JPH04166094A - Anti-crp monoclonal antibody, hybridoma and method for recovering and purifying human crp - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody CRPB-2(IgG1), CRPB-3(IgG2a) or CRPB-5(IgG1) capable of exhibiting calcium or magnesium-dependent reactivity to a human C reactive protein (hereinafter referred to as CRP). USE:Production of purified human CRP, etc. PREPARATION:A hybridoma CRPB-2, CRPB-3 or CRPB-5 obtained from a spleen cell of mammal and myeloma cell of mammal immunized with the human CRP is separated from a cultured mixture and purified.

Description

[Detailed description of the invention] [Industrial application field]

The present invention relates to human C-reactive protein (Creactiv).
e protein (hereinafter sometimes abbreviated as CRP), a monoclonal antibody that exhibits reactivity in a calcium- or magnesium-dependent manner, a hybridoma that secretes the monoclonal antibody, and a collection of human CRP using the monoclonal antibody. It is related to the purification method.

[Prior art and problems to be solved by the invention]

CRP is a protein discovered by Tilett et al. in 1930 in the serum of pneumonia patients infected with pneumococcus. This protein is called C-reactive protein because it reacts with C-polysaccharide of pneumococci in the presence of calcium to produce a precipitate. The presence and amount of CRP in serum is said to be an extremely important acute-phase protein, as it reflects the presence or absence of acute inflammation and tissue damage, as well as its degree, and the measurement of CRP has become increasingly popular in recent years. It is becoming important. For this reason, it is widely clinically practiced to measure CRP not only qualitatively, but also quantitatively to determine its evolution. As methods for measuring CRP, the capillary tube method, 5RID method, immunoturbidimetry, or latex agglutination method, which utilizes antigen-antibody reactions, are mainly used. Further, with the progress of CRP quantitative technology, it has been required to enable measurement on the order of several hundred ng/ml. Under these circumstances, high specificity and affinity are required for the antiserum used in the measurement, and there has therefore been an increasing need to highly purify CRP used as an antigen in order to obtain the antiserum. Conventionally, CRP purification methods have been carried out using serum or ascites of CRP-positive patients as raw materials, using a combination of ammonium sulfate salting out, phosphorylcholine (or phosphorylethanolamine)-Sepharose affinity chromatography, ion exchange chromatography, or molecular sieve chromatography. However, the purification process is complicated and the recovery rate is low (approximately 30 to 40%). On the other hand, antibody affinity columns are sometimes used as a general method to specifically purify antigens, but high-concentration chaotropic ions such as 3M sodium thiocyanate (pH 8,0) or glycine- Strongly acidic conditions such as hydrochloric acid (pH 2°0) are used. However, CRP has a pentameric structure due to non-covalent bonds, and this pentameric structure dissociates or denatures under the above elution conditions. It is not appropriate to use it. Therefore, it has not been conventional to use an antibody affinity column for purifying CRP. On the other hand, as a result of various studies, the present inventor has developed monoclonal antibodies CRPB-2, CRPB-3 and CRPB-2, which react with human CRP in a calcium- or magnesium-dependent manner.
We discovered PB-5. Furthermore, this monoclonal antibody C
RPB-2, CRPB-3 and/or CRPB-5
An immunoadsorbent with CRP as a ligand is prepared, and human CRP is first specifically adsorbed and recovered in the presence of calcium or magnesium, and then human CRP is adsorbed under mild conditions using a chelating agent such as citric acid or ethylenediaminetetraacetic acid (EDTA). It has been found that human CRP can be efficiently recovered and purified by elution of human CRP.

[Means to solve the problem]

