JPS61114165A - Measuring method of immune complex - Google Patents

Abstract

PURPOSE:To measure the immune complex in the human bodily fluid with high sensitivity by bringing a mouse monoclonal rheumatoid factor into reaction as an antibody reagent to conjugate specifically with the immune complex in the human bodily fluid sample. CONSTITUTION:The mouse monoclonal rheumatoid factor (abbreviated as mRF) is obtd. by a plasmogamy method and is fixed as a solid phase to the inside wall of a test tube or the wall of a reaction vessel such as microplate. The solid phase mRF and the human bodily fluid (serum, etc.) sample are brought into reaction and thereafter the antihuman IgG antibody labeled by Western horseradish peroxidase or the fragment thereof is added to the resulted product of reaction. A substrate soln. contg. 4-aminoantipyrine, phenol and H2O2 is added to the labeled quantity conjugated to the solid phase mRF and the absorbancy at 550nm is measured. Enzyme labeling activity is then calculated and the immune complex in the bodily fluid is quantitatively determined by using the calibration curve obtd. preliminarily therefrom. The diagnosis of the diseases such as cancer, general lupus erythematodes and chromic articular rheumatism is thus made possible with high accuracy.

Description

Detailed Description of the Invention [Industrial Application Field] O The present invention provides a novel immune complex (1 mmunslfomp
lex.

Hereinafter, it will be abbreviated as 1'-ICJ. ) relates to an immunoassay method.

[Prior art and its problems to be solved]

Diseases where IC is detected in body fluids such as patient serum can be broadly defined as immune complex diseases, including serum sickness, glomerulonephritis,
Vasculitis, infective endocarditis, infectious disease (leprosy), systemic lupus erythematous disease (SLE), rheumatoid arthritis (
RA), other collagen diseases, liver diseases, inflammatory bowel diseases, lung diseases, blood diseases, neurological diseases, cancer, and many other diseases. Among these diseases, high concentrations of IC in patient serum
is frequently detected in SLE. In SLE, there are many reports that show a correlation between blood IC levels and renal damage.
Although the amount of IC in blood is not large, it has been shown to be correlated with disease activity. - Deposition of JC in the lesion area has not been proven in cases of osteoporosis or cancer, and I
The nature of the antigen that forms C is also not clear.

Conventionally known methods for measuring IC in serum include: ■ Physicochemical methods such as analytical ultracentrifugation, sucrose density gradient ultracentrifugation, and polyethylene gelcol precipitation; ■ Complement methods such as CI- 4 methods: 1) a method that utilizes a reaction with anti-globulin antibodies such as monoclonal rheumatoid factor (mRF), and 2) a method that utilizes attachment to cell surface receptors such as the RaJl cell method. It is broadly divided into

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 it is not possible to analyze a large number of specimens simultaneously.

Furthermore, although the polyethylene gelcol 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 involves the use of IgG bound to an antigen.
This method takes advantage of the property of RgM antibodies to bind complement in the case of multiple antibodies. Complement 1 and 2 solid-phase radioimmunoassay (RIA) and C11L antibodies also have the disadvantage of deactivation over the long term. In addition, since substances bound to IC or autocomplement in the blood cannot be measured as they are, samples should be treated with EDTA or 55% before measurement.
Heat treatment at 0°C for 130 minutes is required.

Regarding antiglobulin antibody methods, for example, methods that utilize RF present in the serum of rheumatoid arthritis patients have low specificity because the RF is polyclonal RF (pRF), and RF-containing serum has low IC. Detection rate is poor. On the other hand, Waldenström macroglobulinemia (Waldenstr6mmacroglobulin)
emia, 1 g M x o -v) (7)
Monoclonal RF (mRF) exists in patient serum, and RIA using immobilized mRF has a high sensitivity,
Although it has excellent specificity, it has the disadvantage that it is difficult to obtain mRF and that mRF cannot be obtained in large quantities.

As a receptor method, Burkitt's lymphoma patients (Ra
Raji obtained from the tumor cells of
Methods using cells are known.

Complement (:ab, Cac) present on the surface of Raji cells
This method is based on the principle of measuring IC through complement bound to IC using T receptors, and although it is sensitive, it has the problem of requiring special equipment because cells need to be maintained.

As mentioned above, various IC measurement methods have been developed to date, but none satisfy all of the requirements in terms of sensitivity, specificity, simplicity, and the presence or absence of variation in measured values between 1-ot measurement reagents. There was no.

