JPH04507285A - Anti-idiotope immunoassay - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Anti-idiotope immunoassay Korigo! 1 Recent developments in monoclonal antibody technology have enabled the production of homogeneous antibodies with a single specificity. It became possible to produce preparations in unlimited quantities. Anti-11 (present in Rh blood type) Evans et al., 140:941 (1988) ], human immunodeficiency virus [Thompson et al, σ, 58:157 (1986)], malaria parasite [Udomsangpe Lch et al, 5science, 32231 (1986)], Endotoxin [Teng et at, PNAS, 80 near 30 P11 (1 985)], human platelets (Nugent et at, Blood, 7) 0:16 (1987)], and human tumor cells, e.g. maguchi et al., PNAS, 84:2416 (1987) J, Breast Cancer [Schlom et al, PNAS, 77:6841 ( 1980)] and colon cancer [)1aspel eL al,, Canc■■ Research, target: 3951 (1,985) 1-specific human moiety Many noclonal antibodies have been developed. These monoclonal antibodies are therapeutic. It may be useful as a therapeutic agent. Furthermore, compared to the use of pooled human hyperimmune sera, When viewed, they have the advantages of both safety and product standardization. in addition, Their use is probably more immunogenic than using murine monoclonal antibodies. There are likely to be fewer problems with origin.

Clinical trials involving human monoclonal antibody products will improve the drug kinetics of such therapeutic agents. It would require a thorough analysis. These analyzes determine the dosage and dosing regimen of therapeutic agents. Enables rational design. Drug kinetics of monoclonal antibody products in humans To examine the kinetics, radiolabeled antibodies were used [5ears et al. , J. Biol.

A box of animals! Hyoshi, 3:138 (1984) and Meeker et al. , Blood, 65:1349 (1985) 1, but this method does not modify the molecule. and the need to use radiolabeling methods, which may result in changes in pharmacokinetics. It has the disadvantage of being necessary. Additionally, the pharmacokinetics of the injected dose are It can also be determined by quantifying circulating antibodies at different time points (Nihazae li et al., Cl1n, Res., 35:615A (1988) ], this technique is used to monitor the presence of a large excess of normal human immunoglobulin in the blood groove. We need a specific and sensitive assay that can detect noclonal antibodies. would be.

Vertical 9 summer η The present invention provides a method for detecting preselected antibodies in biological fluids such as blood samples. Provides immunoassays. This assay uses a first monoclonal antibody to be labeled, a predetermined antibody and a second monoclonal antibody, and in a biological fluid. detecting the amount of label associated with said complex as an indicator of the presence or amount of a given antibody of consists of doing. The first and second monoclonal antibodies are Specific for idiotopes (antigenic determinants). The preferred assay method is solid-phase assay. method in which at least one antibody component of the complex is is later bound to a solid phase. The first antibody can be labeled before or after complex formation. can.

Brief description of the drawing Figure 1 shows the purified anti-idiotype murine monoclonal antibody 15B2.2. HPLC profile is shown. (A) Blo-5il TS with 250 columns ( Bio-Rad Quick Check with 300x 7.5 m11) Analysis using Analyzer. The sample was 0.3M NaCl, 10% dimethylene. using 10mM phosphate pH 6,8 buffer containing Rusulfoxide (V/V)] , , Om+/min. (B) Radioisotope for detecting radioactivity 1582-2 after radiolabeling using HPLC as described above, with a monitor. analysis. 1 Figure 2 shows the cross-reactivity of normal human immunoglobulins. Using solid phase radiometric assay method , gradually increasing concentrations of HA-I A normal human IgG % IgM, IgA, Ig E and IgD in ink assays as described in Materials and Methods. It was incubated. (0) HA-IA, (O) other normal human immunoglobulins. cross anti The response was calculated to be less than 0.061%.

Figure 3 shows the dose-response curve of HA-IA in a solid-phase radioassay. gradually increase Concentration of HA-IA was incubated with 9B5.5 coated beads for 2 hours and washed. , incubation was continued for 1 hour with ``l-15B2.2''. The performance was measured and a standard curve was created using Iogit-1og transformation regression analysis.

Figure 4 shows human IgM monoclonal antibodies in a patient who received 100 mg of antibody. Serum concentrations of HA-IA are shown. Serum samples obtained from patients are assayed using solid-phase radioactivity. The assay was performed using the method at appropriate dilution rates. Values are the average of three measurements ± 1 standard deviation. be.

It's been a long time since I've been in the middle of a long time since I've been in the middle of a long time. The present invention provides a novel immunoassay for a given antibody in a liquid sample. Ru. In the immunoassay of the present invention, a labeled first monoclonal antibody, a A complex consisting of the target antibody (“antibody”) and a second monoclonal antibody is formed. Ru. The first and second monoclonal antibodies are the idiotopic sites of the antibody to be measured. reacts. Preferably they are derived from the same hybridoma cell line. , and reacts with the same idiotopic site on the membrane. This immunoassay is It can be done in reverse, simultaneous, or forward 7 formats. After the formation of the complex, a given The amount of monoclonal antibody is determined by detecting the amount of label associated with the complex. quantified. A preferred assay is one in which the complex is immobilized on a solid phase.

