JPH0799372B2 - Non-specific reaction absorbent for reverse passive agglutination - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention improves the reverse passive agglutination reaction for measuring a specific antigen present in human blood or the like.

[Conventional technology]

The reverse passive agglutination reaction is a method in which an antibody against an antigen to be measured is bound to carrier particles such as red blood cells, and the antigen is measured using a reaction in which the particles agglutinate due to the presence of the antigen. Polyclonal antibodies obtained by immunizing rabbits, goats, etc. have been used.

However, in order to obtain a polyclonal antibody, a large amount of the antigen is used as an immunogen, and the antigen is also used in the affinity chromatography column in the step of purifying the polyclonal antibody. Cost was a problem. Polyclonal antibodies also have the problem of individual differences among immunized animals, further denaturation in the purification step such as affinity chromatography, and the problem of product lot differences, as well as the problem of antibody activity reduction or variation. These not only increase the burden of product control or process control, but also lower the detection sensitivity because the amount of antibody sensitized to carrier particles is limited to the particle area.

On the other hand, as a method for solving these problems, a method utilizing a monoclonal antibody as an antibody has been developed (JP-A-57-86051, JP-A-57-118159 and JP-A-58-127167). Gazette).

[Problems to be solved by the invention]

However, when this monoclonal antibody is used, a non-specific reaction frequently occurs, so that this method has not been put to practical use.

For example, when a conventional serum-diluting solution or the like is used in measuring human α-fetoprotein by sensitizing blood cells with a mouse monoclonal antibody, it is 20 to 50% in healthy human serum and 50 in rheumatism patient serum. It has been confirmed by the present inventors' experiments that non-specific reactions occur at a frequency of ˜90%.

[Means for solving problems]

The present inventor has conducted extensive studies to solve the above problems and put into practical use a method using a monoclonal antibody having various advantages. As a result, an antibody against an antigen different from the measurement target is absorbed by this nonspecific reaction absorption. The inventors have found that they are extremely excellent as agents and have completed the present invention.

That is, the present invention is derived from an animal of the same species as the monoclonal antibody sensitized to carrier particles that undergo agglutination,
The present invention relates to a non-specific reaction absorbent for reverse passive agglutination reaction using a monoclonal antibody, which comprises a monoclonal antibody against an antigen different from the antigen to be measured.

The monoclonal antibody used for the non-specific reaction absorbent of the present invention (hereinafter, referred to as “the monoclonal antibody of the present invention”) is a monoclonal antibody sensitized to carrier particles that undergo agglutination reaction (hereinafter, “sensitized monoclonal antibody”). It is derived from an animal of the same species as "antibody".). This means that if the sensitized monoclonal antibody is of mouse origin, the monoclonal antibody of the present invention must also be of mouse origin. The animals include rats, humans and the like in addition to mice, and the type thereof is not particularly limited.

Next, the monoclonal antibody of the present invention is directed to an antigen different from the antigen to be measured. The type of antigen is not particularly limited, and examples thereof include α-fetoprotein (AFD) carcinoembryonic antigen (CEA) immunoglobulin (IgE, IgA, etc.), albumin, plasma proteins such as ferritin, HB ₈ antigen, herpesvirus, etc. Viral antigens, fibrin degradation products, prostaglandins, testosterone, brogesterone, thyroxine and other hormones, digoxin, theophylline, phenobarbital, phenytoin, penicillin, amikacin, various organs such as drugs, antigens present in blood or urine, etc. And so on.

Although the antigen specificity is not clear, a myeloma-derived monoclonal antibody is also included in the monoclonal antibody of the present invention.

Such a monoclonal antibody can be obtained according to a general method for producing a monoclonal antibody. For example, any antigen different from the antigen to be measured is injected into the abdominal cavity of the mouse several times together with an adjuvant, and spleen cells are taken out and fused with mouse myeloma cells using polyethylene glycol or the like. Then, among the fused cells, those producing the antibody are proliferated as monoclonal cells by cloning, and proliferated in mouse abdominal cavity.

The propagated monoclonal cells produce a large amount of a single antibody, that is, a monoclonal antibody in the peritoneal cavity and accumulate in the ascites. It is found in the case of polyclonal antibody from this ascites by gel chromatography, ion exchange chromatography and the like. The globulin fraction, that is, the monoclonal antibody is purified with good reproducibility without problems such as lot difference of individual difference products of immunized animals and reduction of antibody activity.