On the other hand, as mRF, mice with spontaneous arthritis (MRL/
It has been reported that hybridomas with myeloma cells were created using mRF-producing tile cells produced from Mp-1pr/lpr), and mouse mRF was obtained from the hybridomas (J, EXI), Med, Vol.

158, pp 901-919 (1988)), 1b
id Vol, 159 pp 1429-144
0 (1984)). However, the binding reactivity of such mouse mRF with human IC has not been confirmed, and its application to IC measurement has not been suggested at all.

[Means to solve the problem]

Although the measurement of IC is extremely important in the clinical diagnosis of autoimmune diseases such as SLE and RA, and cancer, the present inventors pointed out that although the measurement of IC is extremely important in the clinical diagnosis of autoimmune diseases such as SLE and RA, In order to overcome the current situation where it is not possible to objectively compare IC measured values due to non-uniformity (heterogeneity, etc.) and unestablished measurement methods, we are focusing not only on sensitivity and specificity, but also on the stability of measurement reagents and the possibility of large-scale supply. We have made earnest efforts to develop a measurement method that satisfies these requirements, and have finally completed the present invention.

That is, the present inventors obtained a mouse InRF-producing hybridoma by fusing the tile cells of a spontaneously arthritic mouse with mouse myeloma cells, and succeeded in producing a large amount of uniform mouse mRF. As a result of a detailed study of the properties of mouse mRF, we found that mouse mRF strongly reacts with human heat-denatured 1gG (HAG) or only reacts with human monomer IgG at high concentrations, indicating that mouse mRF is IC-specific. It was presumed that he had sex. Therefore, we used mouse mRF as an antibody reagent to detect human IC.
When applied to the immunoassay method, it was found to have the lowest sensitivity and specificity among the conventional methods. It was found that IC measurement was possible.

The method of the present invention was completed based on such knowledge, and is a method for measuring IC in a human body fluid sample by immunoassay, in which an antibody reagent that specifically binds to IC in a sample is used. The present invention provides a method for measuring IC characterized by using mouse mRF as a method.

The present invention will be explained in detail below.

The mRF used in the method of the present invention can be obtained by known monoclonal antibody production methods. 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 detailed information on the general technical means of the cell fusion method can be found in reviews and books, such as those written by Tatsuo Iwasaki et al. ELISA-J, Kodansha Co., Ltd., 1982
Please refer to the statement issued on February 20, 2016.

(1) Preparation of antibody-producing cells Mouse RF has symptoms similar to rheumatoid arthritis (RA) and is used as an animal model for RA.
7M p-1pr/Ipr 7 Usuno bloodf'f
Produced during aging with age. RF-producing cells may be prepared from mouse tile cells and lymph node cells that produce such RF.

For example, serum was collected from MRL/Mp-1pr/lpr mice (female, 20 to 40 weeks old), and heat-denatured human I
Enzyme labeled immunoassay (mmu
no 5orbent As5ay, E L I
The titer of RF in serum is measured by the S A ) method, a mouse with a high titer is selected, and tile cells or lymph node cells are extracted from the mouse by a conventional method to obtain RF-producing cells.

(2) Preparation of myeloma cells As myeloma cells, established cell lines derived from various animals such as mice, rats, rabbits, and humans, and commonly available to those skilled in the art can be used. The cell line used is preferably drug resistant and has the property that it cannot survive in the selective medium in an unfused state and can only survive in a fused state. Usually has resistance to 8-azaguanine, hypoxanthin-phosphoribosyl) 5 female 7 engineering 5-41 (hypoxa@1t
hine phosphoryltransferas
A cell line that is deficient in e) and cannot grow in hyboxanthin-aminopterin-thymidine (HA T ) medium is used. 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 P8-NSI-1-Ag4-1 (NS-
1), P8-X68-Ag8 (P8), P8-X68-
Ag8-Ul (, P8U1), P8-X68-Ag
8-653 (X63.6.5.8), 5P210.Ag
Mouse myeloma cell lines such as 14 (SF3), 21 (
Rat myeloma cell lines such as lRcY8・Agl・2・8 (Y8・1・23), U-266-ARl, G
M1500, GM15006TG-A12.5KO-0
Examples include human myeloma cell lines such as 07. 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 was performed using Eagle's minimal essential medium (MEM, l, Dulbecco's modified Eagle's medium (DME),
M), by mixing 107-108 myeloma cells and RF-producing cells at a mixing ratio of 1:4-10 in an animal cell culture medium such as RPM11640. To promote cell fusion, use an average molecular weight of 111,000 to 60 as a fusion promoter.
00 polyethylene glycol (PEG), polyvinyl alcohol, virus, etc. are used.