The invention is based on the recognition that antibodies themselves are antigenic. Therefore, exemption Antibiotics that recognize antigenic determinants present in both the constant and variable regions of epiglobulin chains. It is possible to induce the body. L chain and H chain related to antigen binding site of antibody The antigenic determinants present in the variable region of the chain are called idiotopes. It is. The group of idiotopes in each antibody molecule is the idiotope of that antibody. Specify the type.

The first and second monoclonal antibodies of the invention are anti-idiotypic. Sunawa They contain antigenic determinants associated with the antigen-binding site of a preselected antibody molecule. biotope). Preferably, but not necessarily , these monoclonal antibodies are specific for “unique idiotopes”. Ru. The term “unique idiotope” refers to the fact that some idiotopes are immunoglucose. It is based on the fact that it constitutes the Roblin idiot type. Therefore, two types The above antibodies bind to different antigens and are derived from separate vN and ■, genes. each individual idiotope is “fixed” to one of several antibodies. However, if the vnt is far from the antigen binding site, In some cases, the vL region portion has a common di-idiotope among several types of antibodies.

This particular antibody has a "public" idiotope. The “uniqueness” of the antibody of the present invention is The more general anti-idiotypic “public” antibodies [ Kiyotaki, M. at, J. Immunol, 138: 4150-4158 (1987)] may also be effective, but these and normal immunity Potential for cross-reactivity with globulins is higher than with “unique” anti-idiotypes right.

Antibodies useful in the present invention can be obtained by immunizing an animal (preferably a mouse) with a given antibody. It can be obtained by Cells that produce antibodies are produced using antibodies from the immunized animal. It is formed by the fusion of body-producing cells and immortal cells such as myeloma.

Particularly preferred monoclonal anti-idiotype antibodies of the invention are "unique" anti-idiotype antibodies. Otype antibodies and are produced as described herein. Monochrome like this An example of a null anti-idiotype antibody is the human monoclonal antibody HA-IA. It specifically reacts with diotopes.

In a preferred embodiment, the anti-idiotypic monoclonal antibody 1 of the invention, a) Mouse myeloma that does not secrete antibodies and b) pre-selected human monochrome anti-ideal antibodies obtained from mice immunized against natural antibodies (e.g., HA-IA). I/ Produced from 1 hybridoma.

Mice received a primary immunization injection of a monoclonal antibody, followed by several secondary injections of the same antibody. Immunize by subsequent immunization injection. During or after the immunization method, antibodies (two In order to identify mice in which a specific immune response was induced, mouse serum was collected by ing. Tile cells are collected from the selected mice and fused. Appropriate fusion technology i Sendai virus method [Kohler, G. and Milstein, C. , Nature 256:495 (1975)], or polyethylene glycol Cole method [Kennet, R.H., “Monoclonal Antibod ies.

Hybridomas--A New Dimension in Biolo soot B T, J, McKearn and B, Bechtol, Plenu*P ress, N.Y., 1980]. Matco, Den Membrane B io, 67:165 (1982).

Hybridomas are then screened for production of anti-idiotypic antibodies. do. Suitable preliminary screening methods include monoclonal antibodies of interest and control immunizations. Enzyme-linked immunosorbent assay (ELISA) using globulin-coated plates It is. That is, solid-phase immunoadsorbents, for example, contain HA-IA in an insoluble matrix. It is made by coupling. This solid-phase immunosorbent material is used as a hybridoma. of culture supernatant. Reacts with a given monoclonal antibody but does not react with other immunoglobulins Hybridomas that secrete antibodies that do not react with Brin are selected and subcloned. be logged. Anti-idiotype antibodies are then used to identify “public” and Can be tested for "inherent" specificity. Kiyotaki, M, e tal, , J, I+a+muno1. Jikage 4150 (1987).

Monoclonal anti-idiotype antibodies for use in assays are Inject the hybridoma cells into the peritoneal cavity of mice, and after an appropriate period of time, Collect ascites fluid from mice that consistently contains high titers of uniform anti-idiotype antibodies. It can be obtained in large quantities. Monoclonal antibodies are then isolated.

Separately, anti-idiotype producing cells were cultured in vitro and secreted Alternatively, monoclonal anti-idiotype antibodies can be isolated directly from the culture medium. Antibodies can be obtained.