Accordingly, the present invention relates to monoclonal antibodies that exhibit calcium- or magnesium-dependent reactivity against human C-reactive protein. The present invention also provides a monoclonal monoclonal cell that is formed by fusion of a mammalian spleen cell immunized with human C-reactive protein and a mammalian myeloma cell, and exhibits calcium- or magnesium-dependent reactivity to human C-reactive protein. It also relates to hybridomas or cell lines derived therefrom that secrete antibodies. -5= Furthermore, the present invention provides a method for recovering human C-reactive protein or Relating to a purification method. Monoclonal antibodies of the present invention CRPB-2, CRPB-
3 and CRPB-5 are secreted from hybridomas (or cell lines derived therefrom) formed by the fusion of mouse spleen cells immunized with human CRP and mouse myeloma cells, as detailed in the Examples below. ,
It is a monoclonal antibody that shows calcium- or magnesium-dependent reactivity against human C-reactive protein, and is classified into classes IgG+, IgG*-1 and I, respectively.
gG, is. Hereinafter, the monoclonal antibodies CRPB-2 and CR of the present invention will be described.
A method for creating PB-3 and CRPB-5 will be explained. Human CRP used as an immunogen can be prepared, for example, by the following method. That is, CRP-positive human serum or ascites fluid is used as a raw material, and cells and insoluble matter are removed by centrifuging and filtering these. The upper groove thus obtained is subjected to, for example, adsorption chromatography using phosphorylcholine-Sepharose, ion exchange chromatography using DEAE-cellulose, Sephacryl 5
Purified human CRP is obtained by a purification operation such as molecular sieve chromatography using -aOO. The human CRP thus obtained was subjected to Equine Brilliant Blue R-250 (CBB) after 5DS-PAGE treatment.
R-250) staining shows a single band. The purified human CRP immunogen solution is then used to test mammals (e.g., mice, rats, rabbits, goats, or horses).
is immunized by in vivo immunization. Specifically, for example, a purified human CRP immunogen solution is mixed with an equal volume of Freund's complete or incomplete adjuvant until emulsified. This mixture is administered, for example, subcutaneously to a mouse (first immunization). Thereafter, similar operations were performed at intervals of 2 to 4 weeks.
Immunize several times. A few days after the final immunization, the spleen is removed aseptically and crushed with a stainless mesh to prepare spleen cells, which are used in the cell fusion step. As myeloma cells (myeloma cells), which are the other parent cells used for cell fusion, various known cell lines, such as p8 (p3/x63-Ag8) (Nature,
256.495-497 (1975) L p3-Ul
(Current Topics in Micr
obiology and immunology,
81; 1-7 (1978)), N5-1 (Eu
r, J, Immuno1. ．． 6;511-519 (1
976)), MPC-11 (Cell, 8:405-4
15 (1976)), 5P210 (Nature,
276:269-270 (1978)), FO (J
. Immunol, Meth, 85; 1-21 (1
980)), X68.6.55,3 (J,, Immu
nol, 123; 1548-1550 (1979
) L 5194 (J, Exp, Med., 148.313-323 (1978) L or R210 in rats (Nature, 277:13
1-133 (197,9)), etc. can be used. Cell fusion between immune spleen cells and myeloma cells can be carried out in a conventional manner, for example, by using a known fusion promoter (polyethylene glycol or Sendai virus, etc.) and optionally using an adjuvant (dimethyl sulfoxide, etc.). You can also do it. The ratio of immunized spleen cells to myeloma cells may be the same as in the conventional method; for example, spleen cells are used at about 1 to 10 times the amount of myeloma cells. As a fusion medium,
For example, Dulbecco's Modified Eagle Medium (DMEM) containing 40% (w/v) polyethylene glycol can be used. Fusion is performed by thoroughly mixing the immunized spleen cells and myeloma cells in the medium described above. Subsequently, cells other than hybridomas are removed using a selection medium (e.g., HAT medium), and the presence or absence of antibody production in the hybridoma culture supernatant is measured by, for example, ELISA method, and the target hybridoma is isolated. . Monoclonal antibody CRP of the present invention thus obtained
B-2, a hybridoma secreting CRPB-3 or CRPB-5 CRPB-2, CRPB-3 or CRP
B-5 can be subcultured in a normal medium and can be easily stored for a long period of time in liquid nitrogen or the like. From a culture solution obtained by culturing the above-mentioned hybridoma CRPB-2, CRPB-3 or CRPB-5 by a conventional method, or from the ascites of a suitable mammal (e.g. mouse or rat) to which any of the above three hybridomas has been administered, It is possible to separate and purify the desired monoclonal. Such separation and purification methods include ammonium sulfate salting out, DEAE
- Ion exchange column chromatography using cellulose, molecular sieve column chromatography using molecular sieve gel, affinity column chromatography using protein A-binding polysaccharide, dialysis, or freeze drying. Monoclonal antibody CRPB of the present invention thus obtained
-2, CRPB-3 or CRPB-5 is human CRP
It is possible to recognize the change in steric structure caused by the binding of calcium or magnesium to. In addition, monoclonal antibodies CRPB-2, CRPB=3 or CRP
The calcium- or magnesium-dependent reactivity of B-5 to human CRP is not inhibited even in the presence of phosphorylcholine. Furthermore, monoclonal antibody CRPB-2 can cause an in-gel precipitation reaction. This in-gel precipitation reaction is possible because the antigenic determinant is human C.
This is probably because there is little steric hindrance because it exists near the periphery of the pentamer plane of RP. By the way, it has already been reported that when human CRP binds to calcium, its tertiary structure changes (Yo et al.
ung, N, M, and qWjl 1 i ams, R,
E,,J,Immun. 121.1893-1898 (1978)). In addition, it has already been reported that monoclonal antibodies that recognize changes in the three-dimensional structure have been established (Kilp
atrik, J, M, et al+M. 1, Immun, 19.1159-1165 (198
2)). However, it has been reported that the binding of these monoclonal antibodies to CRP is inhibited in the presence of phosphorylcholine, and the monoclonal antibodies of the present invention CRPB-2, CRPB-3 and CRPB-5
and have different reactivity. Furthermore, there has been no known example of applying a monoclonal antibody that binds to CRP in a calcium- or magnesium-dependent manner to the purification of human CRP. Monoclonal antibodies of the present invention CRPB-2, CRPB-
Human CRP can be recovered and purified in high yield by affinity chromatography using CRPB-3 and/or CRPB-5. A monoclonal antibody-immobilized gel used for affinity chromatography can be prepared as follows. After swelling and gelling CNBr-activated Sepharose 4B (Pharmacia) by a conventional method, monoclonal antibody CRPB-2 was isolated and purified as described above.
, CRPB-3 and/or CRPB-5, respectively, are coupled to the gel. Thereafter, excess active groups are blocked and unreacted monoclonal antibodies are removed. By filling the thus obtained monoclonal antibody-immobilized gel into a suitable column, a monoclonal antibody-immobilized affinity column can be prepared and used in a method for collecting and/or purifying human CRP. CRP-positive human serum or ascites fluid, etc. from which insoluble matter has been removed by centrifugation and filtration, is mixed with a buffer (e.g., 50 mM) containing 2 to 10 mM (preferably 5 mM) calcium or magnesium, Lis-HCl, 150 mM NaCl.
; pH 8.0) and added to the aforementioned monoclonal antibody-immobilized affinity column to bind CRP. Next, after thoroughly washing the column with the buffer containing calcium or magnesium,
A buffer containing 5-20mM (preferably IOmM) of DTA (e.g., 50mMh+J-HCl, 150mM
Elute human CRP with NaCl (pH 8,0). When human CRP thus purified was fractionated by 5DS-PAGE and silver stained, only a single band of 26Kd was detected in the presence of a reducing agent such as 2-mercaptoethanol, and 22Kd in the absence of a reducing agent. Only a single band of C
You can get RP. Anti-human CRP antiserum, which can be obtained by immunizing rabbits etc. with the purified human CRP obtained in this way, shows only a single precipitation line in the γ chain region compared to CRP-positive human serum in immunoelectrophoresis. It can also be used as an immunologically homogeneous human CRP antigen.

