and for a period of time sufficient for the labeled antibody to bind any preselected antibody; - *■ •- f. separating the solid phase immunoadsorbent from unbound labeled anti-idio ypic an ibody; g. detecting the amount of label bound to the immunoadsorbent or the amount of unbound, 30 label; and h. relating the amount of bound label or unbound label detected to a quantitative relationship between the amount of label and the amount of preselected monoclonal antibody to determine the amount of preselected antibody in the sample. 6. An assay of Claim 5, wherein the soluble labeled anti-idiotypic monoclonal is labeled with materials selected from the group consist- ing of radionuclides , enzymes and fluorescent agents . 7. An immunoassay of Claim 5, wherein the anti-idiotypic monoclonal antibodies are anti-idiotypic antibodies specific for the same private idiotope on the preselected monoclonal antibody.* 8. An assay of Claim 5, wherein the preselected antibody Is antibody HA-lA. 9. An assay for a preselected antibody in a liquid sample, comprising the steps of: a. forming an incubation mixture of the sample and a soluble, labeled anti- idiotypic monoclonal antibody that is specific for an idiotope on the pre- selected monoclonal antibody; 27 - b. incubating the incubation mixture under conditions and for a period of time sufficient for preselected monoclonal antibody in the liquid sample to bind to the labeled, soluble monoclonal antibody; c. contacting a solid phase immunoadsorbent containing anti-idiotypic monoclonal antibody affixed to a solid phase, this antibody specific for an idiotope on the preselected monoclonal antibody with the incubation mixture; d. incubating the components of step (c) under conditions and for a period of time sufficient for preselected monoclonal antibody bound to the labeled, soluble anti-idiotypic monoclonal antibody to bind the immunoadsorbent; e. separating the solid phase immunoadsorbent from the incubation mixture; f. detecting the amount of label bound to the solid phase immunoadsorbent or the amount of unbound label; and g. relating the amount of label detected to a quantitative relationship between the amount of label and the amount of preselected monoclonal antibody to determine the amount of preselected monoclonal antibody in the liquid sample. 10. An immunoassay of Claim 9, wherein the anti- idiotypic monoclonal antibodies are specific for the same private idiotope of the pre¬ selected antibody. 05 11. An immunoassay of Claim 9, wherein the pre¬ selected monoclonal antibody is HA-lA. 12. An Immunoassay of Claim 9, wherein the soluble labeled anti-idiotypic monoclonal antibody is labeled with materials selected from the group 10 consisting of radionuclides , enzymes and fluorescent agents. 13. An immunoassay for preselected monoclonal antibody in a liquid sample, comprising the steps of: 15 a. forming an incubation mixture of I. liquid sample; li . a solid phase immunoadsorbent containing immobilized monoclonal antibody that is specific for an. idiotope of the 20 preselected monoclonal antibody; and iii. labeled soluble monoclonal antibody that is specific for an idiotope of the preselected monoclonal antibody; b. incubating the mixture under conditions 25 and for a period of time sufficient for the preselected monoclonal antibody in the liquid sample to complex with both the immobilized monoclonal antibody and the labeled, soluble, monoclonal antibody; c. thereafter separating the solid phase immunoadsorbent from the incubation mixture ; d. detecting the amount of label bound to the solid phase immunoadsorbent or the amount of unbound label; and e. relating the amount of label and the amount of preselected monoclonal antibody to determine the amount of preselected monoclonal antibody in the liquid sample. 14. An immunoassay of Claim 13, wherein the im¬ mobilized and soluble monoclonal antibodies are specific for the same private idiotope of the preselected monoclonal antibody. 15. An immunoassay of Claim 13, wherein the pre¬ selected monoclonal antibody is HA-lA. 16. A forward sandwich immunoradiome rie assay for preselected monoclonal antibody in a liquid sample comprising the steps of: a. forming an incubation mixture of the liquid "sample and solid phase immuno¬ adsorbent comprising polystyrene beads to which is affixed anti-idiotypic monoclonal antibody specific for an idiotope of the preselected monoclonal antibody; b. incubating the mixture at room tempera¬ ture; c. thereafter separating the immunoadsorbent from the liquid sample; d. forming an incubation mixture of the 125 immunoadsorbent and soluble I-labeled anti-idiotypic monoclonal antibody specific for an Idiotope of the pre- selected monoclonal antibody; e. incubating the mixture at room tempera¬ ture ; f. separating the immunoadsorbent from 125 unbound I-labeled monoclonal antibody; g. detecting the amount of label bound to the immunoadsorbent; and 125 h. relating the amount of bound I-label and the amount of preselected monoclonal antibody to determine the amount of preselected monoclonal antibody in the liquid sample. 17. An assay. of Claim 16, wherein anti-idiotypic monoclonal antibodies are anti-idiotypic antibodies specific for the same private Idiotope on the preselected monoclonal anti¬ body. 18. An assay of Claim 16, wherein the preselected monoclonal antibody is HA-lA. 19. An assay kit for preselected monoclonal anti- body in a biological sample from a human, Including: a. an immunoadsorbent containing anti- idiotypic monoclonal antibody specific for an idiotope of the preselected monoclonal antibody; and b. labeled anti-idioty ic monoclonal antibody specific for an idiotope of the pre¬ selected monoclonal antibody. 20. An assay kit of Claim 19, wherein the anti-idiotypic monoclonal antibodies are anti-idiotypic antibodies specific for the same private idiotope of the preselected monoclonal antibody. 21. An assay kit of Claim 19 further including: c. a preselected monoclonal antibody standard. 22. An assay kit for a preselected monoclonal antibody in a biological sample from a human including : a. an immunoadsorbent comprising polystyrene beads with anti- idiotopic monoclonal antibody specific for an idiotope of the preselected monoclonal antibody affixed thereto ; 125 b. I-labeled anti-idiotypic monoclonal antibody specific for an idiotope of the preselected monoclonal antibody; and c. preselected monoclonal antibody standard. 23. A kit of Claim 22, further comprising: c. preselected monoclonal antibody standard. 24. A kit of Claim 22, wherein wherein the anti- idiotypic monoclonal antibodies are specific for the same private idiotope of the pre¬ selected antibody. 25. An assay of Claim 22, wherein the preselected monoclonal antibody Is HA-lA.

Inventors: LOBUGLIO ALBERT F, LOBUGLIO, ALBERT, F.
Applicants: CENTOCOR INC, CENTOCOR, INC.
Application year: 1989
Main classification: G01N33
IPC codes: G01N33