The anti-idiotype antibody of the present invention is a combination of a preselected antibody and normal human immunoglobulin. They allow sensitive immunization with preselected monoclonal antibodies because they identify Enable academic examinations. A particularly preferred format for immunochemical assays is anti-jlj [(i.e. , natural or recombinant monoclonal antibodies) labeled or labelable. A sandwich that can directly measure antigens by reacting with idiotype antibodies. This is a patch immunoassay.

In the sandwich assay method of the present invention, 1) a predetermined monoclonal antibody ideal a first antibody specific for the antigen; 2) a predetermined antibody (“antigen”); and 3) A complex is formed consisting of a second antibody specific for the idiotope of a given antibody. It will be done. This first antibody is labeled either before or after complex formation. The sign is It can be attached directly or indirectly to the antibody. For example, combine antibodies with biotin and a label is attached to this via avidin binding.

The complex can be formed before it is immobilized on a solid phase. Other embodiments Alternatively, the complex can be immobilized on a solid phase at the same time as it is formed.

In a preferred assay, the antigen is used to specifically "capture" or "capture" a given antibody (the "antigen"). is immobilized on the binding immunoadsorbent. This immunoadsorbent is a predetermined monoclonal formed by binding an antibody specific for the idiotope of the antibody to it Ru. The preferred Sand Inch assay of the invention involves detecting identical idiotopes on the antigen. Two anti-idiotype antibodies that recognize are used. Therefore, in most cases , the same anti-idiotype antibody used to form the immunoadsorbent was labeled. Used as an antibody.

Sandwich assays can be performed in forward, reverse or simultaneous formats. About antibodies In all forward sandwich assays, a model directed against the idiotope of the antibody is A noclonal antibody is bound to a solid phase. The liquid sample to be tested is an immunoadsorbent and Let it incubate. Incubation immunoadsorbs antibodies in liquid samples Continue for sufficient time to allow binding to the immobilized anti-idiotype antibody on the material. This most After the initial incubation, the solid phase immunoadsorbent is separated from the sample. This immunity Wash the adsorbent to remove unbound monoclonal antibodies that may be present in the liquid sample. Remove interfering substances such as non-specific binding proteins. Subsequently, conjugated to the immobilized antibody The antibody-containing immunoadsorbent is a labeled antibody specific to the idiotope of the monoclonal antibody. Incubate with idiotype antibody. Incubation is used to target antibodies. for a time and under conditions sufficient to ensure binding of the identifying idiotype antibody. Implemented. After the second incubation, unbound label is removed from the solid-phase immunosorbent. Wash once more to remove. Then, the labeled antibody bound to the solid-phase immunosorbent Measures the idiotype antibody and the amount of detected label present in the liquid sample Provides a direct measure of the amount of antibody.

The anti-idiotype clonal antibody is HA-IA (human 1g against endotoxin). A highly sensitive forward sandwich for antigens such as M monoclonal antibodies) It can provide the basis for immunoassay. In some configurations, mouse anti-id Biotypic monoclonal antibodies are used to form immunoadsorbents, and Also useful as labeled antibodies. This assay is performed as previously described. These two With one antibody, this assay is specific for HA-IA and is very sensitive. be. HA-IA in serum or tissue culture fluid can be detected at a limiting concentration of approximately 25 ng/m+. can be released.

Sandwich immunoassays can also be performed in reverse or simultaneous formats.

In the reverse format, the incubation mixture contains the liquid sample to be tested and the HA-IA A soluble labeled anti-id directed against a given antibody idiotope, such as Made from otype antibodies. After incubation, this mixture is Anti-idiotype monochromes directed against the same or different idiotopes contact with an in-phase immunoadsorbent containing a null antibody. After another incubation, The immunoadsorbent is separated from the mixture and the label bound to the immunoadsorbent is added to the liquid sample. Measured as an indicator of the amount of a given monoclonal antibody.

In the simultaneous format, the incubation mixture contains the monoclonal antibody to be measured. A liquid sample containing the body (e.g. HA-IA), a labeled anti-idiotype antibody and made from solid-phase immunosorbent material. After appropriate incubation, solid-phase immunosorbent to separate the immunoadsorbent and associated label from the mixture, and to isolate the monochrome immunoadsorbent and associated label in the liquid sample. measured as an indicator of the amount of null antibodies.

Each incubation step of the various assay formats requires immobilization. antigen to anti-idiotype antibodies and labeled anti-idiotype antibodies (i.e., The time and incubation regime should be adjusted to ensure optimal binding of the monoclonal antibody). condition is selected. Forward sandwich requiring two incubation steps Anti-Immunoa 7sei uses a solid-phase immunosorbent containing immobilized anti-idiotype antibodies and Optimal binding is achieved by incubating the liquid sample for several hours at room temperature. monochrome The parameters that result in optimal binding of the antibody reagents are similar to other aspects of the immunoassay. In the case of formats, too, it will be established only by routine experimentation.