【Example】

The present invention will be explained in more detail with reference to the following examples, but these are not intended to limit the scope of the present invention. The buffers used in the following examples are as follows. Buffer y-A: 20mM) Lis-HCl, 150mMNa
c1.5mMCaCl2 (pH 8,0) Buffer B: 20mM) Lis-HCl, 150mMNa
cl, 10mM EDTA (pH 8,0) Buffer C: 10mM sodium phosphate, 90mM
Nacl (pH 7,2) Buffer-y-D: 20mM) Lis-HCl, 150
mMNaCl (pH 8,0) Buffer E: Buffer A containing 0.05% Tween20 Buffer F: 20mM Lis-HCl containing 5mM EDTA, 150mM NaCl (pH8,0) Buffer G: Buffer F containing 0.05% Tween20 Buffer- H: 10mM sodium phosphate (pH 8.0
) Buffer-y-I: 50mM Tris-HCl, 150mN
acl, 5mM CaCL, 0.05% NaNs
(pH 8,0) (1) Purification of human CRP antigen Method of Volanakis, J.E., et al. (J. Immun, Meth, 23,285
-299 (1978)). All operations are 4
It was carried out at ℃. CRP-positive human ascites 50 (7) was centrifuged at 12,000 x g for 60 minutes to obtain a supernatant 50 (7). To this supernatant, Hyflow-Purcell (Wako Pure Chemical Industries) was added to a concentration of 1%. Then, filtration was performed using No. 5G filter paper (Atovanchik) to obtain a filtrate of approximately 500 μm. Next, a phosphoethalamine-agarose column (diameter 2° 5 cm x length Add the above filtrate to 50 ann) and calculate the OD of the flow-through fraction,
1. Buffer A was allowed to flow until o was 0.02 or less. Elution was performed with buffer B, and the purity of CRP was approximately 5.
A 0% elution fraction was obtained. The eluted fraction was concentrated by ultrafiltration and thoroughly dialyzed against buffer C. The fraction after dialysis was transferred to a DEAE-cellulose column (diameter 2.6 cm x length 3
5au), and the 0D211 of the flow-through fraction was 0.02.
Buffer C was flowed until the concentration was below. Elution was performed using buffer C1 and 10 mM sodium phosphate and 1.0
The concentration gradient elution method using MNaC1 (pH 7.2) was used. The second protein peak in this elution method was collected as a CRP fraction. The fraction thus obtained was concentrated by ultrafiltration, thoroughly dialyzed against buffer A, and then added to a phosphorylethanolamine-agarose column in the same manner as above to obtain an adsorbed fraction. After concentrating this fraction by ultrafiltration, a Sephacryl 5-soo column (diameter 1.
Molecular sieve chromatography was performed using a 6 cm x 90 ann (length). The second protein peak was collected as a CRP fraction, concentrated by ultrafiltration, and separated from human CRP.
And so. Human CRP thus prepared was subjected to 5DS-PAGE (7,
After fractionation with 5-20% gradient gel), Coomassie Brilliant Blue R-250 (CBBR-250)
When stained with , it showed a single band with a reduced molecular weight of 26 Kd and a non-reduced molecular weight of 22 Kd. Also, human C
Latex agglutination reagent for RP measurement (Elpia Ace CRP
When the concentration of human CRP in the thick film water was measured using a method (manufactured by Yatron), it was 29.5 μg/−. Therefore, it can be seen that 14.7 μm of human CRP was contained in 500 μm of thick film water. On the other hand, when human CRP obtained by the above purification method was similarly measured, the recovered amount was 5.6
The recovery rate was 38%. ELI using this human CRP as an immunogen and selecting anti-CRP monoclonal antibody-producing hybridomas
It was used as an antigen for SA. (2) Preparation of immunized spleen cells Human CRP immunogen solution 1.0/7d! was mixed with an equal volume of Freund's complete adjuvant until emulsified, and 200 μl of the mixture was intraperitoneally administered to BAL B/c mice (first immunization).
. After 30 days, the mouse was given 20% of the same mixture as above.
0 μl was administered intraperitoneally (second immunization). After 21B from the second immunization, human CRP immunogen solution (1,0 /
-) with an equal volume of physiological saline, and 200μ of the diluted solution
1 was administered intravenously to the mice (final immunization). Three days after the final immunization, the spleen was aseptically removed from the mouse and used in the next cell fusion step. (3) Cell fusion step The aseptically removed spleen was placed in a petri dish containing DME medium 5- containing 15% fetal bovine serum. next,
After the spleen was perfused with approximately 15 ml of DME medium containing 15% fetal bovine serum to drain out the spleen cells, the spleen cell suspension was passed through a nylon mesh. The spleen cells were collected in a 50-centrifuge tube and centrifuged at 500×g for 10 minutes. Hemolizing solution (155mM) was added to the pellet thus obtained.