(4) Selection of hybridomas in a selective medium As a means for selecting hybridomas from cells after cell fusion treatment, a method utilizing selective proliferation of cells in a selective medium is used. For example, dilute the cell solution appropriately with RPMI]640 medium containing 15% fetal bovine serum (Fe2), and dilute it in a microtiter plate.
A selection medium (for example, HAT medium, etc.) is added to each well, and the selection medium is then appropriately exchanged for culturing. When using an 8-azaguanine resistant strain as myeloma cells and HAT medium as the selective medium, unfused myeloma cells died within 10 days of culture, and normal RF-producing cells also died in vitro.
Since RO cannot grow for a long period of time, hybridomas can be obtained as cells that grow from the 10th to 14th day of culture.

(5) Search for mouse mRF-producing hybridomas Search for mouse mRF-producing hybridomas by using ELISA%R
This can be done by rA or the like.

For example, a 96-well E LIS with heat-denatured IgG adsorbed
A culture supernatant containing hybridomas is added to the plate for A, and a specific antibody is allowed to react.Then, the bound specific antibodies are reacted with an enzyme-labeled anti-mouse immunoglobulin antibody, and an enzyme substrate is added to each well to develop color. In this way, wells containing culture supernatants of hybridomas producing antibodies (RF) that have binding strength to heat-denatured 1gG 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 From the RF-producing hybridoma thus obtained
As a method for producing F, a normal cell culture method or a method for collecting it from ascites can be adopted.

In cell culture methods, 10-15% hybridoma
It is possible to culture in an animal cell culture medium such as FC5-containing RPMI 1640 medium or serum-free medium, and to obtain RF from the culture liquid.

In the method of recovery from ascites, animals whose major histocompatibility matches that of No. 1 hybridoma, such as nude mice, are immunized with minerals such as pristane (2,6,10,14-tetramethylpentadecane) intraperitoneally. The hybridoma is administered approximately 107 times intraperitoneally. Hybridoma is 1
Ascites tumors form in the throat from 0 to 18 days and produce high concentrations of RF in the serum and ascites.

If purification of RF is required, ammonium sulfate salting out method, DE
Appropriately select and combine methods such as ion exchange chromatography using an anion exchanger such as AE cellulose, affinity chromatography using Sepharose 4B bonded with heat-denatured IgG, molecular sieve chromatography, etc. By doing so, you can achieve your goals.

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 utilizes mouse mRF as an antibody reagent in various immunoassay methods for antigens by taking advantage of the property that mouse mRF can specifically react with human IC. . Therefore, this mouse m
An immunoassay method that uses RF as an antibody reagent may be one that is based on the principle that the antigen to be measured by basic IC is measured using an antibody specific to the antigen. Many such immunoassay methods have been developed to date. For example, measurement systems include agglutination reaction method, agglutination inhibition method, immunodiffusion method, laser nephrometry, specific hydration method, competitive reaction method (solid phase method, secondary antibody method, etc.), Sand-Deutsch method, immunometric method, etc. Depending on the type of labeling substance, RIA, EIA, fluorescence immunoassay (FIA), chemiluminescence immunoassay, metal immunoassay (MIA), spin immunoassay, etc. are known as measurement systems that use labeled substances. As long as the measurement method is based on the measurement principle described above, the I
It can be applied to the measurement method of C. One application of each of these immunoassay methods may be particularly specified in the present invention. For details of these general technical means, please refer to reviews and books (for example, Irie Prize, ed., "Radioimmunoassay", Kodansha Co., Ltd., published April 10, 1970, Irie 1 ed.) , [Radioimmunoassay continued], Kodansha Co., Ltd., published on May 1, 1980, edited by Eiji Ishikawa et al., "Enzyme immunoassay" 2nd edition, Igakushoin Co., Ltd., published on December 15, 1980, Japan Clinical Immunology Ha
nd-book J, Nippon Rinrinsha Co., Ltd., August 20, 1980, pp. 1198-1227, H. V. Bunakis et al., [Methods in Enzymolo
gy J Vol, 70.

[(mmunochemical 'l'echniq
ues part AJ, 7 Kademik Press,
1980, J. J. Lambon et al., [Methods i
nEnzymology J Vol, 7B,
[Jmmunochemical Technique
es part 3 J, Ibid., VOl, 74 [
(mmunochemical 'l'echniqu
ea part CJ, Academic Press, 1981).