The immunoassay of the present invention detects and defines monoclonal antibodies in liquid samples. used to quantify. Liquid samples include blood, or blood such as plasma or serum. Essentially any biological fluid such as fluid derived from blood, fluid, lymph, etc. included. Liquid samples can also be used in liquid media containing lymphocytes or other mammalian cells. It may also be a ground sample. They are also extracts or supernatants of microbial cultures. There may be.

The assay of the present invention involves combining an anti-idiotype monoclonal antibody with an appropriate conjugate (all Namely, labeled anti-idiotype antibodies: predetermined monoclonal antibodies, unlabeled monoclonal antibodies capable of forming anti-idiotype antibodies), mammals, Preferably used to detect human monoclonal antibodies. these things Clonal antibodies are generally useful as therapeutic agents. The assay method of the present invention uses cancer cells. , lymphoma cells, buiprin (e.g., T2G1, Kudryk, B., atal ,,, Mo1. Immun.

L company name (1984)), platelets (e.g. 7E3, European Patent Application No. 205207) ), monoclonal antibodies induced against endotoxins (e.g. HA-IA), etc. used for giving out. Chimeric (i.e., antibody variable region derived from a mammal) monoclonal (linked to the constant region of an antibody from a different mammal) Antibodies can also be detected in the assays of the invention.

Selected solid-phase immunoassays of the invention first involve the use of a predetermined monoclonal antibody. By immobilizing an anti-idiotype monoclonal antibody reactive with Therefore, the anti-idiotype antibody is a ternary complex. (i.e., labeled anti-idiotype antibody: predetermined monoclonal antibody: unlabeled (identical anti-idiotype antibodies) are bound to a solid phase before formation. This three-component complex The conjugate may also be attached to a solid phase after formation of the complex. For example, this can be applied to solid phase. Bind the 3-component complex (one antibody of this complex is labeled with biotin) in solution. .

This is achieved by forming a

Many types of solid phases can be used. Well-known solid phases include glass and polysilicon. Pieces made of tyrene, polypropylene, dextran, and other materials; Tubes formed from or branched from materials such as included. Anti-idiotypic antibodies are covalently linked via amide or ester bonds. or bound covalently or non-covalently to a solid phase by techniques such as adsorption. . Those skilled in the art will be aware of many other suitable solid phases on which to immobilize antibodies. know about the law and verify it simply by using routine experimental procedures will be possible.

In various solid-phase assay methods of the present invention, the immunoadsorbent material is a liquid sample, a labeled antibody. or separated from an incubation mixture containing both. Separation is normal separation , filtration or centrifugation steps. Preferably, the immunoadsorbent incubation medium (e.g., labeled anti-idiotype antibody and the antibody of interest) solution) and before measuring the amount of label associated with the immunoadsorbent. be purified. This wash may be non-specific, which can affect the accuracy and sensitivity of the assay. remove any interfering substances or excess labels.

In each immunoassay of the invention, a given mammalian monoclonal Monoclonal anti-idiotype antibodies directed against the idiotope of the antibody is used as a labeled antibody (tracer). This type of antibody is l! Like l　l direct labeling by radioactive substances; optical labels such as fluorescent substances; enzymes; or other techniques. can be understood. These antibodies may also be used indirectly (i.e., by another labeled antibody). can also be labeled (by forming a complex with).

Associated with an immunosorbent to determine the amount of monoclonal antibodies in a liquid sample The amount of labeled label or unbound label (i.e., label that remains in soluble form) The amount of anti-idiotype antibodies) is measured. typically bound to an immunoadsorbent. It is desirable to measure the labeled label. Because the monoclonal in the sample The concentration of antibody is very low, and a small amount of labeled anti-idiotype antibody on the membrane is used as an immunoadsorbent. This is because it simply combines with. Therefore, for accuracy, immunoadsorbents and The labeled label should be measured directly. For example, if the label is a radioactive gamma emitter Check the label with a gamma counter if the label is fluorescent, or with a fluorometer if the label is fluorescent. can be released. In the case of enzyme labeling, the enzyme's substrate is used (colorimetric method). Detection is performed by

In that case, the measured amount of detected label is determined by the preselected amount of label and the monoclonal antibody. It is compared with the quantitative relationship with body mass. Quantitative relationships are based on standards (i.e., known amounts of We decided to perform an immunoassay using a liquid sample containing monoclonal antibodies. You can read more. Several species containing varying amounts of a given monoclonal antibody Assays are performed on the sample to determine whether or not the label is bound to the immunosorbent. measure the amount of monoclonal antibody; create a curve that defines the quantitative relationship between the amount of label and the amount of monoclonal antibody. create. By referring to this curve, you can include unknown amounts of monoclonal antibodies. The amount of monoclonal antibody in the liquid sample is determined from the amount of label detected .