NH4C1, 10mM KHCOs, 1mM Na2ED
TA; pH 7,0)4- was added and suspended. The suspension was left at 0° C. for 5 minutes to destroy red blood cells in the suspension. 10 - of DME medium containing 15% fetal bovine serum was added and centrifuged. The cell pellet thus obtained was washed with DME medium by centrifugation, and viable spleen cells were measured. On the other hand, about 2 x 107 mouse myeloma cells (myeloma cells) SP210-Agl 4 (RIKEN Scene Bank Cell Bank), which had been cultured in advance, were added to the spleen cells IX.
10" were added, mixed well in DME medium, and centrifuged (500Xg for 10 minutes). The supernatant was aspirated, the pellet was thoroughly loosened, and the pellet was dissolved in 40% polyethylene glycol 4000 solution (3
0.5- (kept at 8°C), and manually opened the centrifuge tube.
Mix the polyethylene glycol solution and cell pellet by gentle rotation for a minute. Next, 1 liter of DME medium kept at 38° C. was added every 30 seconds, and the tube was gently rotated. After repeating this operation 10 times, 20-mL DME medium containing 15% fetal bovine serum was added and centrifuged (500×g, 10 minutes). After removing the supernatant, the cell pellet was mixed with HAT medium containing 15% fetal bovine serum (aminopterin 4X1 in DME medium).
After washing twice by centrifugation with 0-7M, 1.6X thymidine, 10-6M thymidine, and 1X10-'M of hypoxanthin, the cells were suspended in the HAT medium 40-'. 200 μl of this cell suspension was dispensed into each well of a 96-well cell culture plate, and culture was started at 37° C. in a carbon dioxide gas incubator containing 5% carbon dioxide gas. During cultivation,
By removing approximately 100 μl of the medium from each well at intervals of 2 to 3 days and adding 100 μl of the above HAT medium,
Hybridomas growing in HAT medium were selected. 8
From the 8th day, the medium was changed to HT medium containing 15% fetal bovine serum (DME medium added with thymidine 1° = 20-6 × 10-'M and hypoxanthine 1X10-'M), and the hybridomas were grown. ELIS described below on the 10th.
Hybridomas producing anti-human CRP antibodies were screened by Method A. (4) Establishment of hybridoma The presence or absence of produced antibodies in the hybridoma culture supernatant can be determined by ELI.
Measured by SA method. The above human CRP immunogen solution (Immu 1 on II; 8 Hon Dynatic Co., Ltd.) was added to each well of a 96-well ELISA plate (Immu 1 on II;
10°C g/yd: 50 μl (diluted with buffer A above)
Aliquots were dispensed and left at 25°C for 2 hours. Next, 0.05% Tween20-5mMCaC12-
After washing three times with physiological saline (hereinafter referred to as washing solution a), 50 μl of culture supernatant was added to each well and reacted at 25° C. for 1 hour. After washing the wells three times in the same manner as above, 50 μm of peroxidase-conjugated anti-mouse antibody (DAKO: P16■) diluted 200 times with buffer E was added to each well and allowed to react for 1 hour. After the reaction, each well was washed as described above and 0.5 m
M aminoantipyrine and 10m'M phenol and 0.0
200 μm of 20 mM Tris-HCl and 150 mM Na CI (pH 7,4) containing 0.5% hydrogen peroxide solution were added to each well, reacted at 25° C. for 30 minutes, and the absorbance of each well at 490 nm was measured. As a result, 19
Antibody production was observed in 5 out of 2 wells.゛ Transfer the hybridomas in these 5 wells to a 24-well plate and add HT containing 10-15% fetal bovine serum.
The cells were cultured in medium for 4 to 5 days. Thereafter, the presence or absence of production of anti-human CRP monoclonal antibodies was confirmed again by ELISA, and then cloning was performed by limiting dilution. In the limiting dilution method, a cell suspension diluted with HT medium to have 5 hybridomas/d is preliminarily dispensed into each well of a 96-well plate in which 2×10' normal BALB/c mouse peritoneal cells are dispensed per well. 100μβ was dispensed into each. After l0EI, anti-CRP was detected by ELISA method.
Hybridoma clones producing monoclonal antibodies were screened. As a result, from among 20 to 40 antibody-producing clones for each hybridoma, clones with good proliferation, high antibody secretion ability, and stability were selected and cloned again in the same manner as above. Five types of anti-human CRP monoclonal antibody-producing hybridomas were established, and CR
PB-1, CRPB-2, CRPB-3, CRPB-4
