JPH0453263B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to a novel immunoassay method for immunocomplexes (hereinafter abbreviated as "IC"). [Prior art and its problems to be solved] Diseases in which IC is detected in body fluids such as patient serum are broadly defined as immune complex diseases.
Glomerulonephritis, vasculitis, infective endocarditis, infectious diseases (leprosy), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), other collagen diseases, liver diseases,
These include inflammatory bowel disease, lung disease, blood disease, neurological disease, cancer, and many other diseases. Among these diseases, SLE is one in which high concentrations of IC are frequently detected in patient serum. In SLE, blood
Many studies have reported that there is a correlation between IC values and renal damage. Regarding RA too
Although the amount of IC in the blood is not as high as in SLE, it has been shown to correlate with disease activity. On the other hand, in liver disease and cancer, there is no evidence that IC is deposited in lesions, and the nature of the antigen that forms IC has not been clarified. The conventionally known method for measuring IC in serum is
Physicochemical methods such as analytical ultracentrifugation, sucrose density gradient ultracentrifugation, polyethylene glycol precipitation, methods that utilize reactions with complement such as C1g,
A method that utilizes the reaction with antiglobulin antibodies such as monoclonal rheumatoid factor (mRF),
There are four main methods: methods that utilize attachment to cell surface receptors, such as the Raji cell method. Analytical ultracentrifugation and sucrose density gradient ultracentrifugation among physicochemical methods have the disadvantage that it takes several tens of hours for one analysis operation and that a large number of specimens cannot be analyzed simultaneously. In addition, although the polyethylene glycol precipitation method is easy to operate, it has a problem with specificity because it measures macromolecular proteins other than IC as well. The method that utilizes the reaction with complement takes advantage of the property that IgG and IgM antibodies bound to antigens often bind complement. C1g binding test, C1g solid-phase radioimmunoassay (R1A), C1g solid phase enzyme assay (E1A), C1g deviation test, etc. have been developed. The C1g method is an excellent method in terms of specificity and sensitivity, but
The drawbacks are that purification of C1g is time-consuming, C1g has low stability, and deactivation occurs over a long period of time even at -70°C. In addition, since it is not possible to directly measure samples in which IC is bound to self-complement in the blood, samples should be treated with EDTA or heated at 50°C before measurement.
A heat treatment of 30 minutes is required. Regarding the antiglobulin antibody method, for example, RF present in the serum of patients with rheumatoid arthritis
The method to use is that the RF is a polyclonal RF
(pRF), its specificity is low, and IC detection rate is poor with RF-containing serum. On the other hand, Waldenstrom macroglobulinemia (Waldenstro...
Monoclonal RF (mRF) is present in the serum of patients with mmacroglobulinemia (IgM myeloma).
R1A, which uses this immobilized mRF, has a high sensitivity,
Although it has excellent specificity, it has the disadvantage that mRF is difficult to obtain and cannot be obtained in large quantities. As a receptor method, a method using Raji cells obtained from tumor cells of a parkhid lymphoma patient (Raji) and cultured is known. This method is based on the principle of measuring IC through complement binding to IC by utilizing complement C3b and C3d receptors present on the surface of Raji cells. Although it is sensitive, it requires cell maintenance. There is a problem in that special equipment is required because of the necessity. As mentioned above, various IC measurement methods have been developed to date, but none satisfy all aspects such as sensitivity, specificity, simplicity, and the presence or absence of variation in measured values between lots of measurement reagents. Ta. On the other hand, it has been reported that, as mRF, mRF-producing splenocytes prepared from spontaneously arthritic mice (MRL/Mp-lpr/lpr) were used to create hybridomas with myeloma cells, and mouse mRF was obtained from this hybridoma. (J.Exp.Med.Vol.158,
pp901-919 (1983)), ibid Vol.159, pp1429-
1440 (1984)). However, such a mouse
The binding reactivity of mRF with human IC has not been confirmed, and no suggestion has been made of its application to IC measurement. [Means for Solving the Problem] The present inventors have discovered that although the measurement of IC is extremely important in the clinical diagnosis of autoimmune diseases such as SLE and RA, and cancer, the complexity of IC itself (antigen diversity, heterogeneity of IC size, etc.)