The above immunoassays are rapid, sensitive, inexpensive, and reproducible. A method for detecting and quantifying clonal antibodies is provided. This assay is relatively time consuming. Current I assays are highly variable, variable, and have relatively low sensitivity and specificity. It is an alternative. Thus, assay reagents are conveniently supplied in kits. It will be.

This assay detects treatment and/or used in hospitals and clinical laboratories to check levels of monoclonal or diagnostic monoclonal antibodies. be exposed. This assay also monitors monoclonal antibody levels during antibody therapy. It is also used to This controls the course of treatment in various disease states. will have a predicted value in doing so.

Reagents for performing the assay of the present invention are contained in an assay kit. For example, H A kit for performing an immunoassay of A-IA is a kit for performing an immunoassay of HA-IA. A solid-phase immunosorbent containing a tope-specific anti-idiotype antibody, the same as HA-IA. a labeled anti-idiotype antibody specific for one idiotope, and optional components HA-IA standard products.

The invention is further illustrated by the following examples, in which all parts and percentages are All measurements are by weight and temperatures are in degrees Celsius.

Chihoga Materials and methods Gram-negative ribopolysaccharide (LPS) was obtained from the List Biological Institute (ListBi). ological Laboratories, Inc., Campbell, Obtained from CA). Escherichia coli was produced by Sigma Chemical Co. Al Company, SL, Louis, MO). normal trgG.

IgM8 and 1g were purchased from Southern Biotechnology Unshaded (Sout hernBiotechnology As5ociates, Inc. Normal human IgE and IgD Behring Diagnostics cs, LaJolla, CA). 1'■ is DuPont Company (D Obtained from uPont Co+5pan7゜Wil+ningLon, DE) Ta.

And Δ2 Shonin 2 production HA-IA monoclonal antibody stimulates fusion between tile lymphocytes and heteromyeloma cell lineage. [Teng, N, N ,^, ,S,Lam, F,C,Rieva and J,S,Kapla n, Proc, NaLl, Acad, Sci, USA, 80 Near 3O8-7 312 ( 1983) and Bron, D., M.B., Feinberg, N.N.H. , Teng and H,S, Kaplan, Proc, maLl , Acad, Sci, USA, 81:3214 (1985)] - tile cells are It was collected from a patient undergoing splenic removal for Zykin's disease. Patients are all gram negative Escherichia coli (Esch The mice were immunized with the J5 mutant of Chia Co11). HA-IA Monoku Local antibodies cross-react with internal antibodies from a wide range of unrelated Gram-negative bacterial species and [Teng, N', N,) I-, H-S, Kaplan, J-M, Herbert, C, Moo. re, H-Doug! a sword B A, Wunderlich and A, Braude, Proc, Natl , ^cad, sci, UsA, 82:1790 (198T)], H A-IA is human IgM, a molecule. That is Centocor. Malvern, PA) isolated and purified in 0.01M sodium phosphate (13M Nacl, 5 mg/ml solution in pH 7.2 buffer) provided.

Lymph node cells from BALB/c mice immunized with HAIA were used as non-Tg producing cells. fused with the series P3-X63-Ag8.653 [Kearney, J.F. et al., Jlmmunol., 123:1548 (1979))-Ha The culture supernatant of hybridoma-containing wells was prepared using HA-IA or control immunoglobulin. Enzyme-linked immunosorbent assay (Eli sa) was used to test for antibody specificity. Reacts with HA-IA, but other immune systems What is globulin CTgM or IgA (including paraprotein and normal summer gG)? Hybridomas that secreted unreactive antibodies were selected and subcloned using limiting dilution. -ning. Subsequently, anti-idiotype reagents were prepared using two-color immunofluorescence as previously disclosed. Epifluorescence analysis [) [1yoLaki, M, et al, J, Immun.

138:4250-4158 (1987)] and "intrinsic" idiotype specificity. “Inherent” anti-idio Two cell lineages with different types (9B5.5 and 15B2.2)’@ascites production Blistane-sensitized syngeneic mice were injected i.p.

Anti-idiotype monoclonal antibody paste ascites was prepared using 25mM MES buffer pH 5, Bakerb 6nd ABX HPLC column (10x250m m). After elution of unbound protein, o-1oo% L M Na0Ac pH 7.0 gradient was used over 60 min to prepare monoclonal anti- The body was eluted. The recovered antibodies were subjected to Quick-check analysis (B1oRad, Purity was checked using a commercially available product (Richmond, Calif.). This analytical column is Blo -5il TS was -250 (7-5x300w). The elution buffer was 0. O IM phosphate, 0.3M NaCl, 10% dimethyl sulfoxide (V/V) and eluted at 1 ml/min.