and CRPB-5. (5) Transplantation of hybridomas Pristane (2,6,10,14-tetramethylpentadecane) 0.5- to 10-12 week old BALB/
After 14 to 20 days had elapsed, the five types of hybridomas described above, which had been grown in vitro, were inoculated into the intraperitoneal cavity of C mice at 2×10′ cells per mouse. -5 to 15 from 1 mouse for each hybridoma!
ILl ascites was obtained. The antibody concentration is 2-10 /
It was ml. (6) Calcium- or magnesium-dependent reactivity was investigated by ELISA method. That is, the human CRP immunization antigen was diluted with buffer A or buffer F to a concentration of 10 ug/mj, dispensed onto the aforementioned ELISA plate, and left at 25°C for 2 hours. Next, 0 with 5mM CaCL+ or 5mM EDTA
．． After washing three times with 05% Tween 26-physiological saline (hereinafter referred to as washing solution a or washing solution B), 50 μl of each of the five types of ascites diluted 5000 times with buffer A or buffer F was added to the wells and reacted at 25°C for 1 hour. I let it happen. Next, after washing the wells three times with washing solution a or washing solution, the peroxidase-conjugated anti-mouse antibody (DAK) diluted 200 times with buffer E or buffer G.
Company O; P2O3) 50u, il was added to each well and reacted for 1 hour. After the reaction is completed, wash the wells three times with washing solution a or washing solution, and perform a color reaction using the method described in Example 1 (4) above.
The absorbance of each well was measured. The results are shown in Table 1. Sakudojin ELISA (absorbance at 490 nm) -26 ~ As shown in Table 1, monoclonal antibody CRPB-2
, monoclonal antibody CRPB-3 and monoclonal antibody CRPB-5 showed reactivity with human CRP in a calcium-dependent manner. Furthermore, when we conducted a similar experiment using magnesium instead of calcium, we found that the monoclonal antibody CRPB-
2. Monoclonal antibody CRPB-3 and monoclonal antibody CRPB-5 showed reactivity with human CRP in a magnesium-dependent manner. The above monoclonal antibodies CRPB-2, CRPB-3
And when CRPB-5 shows reactivity to human CRP in a calcium- or magnesium-dependent manner, it was investigated whether the reactivity was inhibited by the presence of phosphorylcholine. Dilute human CRP immunization antigen with buffer A to 10μg/-, and add 50μg/- to the above ELISA plate.
The mixture was dispensed into 1/2 m portions and left at 25°C for 2 hours. Next, after washing three times with washing solution a, buffer A or 1mM phosphorylcholine chloride (Sigma, P-0
Add 50 μl each of the three types of ascites diluted 5,000 times with buffer A containing 378) to the wells, and then
The reaction was allowed to take place at °C for 1 hour. Subsequently, the wells were washed three times with washing solution a, and then 50 μm of the above peroxidase-conjugated anti-mouse antibody (DAKO, P2O3) diluted 200 times with buffer E was added to each well and allowed to react for 1 hour. After the reaction, the wells were washed three times with washing solution a, and the wells were washed three times with washing solution a.
A color reaction was performed using the method described in (4), and the absorbance of each well was measured. The results are shown in Table 2. [Margin below] Table 2 Presence or absence of reaction inhibition in the presence of phosphorylcholine (490n
Absorbance at m) As shown in Table 2, monoclonal antibody CRPB-2,
The calcium-dependent reactivity of CRPB-3 and CRPB-5 to human CRP was not inhibited even in the presence of phosphorylcholine. Furthermore, when a similar experiment was conducted using magnesium instead of calcium, the same results were obtained. By the way, Ki 1patrik, J, M, S (Mo
1. Immun, 19.1159-1165 (198
2) Four types of monoclonal antibodies (HD
2-4, EA4-1, FB2-1, HB3-2) are
In both cases, the reaction is inhibited in the presence of phosphorylcholine. As described above, 'monoclonal antibody C in the present invention
There is a difference between the reactivity of RPB-2, CRPB-3 and CRPB-5 and that of the monoclonal antibodies reported by K11patrik, J.M. et al. It can be assumed that the antigenic determinants are different. (7) Ophthalony method hybridoma CRPBI to hybridoma CRPB5
The reaction between ascites and purified human CRP was investigated by the Ophthalony method. Prepare a 1.0% agarose plate (2.0 mm thick) with buffer A (or F) and add purified human CRP (1.0 mg/7 nl) 10tt to the center well.
10 μl of buffer A (or F) as a negative control per well in the surrounding 6 wells.
Buffer A (or F) into the remaining 5 wells.