The monoclonal antibody of the present invention thus obtained can be sufficiently used as a non-specific reaction absorbent as it is, but when the rheumatoid factor or the like is contained in the sample, it can be treated by denaturing the non-specific reaction absorber. The specific reaction absorption capacity can be increased.

As the denaturing treatment, known denaturing treatments for polyclonal antibodies, for example, heat treatment, acid treatment, dimethyl sulfoxide treatment and the like can be used. The degree of treatment is preferably stronger than generally known conditions, for example, heat treatment at 60 to 65 ° C for about 1 to 2 hours, and acid treatment at pH2.
3 to 2.8 is suitable for 1 to 2 days, and in the case of dimethyl sulfoxide treatment, 7 to 8 M is suitable for 30 minutes to 1 hour.

It is convenient to add the monoclonal antibody of the present invention to a serum dilution solution before use. When the sample is normal human serum, the amount added is 1 to 10 μg / ml, usually 2 μg / ml.
If the sample is serum of a rheumatism patient, it may be about 5 to 500 μg / ml of a modified product, or 10 to 100 μg depending on the method of denaturation.
/ ml is enough. This monoclonal antibody may be used alone or in combination of two or more. In the case of two or more kinds, the addition amount may be the above amount in total. On the other hand, this monoclonal antibody may be contacted with the sample separately from the serum dilution solution. In that case, the sample and the sensitized monoclonal antibody are allowed to act on the sample before or during the reaction.

The reverse passive agglutination reaction to which the non-specific reaction absorbent of the present invention is applied does not matter as long as the antibody sensitized to the carrier particles is a monoclonal antibody, and the antigen to be measured includes, for example, the above-mentioned antigen. Carrier particles for sensitizing monoclonal antibodies include erythrocytes such as sheep and chicken (including those fixed with formalin), microbial carriers (Japanese Patent Publication No. 50-2730, Japanese Patent Publication No. 52-48168, etc.), gelatin particles. (Japanese Patent Application Laid-Open No. 57-153658, etc.), synthetic polymer particles such as latex, bentonite, kaolin, etc. may be used so long as they can be used in the agglutination reaction based on the antigen-antibody reaction.

In the measurement reagent kit, a serum diluting solution usually containing the sensitized particles and the monoclonal antibody of the present invention, a reconstitution solution,
Control particles, positive serum for control, particles for absorbing non-specific reaction, etc. are included.

[Action]

As a result of various studies, the present inventor has come to judge that two types of non-specific reaction factors are present in serum. One of them is called xenoantibody, which also recognizes mouse immunoglobulin (monoclonal antibody) and the like and causes non-specific reaction. The other is rheumatoid factor, that is, an antiglobulin factor present in the serum of patients with rheumatism.
It is considered that the monoclonal antibody of the present invention reacts with these factors to prevent nonspecific reaction.

〔Example〕

Example 1 An antigen (human AFP CEA HB ₈ human I was prepared by a known cell fusion method.
The resulting lymphocytes and myeloma cells were fused with polyethylene glycol or the like to establish monoclonal antibody-producing hybridomas, and a large amount of monoclonal antibodies were obtained.

Monoclonal antibody does not react with AFP (anti-CEA, anti-HB _8, anti-human IgE monoclonal antibodies, etc.) was adjusted to 1-2 mg / ml concentration was 1.5 hours of heat treatment at 63 ° C..

Disodium phosphate dodecahydrate 19.2053g Potassium phosphate 2.9089g NaCl 4.383g Healthy rabbit serum (NRS) 10ml NaN ₃ 1.5g Distilled water 1000ml The above monoclonal antibody heat-denatured to a solution of the composition shown in the table above Was added to give a predetermined concentration to prepare a diluting solution.

According to a known method, chicken erythrocytes were fixed with formalin and then treated with a tannic acid solution diluted 100,000 times with PBS. 1 volume part of this 5% PBS suspension of erythrocytes and 1 volume part of 30 μg / ml concentration PBS solution mixed with three kinds of anti-human AFP monoclonal antibodies having different antigen recognition sites (different specificity) were mixed, and the mixture was heated to 37 ° C. The monoclonal antibody was adsorbed on the surface of the red blood cells for 30 minutes. Then, erythrocytes were collected by centrifugation at 3000 rpm for 10 minutes, and washed with PBS three times by centrifugation.

By this operation, monoclonal antibody-adsorbed blood cells were prepared.

The above-mentioned diluent was used to examine the reaction between anti-AFP monoclonal antibody-sensitized red blood cells and rheumatism patient serum.