HA-IA solid phase radiological assay method Polystyrene beads (Precision Plastic) with a diameter of 6.4 mm Ba11, Chicago, IL) in phosphate buffer solution (pBS). 200 μl anti-id 9B5.5 was coated at a concentration of 5 μg/ml. Bee Contains 2% Bovine Serum Albumin (BSA) and 0.002% Tween 20 Washed three times with PBS and left in wash buffer for 1 hour at room temperature. Let the beads air dry and stored at 4°C until use. Human monoclonal antibody HA-IA is normal Diluted with human serum (NH3) to concentrations ranging from 12.55-6400n/ml did. Appropriately diluted standards (controls) and patient serum were placed in triplicate on a laboratory rotating machine. Incubated with coated beads for 2 hours at room temperature (100ul). 4 beads Washed with ml PBS. Each bead was coated with 100 μm of radiolabeled anti-ideal Type antibody 15B2.2 was added at a concentration of IgG/m[. incubation It continued for another hour. Wash the beads again with 4 ml of PBS and place in a clean tube. Microm transfer and bead-associated radioactivity interfaced with the 18M System 2. edic automatic gamma counter (manufactured by Micromedic System, H orsham, PA). Create a standard curve and compare control and patient samples. To obtain the numerical value of the sample, use the logit data reduction program (logi t-daLa reduction program) was used.

Protein iodination Purified monoclonal antibodies were prepared by Greenwood et al., 196 Labeled with III■ using a modified method of 3. Radiolabeled monoclonal antibodies are as described above. The analysis was carried out using HPLC, but in conjunction with a UV monitor to detect radioactivity. A radioisotope monitor was used. Proteins are Lovry, O, H, etc. al,, J, Biol, Chem,, 193:265 (+951) method in the final preparation.

Reacts with HAIA in ELrSA but with other myeloma proteins or normal IgG Of the 11 hybridomas that produced antibodies that did not react with Only B5.5 and 15B2.2) were detected by two-color immunofluorescence analysis. It was found to be non-cross-reactive with plasma cells derived from togen-stimulated blood lymphocytes. (0.1%). The other nine antibodies targeted plasma cells in the range of approximately 0.1-2.0%. It was cross-reactive. Thus, antibodies 9B5.5 and 1.5B2.2 These monoclonal Antibodies were purified as described above. The final product is filtered through a 8050 stirred cell. It was concentrated to 5.0 mg/ml using a device and stored at 4°C. Figure 1 shows purified monochrome of null anti-1d antibody 15B2.2 (A) and radiolabeled 15B2.2 (B). Shows HP L and C profiles. No aggregates or decomposition products were observed, H A-IA solid phase radiation standard method Many experiments were performed to establish standard conditions for this assay. polystyrene vinyl The beads carry variable amounts of 9B5.5 from 0.1 gg to 16 μg/bead. Ta. Maximal binding occurred at 1.0 μg/bead. Radioactive label used in this assay The amount of 15B2.2 was determined by incubating 9B5.5 analyte beads with HA-IA standards. by incubating with various amounts of "J-15B2.2". It was measured. O, 1gg of 1”l-15B2.2 is available in all standard concentrations. was sufficient to provide a functional excess of 15B2.2. Both room temperature and 37°C Two incubations were performed at intervals of 30 minutes to 18 hours. 90% Maximum binding of more than 2 hours with serum and radiolabeled 15B This occurred after an hour of incubation with 2.2. Bonding occurs at room temperature and at 37°C. It was similar. For convenience, incubation at room temperature was employed. as above Using a standard HA-IA assay, increasing concentrations of HA-IA, normal human Ig. G, IgM, IgA, IgE and IgD were assayed (Factor 2) I, these The cross-reactivity of various immunoglobulin preparations was calculated to be less than 0.1%. . The sensitivity of the assay is defined as 2 standard deviations or more of nonspecific binding (normal human serum). It was about 25 ng/ml. The linearity of this test was determined by 1 log analysis. is best shown (Figure 3). This assay is linear between 225-800n/ml. It is the shape.

Recovery and reproducibility experiments were also performed. 0.07.1.6.8.2 to normal human serum. 120 and l9. “Inoculation” with Oμg/mI HA-1A, followed by three separate Each assay was performed at an appropriate dilution rate. As shown in Table 1, the average recovery rate was 1 16 ± 4%, with a range of 113-123% over a wide range of serum concentrations. It was.

Topsoil - Recovery of HA-IA in human serum Added HA-IA Detected HA-IA"'b±SD Recovery rate % 0.07 0.08±0.01 1141.6 1.97±0.06 123 8.2 9.4±0.4115 12.0 13.6±0.87 11319.0 118 22.47±1.52 The assay variance was calculated using three concentrations of H as a positive control in 10 consecutive independent assays. To assay A-IA (200, 1000 and 2500 ng/m+human serum) I investigated. The average of the three types of HA-IA levels was 206 ± 12.9, respectively. 81±65 and 2573±161ng/m! (coefficient of variation 5.8-) Since the antibody (HA-IA) is probably used in patients with Gram-negative sepsis, The effects of bacteria and LPS on HA-IA in human serum were investigated. 20 Human serum containing μg/ml of HA-IA was infected with various numbers of bacteria (E, coli ) or with various concentrations of LPS for 20 minutes at 37°C; These additives were then assayed for HA-IA content (Table 2). It had no negative effect on detection.