Ascitic fluid serially diluted 2-fold (16-fold diluted from the stock solution) 10μ
m was added and allowed to react at 4°C overnight. The results are shown in Table 3. Master Ophthalony method (precipitation line formation) As shown in Table 3, monoclonal antibody CRPB-1
The formation of a calcium-independent sedimentation line was observed in
Formation of a calcium-dependent precipitation line was observed in the monoclonal antibody CRPB-2. Furthermore, when a similar experiment was conducted using magnesium instead of calcium, similar results were obtained. Other monoclonal antibodies CRPB-3, monoclonal antibodies CR
No sedimentation line was observed for PB-4 or monoclonal antibody CRPB-5 under any conditions. As is clear from the above experiments, the monoclonal antibodies CRPB-2, CRPB-3 and CRPB-5 of the present invention
can recognize the conformational change caused by binding of calcium or magnesium to human CRP. Furthermore, the reactivity of each of these antibodies is not inhibited even in the presence of phosphorylcholine. Furthermore, monoclonal antibody CRPB-2 can cause an in-gel precipitation reaction with human CRP in a calcium- or magnesium-dependent manner. In this way, the in-gel precipitation reaction can occur because the antigenic determinant of monoclonal antibody CRPB-2 exists near the periphery of the pentamer plane of human CRP, so there is little steric hindrance. It appears to be. In the following example, the recovery and purification of human CRP by affinity chromatography will be specifically shown.
RPB-2 was used. This may be because it was expected that by using a monoclonal antibody that recognizes an antigenic determinant in a site with little steric hindrance as a ligand, as described above, the amount of CRP bound per column unit volume would increase.53 = It is et al. However, the recovery and purification of human CRP is performed using monoclonal antibodies CRPB-3 and CRPB-5 instead of the monoclonal antibody CRPB-2.
Of course, this can also be carried out when using the column as a ligand. (1) Purification of anti-human CRP monoclonal antibody CRPB-2 An equal volume of saturated ammonium sulfate solution was gradually added to 20 d of mouse ascites containing monoclonal antibody CRPB-2, the mixture was stirred for 30 minutes under water cooling, and then left for 60 minutes. After centrifugation (10,000×g, 10 minutes), the resulting precipitate was dissolved in a small amount of buffer H and dialyzed against 1,000 times the amount of buffer H. This was added to a DEAE-cellulose column (diameter 2.5 cm x length 183 cm) that had been equilibrated with buffer H in advance. Elution of the monoclonal antibody was performed by a concentration gradient method between buffer H and buffer H containing 0.2M Nacl. The eluted monoclonal antibody was concentrated by ultrafiltration, and
0mM) Lis-HCl and 150mM Nacl (pH
Sephacryl S-300 column (8,0) equilibrated with
2.6 cm in diameter x 95 cm in length). 20mM
Tris-HCl and 150mM NaCl (pH 8,0)
The purified monoclonal antibody CRPB-2 (47
■) was obtained. (2) Preparation of monoclonal antibody CRPB-2-immobilized affinity column Weigh out 4.5 μm of dried CNBr-activated Sepharose 4B (Pharmacia), swell with 1 mM HCl to gel, and place on a glass filter. The gel was thoroughly washed. Coupling buffer = (0,
2M NaHCOs, 0.5M NaCl: pH 8,
5) Transfer the purified monoclonal antibody CRPB-2 (3
0■) was added, and the reaction was allowed to proceed at 4°C overnight. After washing and removing unreacted monoclonal antibody CRPB-2, the gel was soaked in 0.1M Tris-HCl (pH 8.0).
The active groups were blocked by transferring the mixture to a solution and leaving it for 2 hours at 25°C. Next, transfer the gel on a glass filter to coupling buffer, as well as 0.1 M sodium acetate and 0.5
Non-specifically adsorbed proteins were removed by alternately washing with MNaC1 (pH 4,0). The gel thus obtained was packed into a column (diameter 1.5 cm x length 8.5 ann; total volume 15 d) to prepare a monoclonal antibody CRPB-2 immobilized affinity column (hereinafter simply referred to as affinity column). (1) Binding amount Hyflow Super-Purcel (Wako Pure Chemical Industries) was added to 1.51% of human CRP-positive human ascites to a concentration of 1.0%, and N
o, 5C/ji paper (Atovanchik). This filtrate was thoroughly dialyzed against buffer Ⅰ, and then centrifuged (12,000×g; 60 minutes) to remove insoluble matter. The human ascites pretreated as described above was pre-mixed with buffer I.