Rheumatism patient serum was tested in advance using another test method (RIA method,
It was confirmed by EIA method) that the AFP value was 5 ng / ml or less.

The reaction with the serum of rheumatism patients was performed by the microplate method. The operation method is shown below.

First, dilute the solution with 50μ in well No.1, and after well No.2.
25 μm was added to each well, and 5 μm of serum was further added to well No. 1. 2 ^{n was} diluted in order from well No. 1 with a diluter (for feeding 25 μ) and left at room temperature for 30 minutes. After 30 minutes, the monoclonal antibody-sensitized hemocyte suspension prepared above was adjusted to a 0.6% cell with a diluting solution, and 25 μl of each was sequentially added from well No. 1. After shaking and stirring, the mixture was allowed to stand at room temperature, and after about one and a half hours, an aggregated image was judged.

The results are shown in the table below.

Only the serum of two rheumatoid patients was shown, but the agglutination titers differed depending on the serum. However, even if the serum has a high agglutination titer, heat-denatured monoclonal antibody
When added to the diluent so that the concentration became 100 μg / ml, the nonspecific agglutination factor could be absorbed at the same time, and the nonspecific agglutination reaction did not occur at all.

On the other hand, when this heat-denatured monoclonal antibody was not added, nonspecific agglutination occurred in 80 to 90% of the serum of rheumatism patients.

Example 2 Monoclonal antibodies other than the anti-AFP monoclonal antibody obtained in Example 1 were adjusted to a concentration of 1 to 2 mg / ml, and dialyzed against 0.1 M glycine / hydrochloric acid (pH 2.3) solution for acid denaturation. After denaturation, the solution was dialyzed with a saline solution to remove the 0.1 M glycine / hydrochloric acid solution.

The above-described acid-denatured monoclonal antibody was added to the same 1% NRS-containing PBS solution as in Example 1 to a predetermined concentration to prepare a diluting solution.

Using this dilution liquid and the same anti-AFP monoclonal antibody-sensitized blood cells as in Example 1, the reaction with the rheumatism patient serum was examined by the microplate method in the same manner as in Example 1,
In the case of any of the monoclonal antibodies, it was possible to remove the non-specific aggregation factor as in Example 1.

Example 3 A non-heat-denatured monoclonal antibody other than the anti-AFP monoclonal antibody obtained in Example 1 was added to 0.15MPBS solution containing 1% NRS having the composition shown in Example 1 to a concentration of 2 μg / ml to prepare a dilution solution. did. Using this dilution liquid and the same anti-AFP monoclonal antibody-sensitized blood cells as in Example 1, the incidence of non-specific agglutination reaction was examined in the serum of healthy persons, and no non-specific agglutination reaction occurred.

On the other hand, the incidence of non-specific agglutination reaction was 20 to 50% when no monoclonal antibody other than the anti-AFP monoclonal antibody was added to the diluent.

〔The invention's effect〕

By using the non-specific reaction absorbent of the present invention, even if the monoclonal antibody is used for the reverse passive agglutination reaction, it is possible to completely prevent the occurrence of the non-specific agglutination reaction not only in the sera of healthy persons but also in the serum of rheumatism patients. The non-specific reaction absorbent of the present invention is practically made possible for the first time by utilizing it for the reverse passive agglutination reaction of a monoclonal antibody, and thereby, the amount of AFP and other generally expensive antigens used is greatly increased. The cost can be reduced by lowering the cost.
Further, since the quality of the monoclonal antibody is stable and there is no problem of denaturation or inactivation in the production stage, the reliability of the measurement result can be increased and the detection sensitivity can be improved. The sensitivity of AFP detection when the conventional polyclonal antibody was used was about 25 to 50 ng / ml, but it could be increased to 5 to 10 ng / ml by using the monoclonal antibody.

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Claims (3)

[Claims] 1. A reverse passive agglutination reaction using a monoclonal antibody, which is derived from an animal of the same species as the monoclonal antibody sensitized to carrier particles that undergo an agglutination reaction and comprises a monoclonal antibody against an antigen different from the antigen to be measured. Non-specific reaction absorbent 2. The non-specific reaction absorbent according to claim 1, wherein a monoclonal antibody against an antigen different from the antigen to be measured is contained in the serum dilution solution. 3. The non-specific reaction absorbent according to claim 1, wherein a monoclonal antibody against an antigen different from the antigen to be measured is treated by protein denaturation treatment.