Table λE Effect of E. coli and LPS on HA-1A expression in human serum Average measured HA-IA Blood +20/ml HA-IA Plus L μml*None 21.9+0.4 9 <　I　CF　U　E, coli　/m　I　21.4±1.810　CF　U E, coli / m 1 20.2 ± 1.880 CF U E, coli /m　1　21.02±1.50.20μg/L　P3　22.95±1.0 42.0μg/LP3 20.5±1.820, hg/ml LPS 19.9 ±1.9X HA-IA from humans (7) 100 mg of HA-I to patients A was administered and serial serum samples were collected for quantification of HA-IA. figure As shown in Figure 4, the average peak serum concentration after infusion was 36.6 μg/m+; This was 101.5% of the predicted value based on the patient's calculated plasma volume. this Data are based on a one-compartment plasma elimination model (one plasma elimination model) with a mean plasma half-life of 24.5 hours. compartment plasma disappearance mod el) (Sisson, 19g3). This means that this assay is effective at drug kinetics. It has been shown that it can be used for degree-based research.

Shio! strength Those skilled in the art will recognize that there are many equivalents to the specific embodiments of the invention described herein. You can understand them and verify them using only routine experiments.8 Dedicated space international search report international search report S^　33574

Claims (25)

[Claims] 1. 1. An immunoassay for a given antibody in a biological fluid, comprising the steps of: a. ．． i. Labeled or labeled specific for the idiotope of a given antibody a first monoclonal antibody capable of ii. Specific to the idiotope of a given antibody a second monoclonal antibody, and iii. given antibody forming a ternary complex of; b. an amount of label associated with the complex formed from the components of step (a) in said liquid; Detecting as an indicator of a predetermined antibody. 2. 2. The first labeled monoclonal antibody of claim 1, wherein the first labeled monoclonal antibody is labeled with iodine-125. Assay method. 3. The second monoclonal antibody is immobilized on the solid phase before or after formation of the ternary complex. The assay method of claim 1. 4. The first and second monoclonal antibodies are identical immobilizers of a given monoclonal antibody. The assay method of claim 1, which is an anti-idiotype antibody specific for an idiotope. . 5. 1. An assay for a given antibody in a blood-derived sample, comprising the steps of: a. ．． A monoclonal anti-idiotypic antibody bound to the sample and solid phase (this The antibody is specific for the idiotope of the given antibody) with a solid-phase immunoadsorbent. preparing an incubation mixture; b. of a given antibody in a liquid sample The incubation reaction is carried out under conditions and times sufficient to bind to the immunoadsorbent. incubating the mixture; c. subsequently separating the immunoadsorbent from the liquid sample; d. This immunoadsorbent and Soluble labeled monoclonal anti-idiotypic antibody (this antibody is an identical antibody for a given antibody) preparing an incubation mixture with the diotope (specific for the diotope); e. The above conditions and time are sufficient to bind the labeled antibody to the predetermined antibody. incubating the mixture; f. separating the solid phase immunosorbent from unbound labeled anti-idiotypic antibody; g ．． Detecting the amount of label bound or unbound to the immunoadsorbent ;and h. Bound label detected in quantitative relationship between label amount and predetermined monoclonal antibody amount or to determine the amount of a given antibody in a sample by relating the amount of unbound label. ; The above test method consists of: 6. Soluble, labeled anti-idiotypic monoclonal antibodies are conjugated to radionuclides, enzymes and 6. The assay method of claim 5, wherein the assay method is labeled with a substance selected from the group consisting of a fluorescent substance and a fluorescent substance. 7. Anti-idiotypic monoclonal antibodies are identical antibodies of a given monoclonal antibody. The immunization according to claim 5, which is an anti-idiotypic antibody specific for a unique idiotope. Assay method. 8. 6. The assay method of claim 5, wherein the predetermined antibody is antibody HA-1A. 9. 1. An assay for a given antibody in a sample comprising the following steps: a. Before a soluble sample and a given monoclonal antibody that is specific for the idiotope. Prepare an incubation mixture with labeled anti-idiotypic monoclonal antibody. to make; b. Soluble monoclonal labeled monoclonal antibody in liquid sample said incubation under conditions and times sufficient to allow binding to the antibody. incubating the mixture; c. Anti-idiotypic mono attached to solid phase Clonal antibodies (this antibody is specific to the idiotope of a given monoclonal antibody) contacting a solid phase immunoadsorbent material containing a target with said incubation mixture. thing; d. bound to a labeled soluble anti-idiotypic monoclonal antibody. conditions and time sufficient to bind a given monoclonal antibody to the immunoadsorbent. incubating the components of step (c) under; e. The incubation separating the solid phase immunoadsorbent from the mixture; f. bonded to solid phase immunosorbent detecting the amount of label or the amount of unbound label; and g. The amount of label detected in the quantitative relationship between the amount of label and the amount of a given monoclonal antibody determining the amount of a given antibody in a liquid sample by relating Statutory law. 10. Anti-idiotypic monoclonal antibodies are monoclonal antibodies that have the same unique identity of a given antibody. 