In order to overcome the current situation where it is not possible to objectively compare IC measured values due to the unestablished measurement method, we aim to satisfy not only sensitivity and specificity, but also the stability of measurement reagents and the possibility of large-scale supply. Through extensive efforts to develop a measurement method, the present invention has been completed. That is, the present inventors obtained a mouse mRF-producing hybridoma by fusing splenocytes from mice with spontaneous arthritis and mouse myeloma cells, and succeeded in producing a large amount of homogeneous mouse mRF. As a result of a detailed study of the properties of this mouse mRF, we found that mouse mRF reacts strongly with human heat-denatured IgG (HAG), but only at high concentrations with human monomeric IgG, indicating that mouse mRF is specific to IC. It was presumed that he had sex.
Therefore, when we applied mouse mRF as an antibody reagent to an immunoassay method for human IC, we found that it achieved a similar level of sensitivity and specificity to the C1g method, which is the IC measurement method that has the best sensitivity and specificity among conventional methods, and the human mRF method. sensitivity,
It was found that IC can be measured with specificity. The method of the present invention was completed based on this knowledge, and is a method for measuring IC in a human body fluid sample by immunoassay.
The present invention provides a method for measuring IC, which is characterized by using mouse mRF as an antibody reagent that specifically binds to IC. The present invention will be explained in detail below. The mRF used in the method of the present invention can be obtained by a known method for producing monoclonal antibodies. Cell fusion methods and spleen fragment culture methods are known as methods for producing monoclonal antibodies. Cell fusion methods are advantageously applied in mass production of antibodies. The method for producing the antibody of the present invention using the cell fusion method will be described in detail below, but more details about the general technical means of the cell fusion method can be found in reviews and books, such as "Monoclonal Antibodies" by Tatsuo Iwasaki et al.
Hybridoma and ELISA”, Kodansha Co., Ltd.
You can refer to the publication on February 20, 1988, etc. (1) Preparation of antibody-producing cells Mouse RF is produced with age in the serum of MRL/Mp-lpr/lpr mice, which have symptoms similar to rheumatoid arthritis (RA) and are used as an animal model of RA. Ru. RF like this
RF-producing cells can be prepared from splenocytes and lymph node cells of mice that produce RF. For example, serum is collected from MRL/Mp-lpr/lpr mice (female, 20-40 weeks old), and enzyme-labeled immunoassay method (Enzyme-linked immunoassay) is performed using a microplate coated with heat-denatured human IgG (HAG).
Measure the titer of RF in the serum using the Linked Immuno Sorbent Assay (ELISA) method, select a mouse with a high titer, and extract splenocytes or lymph node cells from the mouse using a standard method to obtain RF-producing cells. Bye. (2) Preparation of myeloma cells As myeloma cells, mouse, rat,
Cell lines derived from various animals such as humans and commonly available to those skilled in the art can be applied. The bacterial strain used is preferably one that is drug resistant and cannot survive in a selective medium in an unfused state, but can only survive in a fused state. Usually, it has resistance to 8-azaguanine, and hypoxanthine guanine phosphoribosyltransferase (hypoxanthine guanine phosphoribosyltransferase)
A cell line is used that is deficient in transferase and cannot grow in hypoxanthine-aminopterin-thymidine (HAT) medium. Furthermore, it is preferable that the cell line is a so-called non-secreting cell line, which does not secrete immunoglobulin as a result of its cell nature. A specific example of a known cell line that can be used is P3X63Ag8 (ATCC TIB-9).
(Nature. 256 , 495-497 (1975)),
P3X63Ag8U.1 ( _P3U1 ) (ATCC CRL ₋ 1597)
(Current Topics in Microbiology and
Immunology, 81 , 1-7 (1978)),
P3X63Ag8.653 (ATCC CRL−1580) (J.
Immunology, 123 , 1548-1550 (1979)), P2/
NSI/1-Ag4-1 (ATCC TIB-18)
(European J.Immunology, 6 , 511-519
(1976)), Sp2/O-Ag14 (ATCC CRL-
Mouse myeloma cell lines such as 1581) (Nature, 276 , 269−270 (1978)), 210.RCY.Ag1.2.3
(Y3−Ag1.2.3) (ATCC CRL−1631)
(Nature, 277 , 131-133 (1979)), rat myeloma cell lines such as U-266-AR ₁ (Proc.