The affinity column of Example 2, which had been equilibrated at a flow rate of 20. It was added at vJ/h. In order to investigate the amount of human CRP bound to this affinity column, the appearance of human CRP in the non-adsorbed fraction was investigated using a latex agglutination reagent for measuring human CRP (manufactured by Elpia Ace CRP Nyatron). As a result, human CRP was observed to appear in the non-adsorbed fraction after about 11 hours of addition of human ascites, confirming that the affinity column was saturated with human CRP. After the addition of human ascites, the column was washed with 10 volumes of Buffer I. Then, to remove non-specifically adsorbed proteins, 10 volumes of 50mM) Lis-HCl, 1.0M NaCl, 5mM CaCL and 0.
The column was washed with 05% NaN5 (pH 8,0). The column was then re-equilibrated with 10 volumes of buffer ■, followed by 50mM) Lis-HCl, 150mM Na
C1, 10mM EDTA and 0.05% NaN
Human CRP was eluted at pH 8,0. The elution fractions were collected and human CRP was measured using the aforementioned latex agglutination reagent for measuring human CRP (Elpia Ace CRP, manufactured by Yatron), and the recovered amount was determined to be 25.5. From this, it was found that 1.7 μm of human CRP was bound to 1 i of gel packed in the affinity column. The elution pattern of the above experiment is shown in FIG. In Figure 1, A is the non-adsorbed fraction and B is the human CR
P, and a - d are as follows. a: 50mM Tris-HCl, 150mM NaCl,5
mMCaC1z, 0.05% NaN5; pH 8
, 0, b: 50mM) Lis-HCl, 1.0M NaCl, 5m
MCa C1t, 0. 05% NaN5; pH8.0
, c: 50mM) Lis-HCl, 150mM NaCl,5
mMCaC1*, 0.05% = 38-NaNg; pH 8,0, d: 50mM) Lis-HCl, 150mM NaC1,1
0mM EDTA, 0. 05% NaN5: pH8
, 0° (2) Recovery rate The gel for which the bound amount was determined was again packed into another column (diameter 1.0 cm x length 2.0 ann, total volume 1.577 nI) and re-equilibrated with buffer I. Human CRP-positive human ascites, which had been pretreated as described above, was added to the column to reach 80% of the saturated binding amount (approximately 2.1 cm as human CRP). The adsorbed human CRP was eluted in the same manner as above and measured using a latex agglutination reagent for measuring human CRP (Elpia Ace CRP) to determine the recovery rate. The same operation was repeated five times using the same column to determine the recovery rate, and as shown in Table 4, the recovery rate was 90% or more in all cases. [Margin below] (3) Purity test 200 ng of each human CRP obtained in the 5 repeated experiments described in Example 3 (2) above was subjected to 5DS-PAGE (7.5%
After fractionation on ~20% gradient gel), silver staining (
(using a silver staining kit manufactured by Kanto Kagaku). Second result
As shown in the figure. As is clear from Figure 2, both human C
RP was also reduced with 2-mercaptoethanol26
Kd, a single band of 22Kd was observed under non-reduction. Furthermore, in analysis using high performance liquid chromatography (manufactured by JASCO Corporation; column used was Asahipak G5-520), a single sharp peak was observed at a molecular weight of 110 Kd. (4) Immunological specificity Each human CRP obtained by the five operations described in Example 3 (2) above was prepared using veronal buffer (pH 8.6: ionic strength 0) using 1.5% agarose as a support. In immunoelectrophoresis using anti-normal human whole serum rabbit antibody (manufactured by DAKO: A206), no precipitation line was formed with anti-human CRP rabbit antibody (manufactured by DAKO; AO73). A single sedimentation line was formed. In addition, human CRP obtained in the fifth column operation described in (2) above was administered to rabbits in New Zealand white three times every two weeks with Freund's complete adjuvant in an amount of 0.5 μ per time. In the same immunoelectrophoresis as described above, the antiserum obtained by immunizing a female (female) did not form a precipitation line with normal human serum, but formed a single precipitation line with human CRP and CRP-positive human serum used as an immunogen. was formed. From the above experiments, it was confirmed that human CRP recovered and purified by monoclonal antibody CRPB-2 immobilized affinity chromatography was a pure product. In addition, the hybridomas CRPB-2 and CR according to the present invention
PB-3 and CRPB-5 were introduced in 19901, respectively.
It was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology on October 2.3B. The accession number is that the hybridoma CRPB-2 is FERM P-11785.
), the hybridoma CRPB-3 was published in the 117th
No. 86 (FERM P-11786), and hybridoma CRPB-5 was published No. 11787 (FERM P-11786).
ERM P-11787).