Next, some typical immunoassay methods (1) will be briefly explained.

■ Solid-phase mRF method Test sample antibodies (antibodies) are placed in a test tube on which mouse mRF is immobilized.
1251, etc., is added to the reaction, and the amount of the label is measured and quantified. Qlin.

(NVEST, Vol. 65, ppl 86-1.
45 (1980) and measuring the enzyme activity.

■ Latex agglutination method A test sample is reacted with latex beads coated with mouse mRF. If IC is present in the sample, it will aggregate, so IC can be easily measured. It is meaningful as a semi-quantitative method.

(2) Laser nephrometry This is a method in which a certain amount of mouse mRF is added to a sample, and the resulting large particles of IC and mouse mRF are measured for scattered light using a laser beam to quantify IC.

■ Method using RIA inhibition This is a method in which one test tube of mouse mRF is immobilized and the rate at which IC in the sample inhibits the binding of heat-denatured IgG labeled with a radioactive isotope is measured to quantify IC ( Kouan, Rihipusot,, VOl, 56, p458 (1975
)reference).

59, p. 990 (1977)).

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 three types: native state, immobilized product, and labeled product. The native form is prepared in solution or lyophilized form. In addition, the in-phase product is solid-phased on carriers made of various materials such as test tubes, beads, latex, and disks by covalent bonding, crosslinking, or physical adsorption. In addition, 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.) is measured with extremely high sensitivity and specificity by an immunoassay method using mouse mRF as an antibody reagent. In addition, this mouse mRF can be obtained in large quantities with uniform quality using hybridomas obtained by fusing mouse mRF-producing cells and myeloma cells.Thus, the method of the present invention It has all the unique characteristics that none of the conventional IC measurement methods had, and its significance lies in the fact that it has established a general IC measurement method.

〔Example〕

Reference Example Preparation of mouse mRF Washed with EM. Mouse myeloma P3UlwoM E
After washing with M and mixing lung cells and P8U at a ratio of 10:1 and centrifuging, add 50% PEG100OMF and M solution 1 to a pellet.
ml was gradually added to perform cell fusion. Further ME
M solution was gradually added to the final solution of 10 g/bin. Centrifuge again and pellet in RPM11 containing 1596 fetal bovine serum.
I x 105 cells as P8U1 in 640 medium
The suspension was suspended in a volume of 0.1 ml/0.1 ml and distributed in 0.1 ml portions onto a 96-well microplate.

One day later, 0.1 ml of HAT medium was added, and then half of the medium was replaced with fresh HAT medium every 8 to 4 days. From around day 7, hybridoma growth was observed in some wells.

Supernatant of wells where hybridomas have grown 0, 05
ml of human heat-denatured IgG (
HAG) or human monomer 1gG okindase (manufactured by Vector), hydrogen peroxide as a substrate and coloring agent, 4-
E L I using aminoantipyrine, phenol
By the S A method, although it reacts with HAG, the monomer Ig
One that did not react with G was selected and cloned using the limiting dilution method.

As a result, C-26, C-28, D-13,
Eight strains, E-10, E-11, E-21, E-27, and E-86, were obtained.

■ Production of mRF The above hybridomas were incubated at 96 wells in RPM11640 medium containing fetal bovine serum (1591), 2), respectively.
The cells were cultured in scale-up in 5d flask and 75d flask, and the culture supernatant containing mRF was collected.

■ Properties of mRF It is generally said that human RF reacts with human IgG1.1gG2.1gG4 and does not react with IgGg (Kanji Kasuyo, "Immune Complex Disease", Ishiyaku Publishing Co., Ltd., 1982). (Published on February 15, 2015, p. 18).

Further, the recognition site is considered 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 1 gM class RF. Furthermore, most of the RFs did not react with human IgG of the IgGg subclass.

Regarding cross-reactivity with IgG from other animals, most of the RFs reacted with rabbit IgG. Many also reacted with horse IgG, and about half reacted with goats and cows.

These results are shown in Tables 1 and 2.

Table 1 H) Specificity for IgG Table 2 Cross-reactivity with other animal IgG ■ Purification of mRF Agarose gel fB body (CNBr activation - Sepharose 4B, manufactured by Farucia) 1 ml of human IgG
Coupled 3 mg of PB and packed it into a column.
Equilibrated with S. In this column, Ibridoma C-2
8, E-10, E-11, E-27 or E-36 culture supernatant aged 100 to 200 was passed through, thoroughly washed with PBS, and then washed with 0.1 M glinone-HCl buffer 2 (1) H2
, 8) was used to elute the monoadsorbed mRF, and 1/10 volume of 8M Tris-HCl buffer (e.g. 8.2) was added to return the mixture to neutrality, yielding purified mouse mRF.

