JPH0419562A - Diluent for immunoassay - Google Patents

Abstract

PURPOSE:To allow preservation at ordinary temp. and autoclave sterilization without rotting and to provide the excellent diluent which suppresses the nonspecific reaction with various kinds of sera and antibodies by using polyvinyl alcohol (PVA). CONSTITUTION:The average degree of polymn. of the PVA is usually 100 to 10,000, more preferably 200 to 3,000. The rate of saponification thereof is usually 50 to 99.9%, more preferably 70 to 99.9%. The concn. at the time of using this PVA as the component in the diluent is usually 0.01 to 20%, more preferably 0.05 to 10%. The PVA is used by being dissolved in a solvent at the time of using the PVA and the solvent includes water, etc. The immunoassay includes enzyme immunoassay, etc. After the antigens to be analyzed are adsorbed to a solid base, such as plastic plate, the blocking agent is adsorbed thereon and the serum or the like diluted by the diluent is added thereto to conjugate the antibodies in the serum with the antigens fixed to the solid base. The liquid prepd. by diluting a labeling antibody to be conjugated is added to this conjugate and further, a substrate is added thereto. The absorbed light, fluorescence and emitted light are measured.

Description

[Detailed Description of the Invention] [Industrial Technology] The present invention relates to a diluent for immunoassays, and more specifically, a diluent for immunoassays that can be stored at room temperature for a long period of time and that suppresses nonspecific adsorption of various serums and antibodies. It concerns diluents.

[Conventional technology]

In recent years, immunoassay methods have become widely used and are applied to human clinical examinations and diagnosis, animal disease diagnosis, and research in many fields. In this immunoassay, such as enzyme immunoassay or radioimmunoassay, antigens, antibodies, etc. are immobilized on a solid support, such as a microplate made of polystyrene, etc. By contacting an analysis sample containing The free binding group and the analyte in the analysis sample bind nonspecifically, which causes measurement errors. As a means to prevent this non-specific binding, in addition to coating the solid support with a blocking agent to saturate the free binding groups, analysis samples such as serum are coated with a substance that suppresses non-specific binding. A method of diluting with a diluent is used.

The latter method uses phosphate buffer (
PBS) to which bovine serum albumin (BSA) has been added, or to which a surfactant has been further added are used. However, even these diluents are not necessarily effective in suppressing nonspecific binding, and BS
A has the disadvantage that it is easily putrefied, requires sterilization such as filter sterilization, and is not suitable for long-term storage at room temperature.

[Problem to be solved by the invention]

Therefore, the present invention provides an excellent diluent that does not spoil, is easy to store over a long period of time, is easy to sterilize, and suppresses nonspecific reactions of various sera and antibodies.

[Means to solve the problem]

As a result of intensive research to solve the above problems, the present inventors discovered that the above problems can be achieved by using polyvinyl alcohol as a diluent for immunoassays, and have completed the present invention. .

The present invention will be explained in detail below.

The polyvinyl alcohol (hereinafter abbreviated as PVA) used in the present invention usually has an average degree of polymerization of 100 to 1.
0000, preferably 200-3000.

The degree of saponification of PVA is usually 50% to 99.9%, preferably 70 to 99.9%. Also, when using this PVA as a component in a diluent, the concentration is usually 0.01
-20%, preferably 0.05-10%. However, as the molecular weight of PVA increases, its solubility in water deteriorates, and in this case, the upper limit of its concentration is set at the saturated solubility.

When this PVA is used, it is used after being dissolved in a solvent, and examples of the solvent include water, phosphate buffered solution, phosphate buffered saline, etc., and the upper limit of the concentration in these solutions is the saturation solubility.

Immunoassay methods include enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, luminescent immunoassay,
Examples include latex aggregation method.

For example, in enzyme immunoassay, fluorescence immunoassay, and luminescence immunoassay, the target antigen is adsorbed onto a solid support such as a plastic plate or plastic beads, and then a blocking agent is adsorbed and diluted with a diluent. The serum to be measured is added, the antibody in the serum is allowed to bind to the antigen immobilized on a solid support, a solution prepared by diluting the labeled antibody that binds to this antibody with a diluent is added, a substrate is added, and light absorption,
Measurements are made by measuring fluorescence, or luminescence.

In the radioimmunoassay method, the antigen of interest is adsorbed onto a plastic plate or plastic beads, then a blocking agent is adsorbed, and the serum to be measured diluted with a serum diluent is added to this to bind the antibody to the antigen, which then binds to the antibody. A solution of a radioisotope-labeled antibody diluted with a diluent is added, and the amount of bound radioisotope is measured. In the latex agglutination method, antigens are adsorbed onto latex fine particles, then a blocking agent is adsorbed, and serum to be measured diluted with a diluent is added thereto, and diagnosis is made by observing changes in agglutination.