Natl.Acad.Sci.USA, 77 , 5429 (1980)),
GM1500 (Nature, 288 , 488 (1980), KR-4
(Proc.Natl.Acad.Sci.USA, 79 , 6651
(1982)) can be exemplified. These cell lines are maintained and managed by each research institution, and are generally distributed through exchanges between researchers. These cell lines are therefore generally available to those skilled in the art. (3) Cell fusion For cell fusion, select myeloma cells that are compatible with RF-producing cells. Cell fusion is
Mix 10 ⁷ to ^{10 8} myeloma cells and RF producing cells at a mixing ratio of 1:4 to 10 in an animal cell culture medium such as Eagle's minimal essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), or RPMI1640. I will do it. In order to promote cell fusion, polyethylene glycol (PEG) with an average molecular weight of 1000 to 6000, polyvinyl alcohol, Sendai virus, etc. are used as fusion promoters. (4) Selection of hybridomas in a selective medium As a means for selecting hybridomas from cells after cell fusion treatment, a method that utilizes selective proliferation of cells in a selective medium is used.
For example, add 15% fetal calf serum (FCS) to the cell solution.
Appropriately dilute the solution with RPMI1640 containing medium, etc., spread it on a microtiter plate at approximately 10 ⁵ to 10 ⁶ per well, and fill each well with a selective medium (e.g.
HAT medium, etc.), and then culture by replacing the selection medium appropriately. 8-azaguanine resistant strain as myeloma cells, as selective medium
When using HAT medium, unfused myeloma cells die by about day 10 of culture, and RF-producing cells, which are normal cells, cannot grow for long periods in vitro, so they begin to grow from day 10 to 14 of culture. Hybridomas can be obtained as cells that grow. (5) Search for mouse mRF-producing hybridomas To search for mouse mRF-producing hybridomas,
This can be done by ELISA, RIA, etc.
For example, 96-well ELISA with adsorbed heat-denatured IgG
A culture supernatant containing a hybridoma is added to a plate for reaction with a specific antibody, then an enzyme-labeled anti-mouse immunoglobulin antibody is reacted with the bound specific antibody, and an enzyme substrate is added to each well to develop color. In this way, wells containing culture supernatants of hybridomas producing antibodies (mRF) that have binding power to heat-denatured IgG can be identified by the presence or absence of color development. (6) Cloning Hybridoma cloning can be performed by the limiting dilution method, soft agar method, fibrin gel method, fluorescence excitation cell sorter method, etc. (7) Production of antibodies The method for producing mRF from the mRF-producing hybridoma thus obtained is as follows:
A conventional cell culture method or a method of recovering from ascites fluid can be adopted. In cell culture methods, hybridomas are
mRF can be obtained from the culture supernatant by culturing in an animal cell culture medium such as ~15% FCS-containing RPMI1640 medium or serum-free medium. In the method of recovery from ascites, immune suppression is suppressed by intraperitoneally administering mineral oil such as pristane (2,6,10,14-tetramethylpentadecane) to animals whose major histocompatibility matches that of the hybridoma, such as nude mice. and about 10 ⁷ hybridomas
Administer intraperitoneally. Hybridomas form ascites tumors in about 10 to 18 days and produce high concentrations of mRF in serum and ascites. If purification of RF is required, use ammonium sulfate salting out method, ion exchange chromatography using an anion exchanger such as DEAE cellulose, Affinity chromatography using Sepharose 4B coupled with heat-denatured IgG, etc. The objective can be achieved by appropriately selecting and combining methods such as chromatography and molecular sieve chromatography. The mRF used in the method of the invention is characterized by the property of having specificity for human IC. The method of the present invention takes advantage of the property that mouse mRF can specifically react with human IC, and uses mouse mRF as an antibody reagent in various immunoassay methods for antigens.
