JPH0359382B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an aqueous solvent for an antibody detection reagent. As reagents for detecting antibodies, the hemagglutination method, radioimmunoassay method, enzyme antibody method, etc. are sensitive methods and are used in the field of clinical testing. For example, in the hemagglutination reaction method, sheep red blood cells are sensitized using a purified antigen (for example, HBs antigen) as a carrier for an agglutination test to obtain antigen-sensitized sheep red blood cells, which are then mixed with a blood sample. Depending on whether or not an agglutination reaction is observed in an aqueous medium, it can sometimes be determined whether or not an antibody corresponding to the antigen is present in the sample. In addition, if an agglutination reaction is observed, it is better to further dilute the sample in stages to determine the dilution ratio at which an agglutination reaction can no longer be observed.
HBs antibodies can be measured quantitatively. In the radioimmunoassay method or the enzyme-antibody method, antigens are immobilized on polystyrene balls, filter paper, etc., and this is mixed with a sample.After reacting with the antigen labeled with a radioisotope or a certain type of enzyme, By measuring isotope activity or enzyme activity, it can be determined whether or not an antibody corresponding to the antigen is present in the sample. Antigens used in the above reaction reagents have conventionally been extracted and purified from animal blood or tissue. for example,
HBsAg and HBcAg are purified from the serum of HBsAg positive individuals by ammonium sulfate fractionation and ultracentrifugation.
HBc antigens are extracted from liver tissue of hepatitis B patients, but with the recent development of genetic recombination technology, it has become possible to produce these antigens in bacteria such as Escherichia coli and yeast. However, when the present inventors tested antibodies in human serum using an antibody detection reagent using the antigen obtained by this method, some purified antigens showed non-specific reactions. I discovered something. Therefore, it is an object of the present invention to provide an antibody detection reagent that does not cause false positives due to the non-specific reaction even when antigens produced from bacteria based on genetic recombination technology are used. This is a new technical challenge. In order to solve this problem, the present inventors have conducted further research and found that antibodies against the bacteria may exist in human or animal serum, and that antibodies against the bacteria may exist in trace amounts in purified antigens. It was discovered that a non-specific reaction occurs due to reaction with remaining bacterial components. It has also been found that this non-specific reaction is neutralized by bacteria that cannot produce the above-mentioned antigen (ie, host bacteria that have not been genetically modified). The present invention has been completed based on this new knowledge, and is an aqueous extract of bacterial components that can pass through a filter with a diameter of 0.8 microns (the bacteria can be a host in genetic recombination technology).
An aqueous solvent for an antibody detection reagent based on an immune reaction, characterized by comprising: Bacteria that can serve as hosts for the genetic recombination technology used in the present invention include Escherichia coli,
Subtilis blight, yeast, etc. are used as examples, and those that have not been genetically modified (therefore, do not have antigen-producing ability) are used; It is preferable to use bacteria of the same species as those used for production. The bacteria are provided in the present invention as an aqueous extract of a size that can pass through a filter with a maximum diameter of 0.8 microns. When such a size is used, the agglutination reaction product between the bacterial component aqueous extract and the antibody against the bacteria in the sample serum is minute, and does not pose any problem in the intended test. Aqueous extracts of bacterial components of such size can be prepared by, for example, adding bacteria to water (e.g., physiological saline) and treating it with sonication (e.g., 5 ml of water).
The supernatant obtained by centrifugation (for example, 5000 to 15000 rpm for 15 to 60 minutes) is further centrifuged to the maximum diameter. Separation is achieved, such as by passing through a 0.8 micron filter. The bacterial component water extract contains 0.001 to 0.3% (w/
v), preferably in a proportion of 0.01 to 0.1% (w/v) in the aqueous solvent. As the aqueous solvent, those known in this field are used, such as low salt concentration buffers with pH 6 to 8, such as water, physiological saline, various buffers (phosphate buffer, borate buffer, glycine buffer). liquid), albumin solution, dextran solution,
Examples include normal human and animal serum solutions, solutions of synthetic polymer substances, surfactant-containing aqueous solutions, and solutions consisting of combinations thereof. of the aqueous solvent
The pH is preferably about 6.0 to 8.5, and adjustment to such pH is preferably performed with a buffer. The salt concentration in the aqueous solvent is preferably relatively low;
More preferably, it is a physiologically isotonic solution. The aqueous solvent of the present invention is used as a solvent for an antibody detection reagent based on an immune reaction (for example, a reagent based on an agglutination reaction method, a radioimmunoassay method, an enzyme antibody method, etc.). By using the aqueous solvent of the present invention, a unique effect can be obtained in that false positives do not occur due to non-specific reactions even in antibody detection test drugs based on immune reactions using antigens produced by genetic recombination technology. It will be done. The present invention will be explained in more detail below based on experimental examples. Ammonium sulfate fractionation method from Escherichia coli that produces HBc antigen,
Purified HBc antigen was obtained using a general gel filtration method such as ion exchanger adsorption method. Separately, E. coli that does not produce HBc antigen was cultured, 5 times the amount of physiological saline solution was added to the resulting bacterial cells, and the mixture was sonicated with 10Kc sound waves for 30 minutes at a cold temperature (2℃ to 15℃), and further Centrifugation was performed at 10,000 rpm for 30 minutes using a centrifugal separator (Rotor No. 9) manufactured by the company, and an aqueous bacterial component extract was obtained. Add this extract to 0.8μ
It was clarified using a membrane. 0.01% protein in bacterial extract in physiological saline solution (PH7.0)
(w/v) concentration, and further add 0.5% (w/v) bovine albumin and sodium azide.
In addition to the 0.1% (w/v) concentration, an aqueous solvent for HBc antibody detection was obtained. Glutaraldehyde-fixed sheep red blood cells were sensitized with the thus obtained purified HBc antigen and bacterial component aqueous extract to obtain HBc antigen-sensitized sheep red blood cells and bacterial component-sensitized sheep red blood cells. Both sensitized sheep red blood cells were suspended in a phosphate buffer (0.15M, PH7.2, supplemented with 0.5% bovine albumin and sheep red blood cell stroma, hereinafter abbreviated as PBS) to a concentration of 0.5%. 0.1% (w/v) bacterial component water extract in acid buffer
added to the concentration. (Hereinafter abbreviated as PBS containing bacterial aqueous extract.) The reactivity of 11 human serum samples to the above-mentioned sensitized blood cells was examined by the microtitration method. At the same time, HBC antibodies were tested using a different method, and 6 of the 11 samples were positive for HBc antibodies.