Targets for this measurement include tissues, cells, bacteria, molds, mycoplasma, viruses, deoxyribonucleic acids, proteins, polysaccharides, lipids, and various haptens (low-molecular compounds such as antibiotics and hormones) bound to proteins. . As the serum, serum from humans, horses, cows, pigs, sheep, goats, rabbits, chickens, dogs, cats, rats, mice, etc. can be used.

Among these, it is particularly useful for diagnosing diseases using serum from chickens that tend to produce non-specific reactions, such as chicken adenovirus, fowlpox virus, infectious Fabruski's bursal disease virus, infectious bronchitis virus, and infectious rhinotracheitis virus. , avian influenza virus, Marek's disease virus, Newkatus virus, paramyxosis virus, avian reovirus, rotavirus, chlamydia, bordetella, cellular endotheliomatosis virus, avian leukemia respazzella,
mycoplasma, salmonella, chick dysentery, coccidia,
Leukochitozone can be given. In humans, various serological diagnoses, and in cattle diseases such as Ibaraki disease, etc.
It is useful for infectious rhinotracheitis, adenovirus infection, epidemic fever, Akabane disease, bovine viral diarrhea, cowpox, anthrax, swine diseases, swine fever, infectious gastroenteritis, and swine balbovirus.

This diluent is useful not only for diluting serum but also for diluting antibodies and labeled antibodies. The effect of this diluent makes it possible to prevent adsorption of non-specific proteins such as antibodies, allowing for more accurate diagnosis.

〔Effect of the invention〕

According to the present invention, by using PVA, it is possible to provide an excellent diluent for immunoassays that does not spoil, can be stored at room temperature, can be sterilized in an autoclave, and suppresses non-specific reactions of various serums and antibodies. be. By using this PVA, for example, it becomes possible to carry out accurate serology diagnosis.

〔Example〕

Next, examples related to the present invention will be given, but the present invention is not limited to these examples unless it goes against the gist of the present invention.

Example 1 PVA (polymerization degree 500, saponification degree 88%) dissolved in PBS at a concentration of 0.5% was added as a blocking agent to each well of a 96-well microplate (manufactured by Flora Laboratories). It was left at room temperature for 30 minutes. The solution was discarded, and the plate was washed twice with PBS.

PVA shown in Table 1 below was used as an additive in the diluent.

Table 1 Diluents were prepared by dissolving these PVAs in PBS at the concentrations shown in Table 2. Next, dilute female chicken serum 300 times with this diluent, add 50 d of it to each well, and
It was left for 0 minutes. Note that this chicken serum was obtained from the blood of a 22-week-old female chicken that had been administered various vaccines. The liquid was removed from each well and washed three times with PBS. Horseradish perleoxidase-labeled rabbit anti-chicken IgG serum (Miles Laboratories) was diluted 1/2000 with PBS containing 0.7% bovine serum albumin, and 5
07' was added and left for 30 minutes. The liquid was removed and washed six times with PBS containing 0.02% Tween 20. Next, a 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonsan) ammonium salt solution (hereinafter referred to as A
50% of a 1:1 mixture of BTS) and hydrogen peroxide solution.
111 were added to each well and left at room temperature for 1 hour, and the absorbance at 405 nm was measured using a photometer.

The results are shown in Table 2.

Comparative Example 1゜Similar to Example 1, except that PVA was used as the diluent.
The buffer solution, serum albumin, or surfactant shown in Table 2 was added, and the absorbance was measured by the same procedure as in Example 1. The results are shown in Table 2.

The results in Table 2 and above show that sample 3 (0.1%, 0.5%), sample 4 (
0.1%, 0.5%), the absorbance is 0.05 or less,
Other samples showed high absorbance. From these results, it was found that Samples 3 and 4 suppressed non-specific reactions of serum and were preferable as diluents.

On the other hand, when a surfactant or salt is added, the absorbance is 0.
Even serum albumin, which has a high absorbance of 1 or more and has been commonly used in the past, has an absorbance of 0.07 or more, and no preferred diluent has been found.

Example 2 and Reference Example 2 Example 1 and Reference Example 1 were repeated. However, as a blocking agent, 0.7% P of bovine serum albumin was used instead of PVA0.
A BS solution was used. The results are shown in Table 3.

Table 3 Sample 3, sample 4 and sample 5 of the present invention have low absorbance and B
Even when SA is used as a blocking agent, these PVA
was found to be preferable as a diluent because it suppresses non-specific reactions of serum. On the other hand, when albumin, salt, or surfactant is added in the conventional technique, the absorbance is high and the effect of suppressing nonspecific reactions is small.