It uses mRF. Therefore, the immunoassay method that can use this mouse mRF as an antibody reagent is based on the principle that the measurement system basically measures the antigen to be measured using an antibody specific to the antigen. That's fine. Many such immunoassay methods have been developed to date. For example, measurement systems include agglutination reaction method, agglutination inhibition method, immunodiffusion method, laser nephrometry, nephelometric method, competitive reaction method (solid phase method, secondary antibody method, etc.), Sand Deutsch method, immunometric method, etc. Measurement systems that use labeled substances are broadly classified into RIA, EIA, fluorescence immunoassay (FIA), chemiluminescence immunoassay, metal immunoassay (MIA), spin immunoassay, etc., depending on the type of labeling substance. As long as each of these measurement methods is based on the measurement principles described above,
It can be applied to the IC measurement method using mouse mRF of the present invention. When applying these individual immunoassay methods, the present invention does not require special conditions or operation settings, and any method used in each method can be applied in accordance with the conventional method. Mouse mRF as an antibody reagent
The measurement system can be configured using the following. For more information on these general technical means, please refer to the reviews,
You can refer to published books, etc. (for example, Hiroshi Irie, ed., "Radio Immuno Assay", Kodansha Co., Ltd., published on April 10, 1972; Hiroshi Irie, ed., "Radio Immuno Assay", Kodansha Co., Ltd.) ,Showa
Published on May 1, 1954, edited by Eiji Ishikawa et al., "Enzyme Immunoassay" 2nd edition, Igakushoin Co., Ltd., 1988.
Published December 15th, Nippon Rinsai, Volume 42, Special Spring Issue (1984) "Clinical Immunology Handbook", Nippon Rinsai Co., Ltd., Published March 20, 1980, No. 1198~
Page 1227, HV Bounakis et al., “Methods in
"Enzymology", Vol.70, "Immunochemical
Techniques Part A”, Academic Press,
1980, JJ Langone et al., “Methods in
"Enzymology" Vol.73, "Immunochemistry"
Techniques Part B”, same book, Vol.74
(See "Immunoshemical Techniques Part C", Academic Press, 1981, etc.). Next, some typical immunoassay methods will be briefly explained. Solid-phase mRF method The test sample is placed in a test tube with immobilized mouse mRF, and after binding IC to mRF, it is washed.
Human IgGFc produced in rabbit, goat, horse, etc.
Antibodies against fragments or Fab fragments or F(ab')z fragments of these antibodies (anti-human IgG antibodies) labeled with ^125I etc. are added and reacted, and the amount of labeling is measured and quantified (J .Clin.Invest., Vol.65, pp136
~145 (1980), ibid, Vol.66, pp655~663
(1980)). It is also possible to quantify by measuring enzyme activity using an enzyme such as peroxidase or β-galactosidase instead of a radioactive isotope as a label. Latex agglutination method Latex beads coated with mouse mRF are reacted with the test sample. IC in sample
If it exists, it will aggregate, so IC can be easily measured. It is meaningful as a semi-quantitative method. Laser nephrometry is a method for quantifying IC by adding a certain amount of mouse mRF into a sample and measuring the scattered light of the large particles of IC and mouse mRF generated using a laser beam. Method using RIA inhibition Mouse mRF is immobilized on a test tube, and the binding of heat-denatured IgG labeled with a radioactive isotope is detected in the sample.
This is a method of quantifying IC by measuring the inhibition rate of IC (J.Clin.Invest., Vol.56, p458
(1975)). Additionally, mice labeled with radioactive isotopes
There is also a method to quantify IC by applying the fact that IC in a sample inhibits the binding of mRF to immobilized IgG (such as IgG Sepharose 4B) (J.Clin.Invest., Vol.59, (See p990 (1977)). Sand-Deutsch method A test sample is placed in a reaction container such as a test tube or microplate containing immobilized mouse mRF and allowed to react. Then the labeled mouse
mRF is added and reacted, and the amount of bound or free label is measured and quantified. In the method of the present invention, mouse mRF is prepared in the form of an appropriate antibody reagent according to the immunoassay method to be applied. The preparation forms of these antibody reagents include native state, solid phase,
There are three types of labeled products. Native forms are prepared in solution or lyophilized form. Further, the solid-phased product is one that is solidified on carriers made of various materials such as test tubes, beads, latex, and disks by covalent bonding, crosslinking, or physical adsorption. Furthermore, the labeled product is one in which a labeling substance such as a radioactive isotope, an enzyme, a fluorescent substance, a chemiluminescent substance, or a metal is bound by a known method. [Effects] According to the method of the present invention, IC in body fluid samples (e.g. serum, synovial fluid, etc.) can be measured with extremely high sensitivity and specificity by immunoassay using mouse mRF as an antibody reagent. It is possible to do so. Moreover, this mouse mRF
Hybridomas obtained by fusing mRF-producing cells and myeloma cells can be obtained in large quantities with uniform quality. Thus, the method of the present invention has all the excellent characteristics that none of the conventional IC measurement methods have, and its significance lies in the fact that it is the first to establish a general IC measurement method. be. [Example] Reference example Preparation of mouse mRF Obtaining hybridoma Mice with spontaneous arthritis (MRL/Mp-lpr/
lpr, female, 20-40 weeks old) with a high titer of RF in the serum, and splenocytes were removed.