【Effect of the invention】

Monoclonal antibodies of the present invention CRPB-2, CRPB-
3 and CRPB-5 can recognize conformational changes caused by binding of calcium or magnesium to human CRP. Furthermore, each of these reactivities is not inhibited even in the presence of phosphorylcholine. Furthermore, monoclonal antibody CRPB-2 can cause an in-gel precipitation reaction. Furthermore, the monoclonal antibodies CRPB-2, CR
By using affinity chromatography using PB-3 and/or CRPB-5, highly pure human CRP can be obtained from human ascites in just one step, and the recovery rate is also as high as 90% or more. Also, since mild elution conditions using a chelating agent in the neutral range can be used, human CRP and the ligand monoclonal antibodies CRPB-2, CRPB-3, and CRPB-5 will not be denatured or degraded. Therefore, the affinity column of the present invention can be reused for a long period of time.

[Brief explanation of drawings]

FIG. 1 is a graph showing the elution pattern when CRP-positive human ascites was applied to the monoclonal antibody CRPB-2-immobilized affinity column of the present invention and human CRP was eluted. FIG. 2 is an explanatory diagram showing the results of 5DS-polyacrylamide gel electrophoresis of human CRP obtained through repeated experiments five times using the monoclonal antibody CRPB-2-immobilized affinity column of the present invention. Patent applicant Yatron Co., Ltd. Patent application agent Patent attorney Mori 1) Ken

Claims (3)

[Claims] (1) A monoclonal antibody that exhibits calcium- or magnesium-dependent reactivity against human C-reactive protein. (2) Formed by fusion of mammalian spleen cells immunized with human C-reactive protein and mammalian myeloma cells, secreting monoclonal antibodies that exhibit calcium- or magnesium-dependent reactivity against human C-reactive protein. hybridomas or cell lines derived from them. (3) A method for recovering or purifying human C-reactive protein, which comprises using affinity chromatography using a monoclonal antibody that exhibits calcium- or magnesium-dependent reactivity toward human C-reactive protein.