For C-26, agarose gel carrier (A
ffigel-10, manufactured by Bio-Rad) 1 ml
Using IC horse IgG coupled with 101R9, it was eluted with 0.1 M citrate buffer (pH 4, 5), dialyzed against PBS, and purified mouse mR.
It was set as F.

The titer by ELISA was 2000 to 8000 times higher than HAGI per mg of purified mouse mRF. The titer is determined by the dilution rate from the stock solution of the antibody sample that gives a value of 5096, assuming that the absorbance obtained by ELISA is 10096 for a sample in which sufficient antibodies against the immobilized antigen are present. expressed.

Reference Example 125I - Preparation of (Fab')2 fragment of rabbit anti-human IgG Fc fragment antibody The antibody obtained by immunizing a rabbit with a human IgG Fc fragment was purified by ammonium sulfate fractionation - DEAE cellulose ion exchange chromatography.

This purified antibody was dissolved in 0.1 M acetate buffer (1) H4,51
(containing 1,0,1M sodium chloride) was prepared at a volume of 10 μ/ml. The Pepson decomposition solution was subjected to gel filtration chromatography (Sephadex G-150 column).
．． The mixture was developed with 1M sodium borate buffer +)H8,0) to obtain a (Fab')2 fragment fraction.

Next, convert this (Fab')2 fragment to /Ater(
Chloramine T according to the method of Hunter et al.
It was labeled with 125I (Nature, VOl,
194, pI) 495-496 (1962)
).

Example l Purification? +7 y, m Rf? was diluted to 25 μQ/me with PBS-EDTA buffer (containing 015 M sodium chloride, 10 mM EDTA), added to polystyrene tubes in 0.5 me portions, and left overnight at 4°C to coat. Remove the contents from the tube and add 5-5g5m1m
-1N1111t%BSA containing P B S -E DT
A! @.

Blocked with liquid.

Diluted HAG, monomer 1gG, or patient serum samples at 1011. g and further added 1% BSA to PBS-
EDTA buffer was added to bring the volume to 0.5 liters, and the mixture was left at 40C overnight.

0.5g/(approximately 108 c
pm/ng) was added.

After incubation for 2 hours at room temperature, PBS-ED
The plate was washed three times with TA buffer, and the amount of radioactivity was measured using an autogamma counter. Calculation is 1251 from the count number
How many nq of labeled fragments are bound by 108C
This was done using a specific activity of pm/n9.

When measurements were performed on 8 SLE cases, RAS cases, cancer cases, and 10 healthy subjects, the measured values were C-26RA41.
．． 0±86. ] nQ/ml (87.5%), cancer 850 ± 8.5 n97M1 (57.1%), healthy person] 75 soil 6.9 nQ/ml.

From this result, it was found that human IC can be measured by mouse mRF in a manner equivalent to the conventional C11!1 method or Hi-mRF method. In addition, in this example, cancer patient's I
The C positive rate was a little high.

Example 2 0.5 ml of C11 or mouse mRF diluted with PBS-EDTA buffer to the following concentration was added to a polystyrene tube I and left overnight at 4°C.

10C+al 5μg/shouldert C-26A28o=2.811 diluted 800 times D-18
A28o=0.495 diluted 300 times E-11A280
= 0.655 diluted 300 times rabbit anti-human IgGFc antibody (Fab') 2 fragment 5 μQ/ml Remove the diluted solution by aspirating and add to PBS-ED containing 1% BSA.
1 ml of TA buffer was added and allowed to stand at room temperature for 1 hour to block. Aspirate and remove the BSA solution. -/@PBS-EDTA buffer 15-21

Add sample 10111 and add 196B SA-containing P
A BS-EDTA buffer solution was added to make up to 0.5 kg, and the mixture was left at room temperature for 2 and a half hours.

I left it for half an hour.

Aspirate the reaction solution and add PBS-EDTA buffer 1 and 5.
ml 8 times, and the amount of radioactivity was measured using an autogamma counter to obtain the measured value.

Calculate the number of 1251 labeled fragments from the count number.
G-binding was determined using a specific activity of 267 cpm/rl.