Washed with MEM. Mouse myeloma P ₃ U ₁
After washing with MEM, splenocytes and P ₃ U ₁ were mixed at a ratio of 10:1 and centrifuged, 1 ml of 50% PEG1000 MEM solution was gradually added to the pellet to perform cell fusion. Furthermore, the MEM solution was gradually added to the solution to make a final volume of 10 ml. Centrifuge again and transfer the pellet to RPMI1640 medium containing 15% fetal bovine serum _1x as _P3U1 .
The suspension was suspended at 10 ⁵ cells/0.1 ml, and 0.1 ml of the suspension was spread onto a 96-well microplate. After 1 day, add 0.1 ml of HAT medium, then add half of the medium every 3-4 days to fresh HAT.
Replaced with medium. From around the 7th day, hybridoma growth was observed in some wells. Supernatant of wells where hybridomas have grown
0.05 ml of each well was taken, and 50 µ of the supernatant from each well in which hybridomas had grown was added to a 96-well soft plate that had been previously coated with human heat-denatured IgG (HAG) or human monomer IgG. Second
Biotinylated horse anti-mouse IgG antibody (manufactured by Vector) as an antibody, avicin D-peroxidase (manufactured by Vector) as an avidin D-enzyme conjugate, hydrogen peroxide as a substrate and coloring agent, 4
- using aminoantipyrine, phenol
We selected a product that reacts with HAG by ELISA but does not react with monomer IgG, and cloned it by limiting dilution. As a result, C-26, C-
28, D-13, E-10, E-11, E-21, E-
27 and 8 shares of E-36 were acquired. Production of mRF The above hybridomas were cultured in RPMI1640 medium containing 15% fetal bovine serum while scaling up in a 96-well plate, a 25 cm ² flask, and a 75 cm ² flask, and the culture supernatant containing mRF was collected. Properties of mRF It is generally said that human RF reacts with human IgG ₁ , IgG ₂ , and IgG ₄ but does not react with IgG ₃ (Reiji Kasukawa, "Immune Complex Disease", Ishiyaku Publishing Co., Ltd., Showa (Published February 15, 1957, p. 18). The recognition site is said to be the Fc or hinge site. Furthermore, one of the characteristics of RF is that it exhibits cross-reactivity with IgG from other animals. When the properties of mouse mRF used in the present invention were investigated, all eight strains obtained secreted IgM class mRF. Also, most RF
It did not react with human IgG of the IgG ₃ subclass. Regarding cross-reactivity with IgG from other animals, most mRFs reacted with rabbit IgG. Many of them also reacted with horse IgG, and half of them reacted with goats and cows. These results are shown in Tables 1 and 2.

【table】

[Table] Purification of mRF 8 mg of human IgG was coupled to 1 ml of agarose gel carrier (CNBr-activated Sepharose 4B, manufactured by Pharmacia) and packed into a column.
Equilibrated with PBS. Pass 100 to 200 ml of culture supernatant of hybridoma C-28, E-10, E-11, E-27 or E-36 through this column,
After thorough washing with PBS, the adsorbed mRF was eluted using 0.1M glycine-HCl buffer (PH2.3).
1/10 volume of 3M Tris-HCl buffer (PH8.2) was added to return the mixture to neutrality, thereby obtaining purified mouse mRF. C-
For 26, add 1 ml of agarose gel carrier (Affigel-10, manufactured by BioRad) to the column.