As is clear from the results of Examples 1 and 2 and Comparative Examples 1 and 2 above, in general, PVA for the diluent of the present invention has a saponification degree of 9 within the range of polymerization degree of 500 to 2000.
Those with a saponification degree of less than 8.5%, such as those with a saponification degree of 95% or less,
For example, a saponification degree of 90% or less is preferably used.

Example 3 Preparation of antigen and immobilization on microplate Mycoplasma gallisepticum C3F7 strain was cultured in modified Channock medium (
CHANOCH medium) for 3 days, and the culture solution was centrifuged (15,000 rpm). The precipitate was suspended in physiological saline and further centrifuged for washing.

This washing operation was performed three times. 5 times concentration of culture solution 1
mj! Sodium dodecylsulfonate (SO
3) Add 1 mj2 of 0.1% PBS solution and heat at 30°C.
Shake for an hour. Then, this liquid was centrifuged (1500
0 rpm) L, supernatant with bacterial concentration 1/2 of culture solution concentration
An antigen solution was prepared by diluting the antigen with a carbonate buffer to a concentration of 0. This antigen solution was added 504' to each well of a 96-well microplate (manufactured by Flora Laboratories), left to stand at room temperature for 2 hours, and the solution was discarded and washed twice with PBS to prepare a plate on which the antigen was immobilized.

Coating of blocking agent As a blocking agent, PVA (degree of polymerization 1000;
A solution of 0.5% (saponification degree 98.5%) dissolved in PBS was added at 20 OI to each well, left at room temperature for 30 minutes, and then the solution was discarded.

Reaction with serum samples: Add 50% of female chicken serum diluted 300 times with various diluents or serum of male chicken immunized with mycoplasma to each well.
4 was added and left for 30 minutes. The serum from this female chicken was obtained from the blood of a 22-week-old female chicken that had been administered various vaccines, and the serum from a chicken immunized with mycoplasma was obtained 5 weeks after the chicks were immunized with mycoplasma-complete adjuvant. It was isolated from blood collected from male chickens. The liquid was removed from each well and washed three times with PBS.

Horseradish peroxidase labeled rabbit anti-chicken IgG serum (
Miles Laboratories, Inc.) was diluted to 1/2000 with PBS containing 0.7% bovine serum albumin, 50111 was added to each well, and the mixture was left for 30 minutes. Except for that wave,
It was washed six times with PBS containing 0.02% Tween 20.

Next, 50 id of a 1:1 mixture of ABTS solution and hydrogen peroxide solution was added to each well and left at room temperature for 1 hour, and then the absorbance at 405 nm was measured using a photometer. The results are shown in Table 4.

Comparative Example 3 The absorbance was measured in the same manner as in Example 2, except that the buffer solution, serum albumin, or surfactant shown in Figure 4 was added instead of PVA as a diluent, and the absorbance was measured in the same manner as in Example 3. .

The results are shown in Table 4.

As a result of Table 4 and above, Sample 3 (0.1%, 0.5%), which was found to be a preferable diluent in Example 1, and Sample 4 (0.1%, 0.
．． 5%), the ratio of the absorbance of the immunized chicken to the absorbance of the unimmunized chicken was as high as about 10, and favorable results were obtained even when the antigen was adsorbed.

On the other hand, for serum albumin, which was relatively effective in Comparative Example 1, the absorbance of immunized chickens and non-immunized chickens showed a high value of about 9, but when surfactant or salt was added, the absorbance ratio was was very small.

Example 4 and Comparative Example 4 Example 3 and Comparative Example 3 were repeated. However, instead of PVA solution, 0.7% of bovine serum albumin was used as a blocking agent.
% PBS solution was used to dilute the serum 100 times, and the absorbance was measured 20 minutes after adding the substrate. The results are shown in Table 5.

As shown in Table 5 and above, Samples 3, 4 and 5, which were found to be preferable diluents in Example 2, had a high ratio of absorbance for immunized chickens to absorbance for non-immunized chickens of about 6, indicating that the antigen Favorable results were also obtained when adsorbed. On the other hand, it was found that when albumin, salt, or surfactant was added, the absorbance ratio was very small, making it undesirable as a diluent.

Example 5 The same PVA as in Example 1 was dissolved in PBS, sealed and allowed to stand at room temperature, and changes in appearance were observed.

All of the PVA used showed no change in appearance even after 6 months. The results are shown in Table 6.

Comparative Example 5 A solution prepared by dissolving the same diluent as in Comparative Example 1 in PBS was sealed and its appearance was observed. After about one month, a precipitate was observed in serum albumin. The results are shown in Table 6.

No. table

Claims (1)

[Claims] 1. A diluent for immunoassay containing polyvinyl alcohol. 2. The polyvinyl alcohol has a degree of polymerization of 200 to 30
00, and a degree of saponification of 75 to 99.9%. 3. The polyvinyl alcohol concentration is 0.01 to 20
%. The diluent for immunoassay according to claim 1.