When measurements were performed on 20 cases of untreated 5LE, 20 cases of untreated RA, and 29 healthy subjects, the proportions were as follows, when an average of normal values plus 25D or more was considered abnormal.

c+j C-26D-18E-11SLE 7
5% 4 096 6096 50%RA 4
0% 50% 50% 8596 Example 3 4.1 pg/ml and 3.5/79/ml of mRF prepared from hybridomas E-27 and D-18 were placed in a 96-well microplate (made of polyvinyl chloride), respectively.
200 µe of PBS diluted was added to the plate, and the plate was left to stand at 4°C overnight to coat.

Next, add 1% BSA-containing PBS, leave it at room temperature for 1 hour to block, remove the BSA solution by suction, add patient serum sample 1011e, and add 1% BSA-containing PBS to 40.1
1 and left at room temperature for 2 hours.

After removing the reaction solution by suction and washing three times with PBS, horseradish peroxidase-labeled ■-gi anti-human IgG was added.
Fc fragment antibody F(ab')2 fragment (manufactured by Capel Laboratories) was added and incubated at room temperature for 2 hours.

The reaction solution was removed by suction, and after washing three times with PBS, a substrate solution containing 4-aminoantipyrine, phenol, and hydrogen peroxide was added, and the absorbance at 0D550 was measured to calculate the enzyme activity, and the IC was quantified. .

8 cases of cancer patients, 8 cases of RAS, 8 cases of 5LE, 8 cases of IC of healthy subjects
As a result of the investigation, the positive rate was 75% for cancer, 12.5% for RA, and 375% for SLE for D-13mRF, and 75% for cancer, 12.5% for RA, and 375% for E-27mRF.
S I-E was 12.5%. In both cases, the positive rate of IC in cancer was high.

Patent Applicant (677) Yamasa Soy Sauce Co., Ltd. Procedural Amendment (Voluntary) February 8, 1985 1. Case indication 1988 Patent Application No. 236909 2. Name of the invention Method for measuring immune complexes 3. Amendment Relationship with the case of a person who makes a patent application Postal code of the patent applicant: 288 Address: 2-10-14 Shinseicho, Choshi City, Chiba Prefecture Date of amendment order: Voluntary action 5 Number of inventions to be increased by amendment: None 6: Specification subject to amendment Detailed Description of the Invention Column 7, Contents of Amendment 1) The following sentence is added between the third and second lines from the bottom of page 18 of the specification.

[■ Sand Inch Method A test sample is placed in a reaction container such as a test tube or microplate in which the mouse mRF is in phase, and the sample is reacted. Next, labeled mouse mRF is added to this and reacted, and the amount of bound or free label is measured and quantified. 2) "Polyvinyl chloride" on page 31, line 2 of the specification is replaced with "
Corrected to "polyvinyl chloride."

3) Add the following sentence from line 6 on page 32 of the specification.

[Example 4 mRF prepared from hybridoma D-13 was added to a 966 microplate (1M polyvinyl chloride) at 6.3x.
/mfi, or mouse IgM without mRF activity
Mouse IgM (for control) prepared from a hybridoma clone producing 13.1 itg/IIIa diluted with PBS was added to each well in an amount of 50 μρ and allowed to stand at 4° C. for coating.

Next, add %BSA-containing solution and leave it at room temperature for 1 hour to block. After removing the BSA solution by suction, add A buffer (0.1M Tris-HCl pH 8°, 0.0.5M sodium chloride, 1% BSA solution).
mRF and mR serum samples diluted 10 times with SA)
50 μΩ in wells coated with IgM without F activity.
The mixture was added in portions and left at room temperature for 2 hours.

After removing the reaction solution by suction and washing three times with PBS, D-13 labeled with 125■ by lactoperoxidase was added.
A solution (0,20μ
g/mQ, 5Q 0cpi/ng) was added at 50μQ,
It was left at 4°C overnight.

After removing the reaction solution by suction and washing three times with PBS, the amount of radioactivity was measured using an autogamma counter, and the number of counts in wells coated with mRF showed that 1 gM of mRF, which does not have activity, was coated. The IC was quantified by calculation by subtracting the Callan 1-number of a given well.

Claims (1)

[Scope of Claims] 1) A method for measuring immune complexes in a human body fluid sample by immunoassay, in which mouse monoclonal rheumatoid factor is used as an antibody reagent that specifically binds to immune complexes in the sample. A method for measuring an immune complex, characterized in that it is used. 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 the bound label, and determine the immune complex. 2. The method according to claim 1, characterized in that: 3) The method according to claim 1, characterized in that the immune complex is measured by reacting mouse monoclonal rheumatoid factor immobilized on microparticles with a sample and measuring the degree of aggregation that occurs.