Using 10ml of 1gG,
Elute with 0.1M citric buffer (PH4.5),
Purified mouse mRF dialyzed against PBS
And so. The titer against HAG per mg of purified mouse mRF by ELISA was 2000 to 8000 times higher. Note that the titer is determined by ELISA for samples in which there is a sufficient amount of antibody against the immobilized antigen.
The absorbance obtained by the method was taken as 100%, and the dilution ratio from the original solution of the antibody sample giving 50% of that value was expressed. Reference Example ¹²⁵ I - Preparation of F(ab') ₂ fragment of rabbit anti-human IgGFc fragment antibody The antibody obtained by immunizing rabbit with human IgGFc fragment was purified by ammonium sulfate fractionation method and DEAE cellulose ion exchange chromatography. did. This purified antibody was dissolved in 0.1M acetate buffer (PH4.5, containing 0.1M sodium chloride) to a concentration of 10mg/ml, 4% pepsin solution was added thereto, and the mixture was incubated at 37°C overnight. An F(ab') ₂ fragment was created. The pepsin-digested solution was subjected to gel filtration chromatography (Sephadex G-150 column),
The F(ab') ₂ fragment fraction was obtained by developing with 0.1M sodium borate buffer (PH8.0). This F(ab') ₂ fragment was then labeled with ¹²⁵ I by chloramine T according to the method of Hunter et al. (Nature, Vol. 194, pp. 495-
496 (1962)). Example 1 Purified mouse mRF was diluted to 2.5μg/ml with PBS-EDTA buffer (containing 0.15M sodium chloride, 10mMEDTA) and placed in a polystyrene tube.
Add 0.5 ml each and leave to coat at 4°C overnight.
Remove the liquid from the tube, containing 1% BSA.
Blocked with PBS-EDTA buffer. After 1 hour, remove the BSA solution and add PBS-
Wash once with 1.5 ml of EDTA buffer, add 10 μl of serially diluted HAG, monomeric IgG, or patient serum sample, and add PBS-EDTA containing 1% BSA.
A buffer solution was added to make 0.5 ml, and the mixture was left at 4°C overnight. Aspirate the reaction solution and add PBS-EDTA buffer 1.5
Rabbit anti-human, washed 3 times with ml and then ^125I
F(ab') ₂ fragment of IgGFc fragment antibody
2 μg/ml) was added at 0.5 ml (approximately 103 cpm/ng). After incubation for 2 hours at room temperature, PBS-
The plate was washed three times with EDTA buffer, and the amount of radioactivity was measured using an autogamma counter. Calculations were made from the count number to determine how many ng of ¹²⁵ I-labeled fragments were bound using a specific activity of 103 cpm/ng. When measurements were performed on 8 SLE cases, 88 RA cases, cancer cases, and 10 healthy subjects, the measured values were of the C-26 strain.
In mRF, SLE21.5±9.9ng/ml (the percentage is 25% if the average of normal values + 2SD or more is considered abnormal),
RA41.0±36.1ng/ml (37.5%), cancer 35.0±8.5n
g/ml (57.1%), and 17.5±6.9 ng/ml in healthy subjects. These results showed that human IC can be measured using mouse mRF in a manner equivalent to the conventional C1g method or human mRF method. In addition, in this example, cancer patients
IC positivity was slightly higher. Example 2 1g of C diluted with PBS-EDTA buffer to the following concentration
Alternatively, 0.5 ml of mouse mRF was added to a polystyrene tube and left overnight at 4°C. C1g 5μg/ml C-26 A ₂₈₀ = 300-fold dilution of 2.311 D-13 A ₂₈₀ = 300-fold dilution of 0.495 E-11 A ₂₈₀ = 300-fold dilution of 0.655 Rabbit anti-human IgGFc antibody F(ab') ² fragment
Aspirate the 5 μg/ml diluted solution and add to PBS containing 1% BSA.
-1 ml of EDTA buffer was added and allowed to stand at room temperature for 11 hours to block. Aspirate the BSA solution and
Washed once with 1.5 ml of PBS-EDTA buffer. Add 10μ of sample and add PBS containing 1% BSA.
EDTA buffer was added to make 0.5 ml, and the mixture was left at room temperature for 2 and a half hours. Aspirate the reaction solution and add PBS-EDTA buffer 1.5
ml three times, then add 0.5 ml of F(ab') ² fragment (2 μg/ml) of rabbit anti-human IgGFc antibody which has been converted to ¹²⁵ I.