10. The immunoassay of claim 9, which is specific for otopes. 11. 10. The immunoassay of claim 9, wherein the predetermined monoclonal antibody is HA-1A. 12. Soluble, labeled anti-idiotypic monoclonal antibodies are 10. The assay method of claim 9, wherein the assay method is labeled with a substance selected from the group consisting of a fluorescent substance and a fluorescent substance. 13. An immunoassay for a given monoclonal antibody in a liquid sample. Then the next step: a. i. liquid sample, li. An immobilized monoclonal antibody specific for the idiotope of a given monoclonal antibody. a solid phase immunosorbent comprising a local antibody, and iii. Predetermined monoclonal antibodies A labeled soluble monoclonal antibody that is specific for the idiotope of preparing an incubation mixture of; b. a given thing in a liquid sample Clonal antibodies, immobilized monoclonal antibodies and labeled soluble monoclonals The mixture is incubated under conditions and for a period of time sufficient to allow the formation of complexes between the two antibodies. incubating; c. Then solid-phase immunoadsorption from the incubation mixture. separating materials; d. The amount of label bound or unbound to the solid phase immunosorbent detecting the amount of a label; and e. Correlate the amount of label and the amount of monoclonal antibody and determine the location in the liquid sample. determining the amount of a specific monoclonal antibody. 14. Immobilized monoclonal antibodies and soluble monoclonal antibodies are 14. The immunoassay of claim 13, wherein the immunoassay is specific for the same unique idiotope of the null antibody. Statutory law. 15. 14. The immunoassay method of claim 13, wherein the predetermined monoclonal antibody is HA-1A. . 16. Advance sandwich for a given monoclonal antibody in a liquid sample An immunoradiological assay comprising the following steps: a. liquid sample and a given monochrome An anti-idiotypic monoclonal antibody specific for the idiotope of the null antibody is attached. Incubation with a solid-phase immunosorbent consisting of combined polystyrene beads. preparing a mixture; b. incubating said mixture at room temperature; c. subsequently separating the immunoadsorbent from the liquid sample; d. The immunoadsorbent and the A soluble 125I-labeled antibody specific for the idiotope of a given monoclonal antibody is Prepare an incubation mixture of the diotype monoclonal antibody and and; e. incubating said mixture at room temperature; f. Unbound 125I labeled moiety separating the immunoadsorbent from the noclonal antibody; g. bound to an immunosorbent detecting the amount of label; and h. The amount of bound 125I label and the predetermined monomer A given monoclonal antibody in a liquid sample is related to the amount of clonal antibody. The above assay method comprises: determining body mass; 17. Anti-idiotypic monoclonal antibodies are identical to a given monoclonal antibody. 17. The anti-idiotypic antibody of claim 16 is specific for a unique idiotope of Assay method. 18. 17. The assay of claim 16, wherein the predetermined monoclonal antibody is HA-1A. 19. Assay for defined monoclonal antibodies in biological samples of human origin A kit with the following ingredients: a. Anti-idiotypic antibodies specific for the idiotope of a given monoclonal antibody an immunoadsorbent comprising a noclonal antibody; and b. Identification of a given monoclonal antibody a labeled anti-idiotype monoclonal antibody specific for the diotope; Including the above kit. 20. Anti-idiotypic monoclonal antibodies are identical to a given monoclonal antibody. 20. An anti-idiotypic antibody specific for a unique idiotope of claim 19. Test kit. 21. The following ingredients: c. The assay kit of claim 19, further comprising: a standard of a predetermined monoclonal antibody. to. 22. Assay for defined monoclonal antibodies in biological samples of human origin A kit with the following ingredients: a. Anti-idiotope antibodies specific for the idiotope of a given monoclonal antibody Immunoadsorbent material consisting of polystyrene beads bound to noclonal antibodies; b. 125I-labeled antibody specific to the idiotope of a given monoclonal antibody idiotypic monoclonal antibodies; and c. Standard for a given monoclonal antibody The above kit including quasi-products; 23. The following ingredients: c. 23. The kit of claim 22, further comprising: a standard of a predetermined monoclonal antibody. 24. Anti-idiotypic monoclonal antibodies are monoclonal antibodies that contain the same unique identity of a given antibody. 23. The kit of claim 22, which is specific for otopes. 25. 23. The kit of claim 22, wherein the predetermined monoclonal antibody is HA-1A.