【table】

[Table] As shown in Table 1, specimens No. 1 to 6 were positive for HBc antibody, and specimens No. 7 to 11 were negative. No.1, 3, 4,
5, 7, 8, and 9 showed a strong reaction with bacterial component-sensitized blood cells, but when this reaction was measured with PBS containing an aqueous bacterial extract that does not contain HBc antigen, the agglutination titer was less than that measured with PBS. It was suppressed to 1/16 or less. Of the HBc antibody-negative specimens No. 7 to 11, 7, 8, and 9 also reacted with HBc antigen-sensitized blood cells. However, this reaction was suppressed to 1:8 to 1:4 by measuring with PBS containing bacterial aqueous extract. HBc antibody-positive specimens No. 1 to 6 showed a strong reaction with HBc antigen-sensitized blood cells, and this reaction was measured with both PBS and PBS containing bacterial aqueous extract and agreed within the margin of error. As mentioned above, antibodies against E. coli body components may exist in human serum, and this antibody reacts with blood cells sensitized with HBc antigen extracted and purified from E. coli. This can be suppressed by using a solvent to which the extract is added, and furthermore, this bacterial cell component extract is used for the purpose
It became clear that this did not affect the measurement of HBc antibodies. Therefore, it was found that HBc antibodies could be tested more specifically by using a solvent to which this bacterial component aqueous extract was added. This solvent is also used when testing HBc antibodies.
It was effective in suppressing non-specific antibody reactions. Example 1 5 times the amount of physiological saline solution was added to the cells obtained by culturing E. coli that does not produce HBc antigen, and the cells were sonicated for 30 minutes with a 10 kc sound wave at a cold temperature (2°C to 15°C). Tomy Seiko centrifuge (rotor No.
9), centrifuge at 10,000 rpm for 30 minutes,
A water extract of bacterial components was obtained. This extract was clarified using a 0.8μ membrane. Physiological saline solution (PH
7.0) contains 0.01% (w/v) protein of bacterial cell component extract.
Mix to a concentrated consistency, then add bovine albumin.
0.5% (w/v), sodium azide 0.1%
(w/v) concentration as well as an aqueous solvent for HBc antibody detection. Example 2 The bacterial cell component extract obtained in Example 1 was added to a phosphate buffer (0.15M, PH7.2) to a concentration of 0.3%, and sheep red blood cell stroma was added to a concentration of 0.1% (w/
v), polyethylene glycol 4000 0.2% (w/
v) and Udialbumin were added to a concentration of 0.2% (w/v).

Claims (1)

[Claims] 1. In the detection of antibodies that react with antigens produced by genetic recombination technology, bacteria that can serve as hosts in the genetic recombination technology and that can pass through a 0.8 micron diameter filter of the same type of bacteria as the bacteria used to produce the antigen. An aqueous solvent for an antibody detection reagent based on an immune reaction, characterized by containing a component water extract.