(approximately 267CPm/ng) and left at room temperature for 1.5 hours. Aspirate the reaction solution and add PBS-EDTA buffer 1.5
ml three times, and the amount of radioactivity was measured using an autogamma counter to obtain the measured value. Calculations were made to determine how many ng of ¹²⁵ I-labeled fragments were bound from the count using a specific activity of 267 cpm/ng. Measurements were performed on 20 untreated SLE cases, 20 untreated RA cases, and 29 healthy subjects, and the average normal value plus 2 SD
If the above were considered abnormal, the proportions were as follows.

[Table] In this example, results equivalent to those of the C1g method were obtained. Example 3 A 96-well microplate (made of polyvinyl chloride) was prepared from hybridomas E-27 and D-13.
mRF to 4.1μg/ml and 3.5μg/ml respectively in PBS
200 μl of diluted solution was added to each plate and left at 4°C overnight to coat. Next, add PBS containing 1% BSA, leave it at room temperature for 1 hour to block, remove the BSA solution by suction, add 10μ of patient serum sample, and add PBS containing 1% BSA.
40μ was added and left at room temperature for 2 hours. After removing the reaction solution by suction and washing three times with PBS,
Horseradish peroxidase labeled goat anti-human
IgGFc fragment antibody F(ab') ₂ fragment (manufactured by Capel Laboratories) was added and incubated at room temperature for 2 hours. After removing the reaction solution by suction and washing 3 times with PBS,
- A substrate solution containing aminoantipyrine, phenol, and hydrogen peroxide was added, and the enzyme activity was calculated by measuring the absorbance at OD ₅₅₀ , and the IC was quantified. As a result of examining 8 cancer patients, 8 RA cases, 8 SLE cases, and 8 healthy subjects, the positive rate for D-13mRF was 75% for cancer, 12.5% for RA, and 37.5% for SLE.
-27mRF: 75% cancer, 12.5% RA, 12.5% SLE
It was hot. In both cases, the positive rate of IC in cancer was high. Example 4 96-well microplate (made of polyvinyl chloride)
mRF prepared from hybridoma D-13
6.3 μg/ml, or 13.1 μg/ml of mouse 1 gM (for control) prepared from a hybridoma clone producing mouse 1 gM without mRF activity.
50μ diluted in PBS to a concentration of ml in each well.
The mixture was added in portions and allowed to stand at 4°C to coat. Next, add a solution containing 1% BSA, leave it at room temperature for 1 hour to block, remove the BSA solution by suction, and add 10x buffer solution A (0.1M Tris-HCl PH8.0, 0.5M sodium chloride, 1% BSA). Serum sample diluted to
50μ of mRF and 1gM without mRF activity were added to each well coated, and the mixture was left at room temperature for 2 hours. After removing the reaction solution by suction and washing three times with PBS,
Add 50μ of a solution (0.20μg/ml, 500cpm/ng) of D-13-derived mRF labeled with ^{lactoperoxidase} and dissolved in A buffer,
It was left at 4°C overnight. After removing the reaction solution by suction and washing three times with PBS,
Measure the amount of radioactivity with an autogamma counter, and calculate by subtracting the count of wells coated with 1 gM, which does not have mRF activity, from the count of wells coated with mRF.
IC was quantified. Measurements were performed on 7 SLE cases and 6 healthy subjects.
If the mean of normal values + 2 SD or more is considered abnormal, the positive rate was 57%.

Claims (1)

[Claims] 1. A method for measuring immune complexes in a human body fluid sample by immunoassay, using mouse monoclonal rheumatoid factor as an antibody reagent that specifically binds to immune complexes in the sample. A method for measuring immune complexes characterized by: 2. React the immobilized mouse monoclonal rheumatoid factor with the sample, then add and bind labeled anti-human IgG antibodies or fragments thereof to the reaction product, measure the amount of bound label, and determine the immune complex. The method according to claim 1, characterized in that the method comprises: measuring. 3. The method according to claim 1, wherein the immune complex is measured by reacting mouse monoclonal rheumatoid factor immobilized on microparticles with a sample and measuring the degree of aggregation produced.