RU2275635C2 - SERUM PANEL WITH STANDARDIZED CONTENT OF IgG CLASS ANTIBODIES TO p17 AND p41 ANTIGENS OF TREPONEMA PALLADIUM AND METHOD FOR PRODUCTION THEREOF - Google Patents

Abstract

FIELD: biotechnology, immunology, in particular production of serum panels to control quality of test systems for lues serotological diagnosis.

SUBSTANCE: claimed panel contains set of samples with standardized content of IgG class antibodies to T. palladium p17 and p41 antigens: namely sample with prevalent content of antibodies to p17 antigen, samples with prevalent content of antibodies to p41 antigen, samples containing antibodies to p17 antigen only, samples containing antibodies to p41 antigen only, and samples containing mixture antibodies to h17 and p41 in equal concentrations. Claimed method includes sampling of positive serums based on ratio (K) of reverse titers of antibodies to T. palladium antigens with mol.w. of 17 and 41 kD (K = T^-1 _p17/T^-1 _p41) and sampling of original serum samples with K>=2, K<=1|2 and K = 1. Said samples are diluted with non-immune donated serum to produce necessary concentration of specific antibodies.

EFFECT: improved method for examination of test system specificity, sensitivity, and other characteristics.

7 cl, 2 tbl, 6 ex

Description

The invention relates to the field of biotechnology and immunology and can be used in the manufacture of serum panels for quality control of test systems for serological diagnosis of syphilis. The normalized content of antibodies to individual proteins of the syphilis pathogen Treponema pallidum in panel samples allows monitoring of a number of quality indicators of test systems made using recombinant antigens at the stages of their production and use.

Syphilis is an infectious venereal disease characterized by a progressive undulating course in which the stages of clinical manifestations (primary syphilis of the genital organs, secondary syphilis of the skin and mucous membranes, etc.) are replaced by asymptomatic periods (latent syphilis). More than half of cases of syphilis occur in a latent, asymptomatic form and are detected only by serological examination of patients. In such conditions, effective laboratory diagnosis of this disease is of great importance.

Laboratory diagnosis of syphilis is based on the detection of nonspecific (reagins) or specific (IgG, IgM) antibodies in the patient's serum (plasma). The profiles of these antibodies are complex and vary significantly depending on the stage of the disease. In the complex of serological reactions used to diagnose syphilis, enzyme-linked immunosorbent assay (ELISA) using recombinant proteins containing specific antigenic determinants of T. Pallidum has become increasingly important in recent years. To date, ELISA systems have been developed for the detection of syphilitic IgG antibodies, IgM antibodies and total (IgG + IgM) antibodies, and in the last two modifications, recombinant antigens are used not only on substrates, but also as an immunoperoxidase conjugate. The sensitivity and specificity of the diagnosis depend to a large extent on what antigens, what quality, in what quantity and in the ratio are used in the test system [1-3].

In the production of test systems are used mainly antigens with a pier. mass of 17 and 41 kDa. Studies have shown that sera from the stage of primary syphilis are characterized by a significant predominance of antibodies to the p41 antigen, and for sera from the stage of latent syphilis - to the p17 antigen. At the stage of secondary syphilis, the ratio of antibody titers to these antigens is diverse. Moreover, at all stages of syphilis, serums with both high and extremely low levels of antibodies are detected. The presence of five fundamentally different situations of the ratio of antibody titers to the indicated antigens was experimentally confirmed:

1. only antibodies to the p41 antigen are detected in serum;

2. in the serum, antibodies to both antigens are determined, but the level of antibodies to p41 antigen is much higher than the level of antibodies to p17 antigen;

3. in the serum, the titers of antibodies to the p17 and p41 proteins are identical or similar in value;

4. in the serum, antibodies to both antigens are determined, but the level of antibodies to p17 antigen is much higher than the level of antibodies to p41 antigen;

5. only antibodies to the p17 antigen are detected in serum.

Obviously, test systems designed to diagnose syphilis should identify all of the above situations. For this, it is necessary to have tablets sensitized with a balanced mixture of p17 and p41 antigens, and in the case of test systems, a balanced mixture of conjugates of these antigens with peroxidase is also needed to detect total antibodies.

The quality of the diagnostic kits is checked on a specific set (panel) of sera. Currently, there are no officially approved domestic panels of syphilitic serums. Foreign analogues are unknown. Manufacturers and consumers of test systems usually use their panels, the selection of sera in which is mostly random and, at best, takes into account the stage of the disease and the sex of the donor.

The Vector-Best production panel is known, however, this panel also represents a blind set of high-volume sera, neither the characteristics of the panel samples nor the criteria for their selection are known [4].

Such a panel cannot provide a sufficiently complete analysis of sera.

The closest method of manufacturing a panel (prototype) is a method of obtaining a standard positive panel of sera for quality control of enzyme-linked immunosorbent assay systems and immunoblots [5, prototype]. The method includes preparing immune sera containing different concentrations of specific antibodies to all major proteins of the test antigen by diluting native immune sera with non-immune donor serum. Moreover, the largest number of panel sera is prepared with antibody concentrations close to their minimum concentration in these sera, which serves as the boundary between positive and negative test sera, and the smallest number of panel sera is prepared with antibody concentrations close to their concentration in native sera.

Unlike the prototype, the present invention involves the targeted selection of sera, the criteria of which is the ratio (K) in the sera of the reverse titers of antibodies to antigens p17 and p41 (K = T ^-1 _p17 / T ^-1 _p41 ).

An object of the invention is to obtain a panel of serums that provides control of the parameters of test systems during their production and operation, as a means of comparing experimental data obtained by different researchers on different test systems and at different times.

The problem is solved by obtaining and using a panel of sera with a normalized content of IgG class antibodies to p17 and p41 antigens Treponema pallidum.

The present invention allows to implement a new approach to the design of panels as an effective tool for monitoring a number of parameters of test systems during their production and operation. Unlike existing panels, the present invention contemplates incorporating into the panel a set of sera reproducing all the situations described above for ratios of antibody titers to p17 and p41 antigens. Such a panel allows consumers to assess the sensitivity and specificity of the test system, and manufacturers, in addition, provides differential control of the adequacy of the loading of tablets with antigens and the optimal ratio of antigens in the “total” conjugate.

The selection of sera is carried out by titration on tablets sensitized with individual antigens (p17 or p41) in a saturating concentration. Source sera are selected:

- serum No. 1 (from the primary or secondary stage of syphilis) with K <1/2;

- serum No. 2 (with a hidden stage of syphilis) with K> 2;

- serum No. 3 (from the primary, secondary or latent stage of syphilis) with K = 1;

- several samples of donor sera that do not contain antibodies to Tr.Pallidum (negative samples);

- donor serum or a mixture of serums that do not contain antibodies to Tr.Pallidum for the preparation of a dilution solution.

Serum No. 1 and 2 are triturated in a diluting solution and the titers of antibodies to individual antigens are determined in the obtained samples in the same way as at the stage of selection of initial sera. At least 2 samples are selected from each sera of the initial sera in such a way that antibodies to one and the other antigen are detected in the first sample, and only the most represented antibodies (antibodies to only one antigen) with an optical signal 1.5 times higher are determined in the second sample OPcrit.

Serum No. 3 is triturated in a dilution solution and the titers of antibodies to individual antigens are determined in the obtained samples in the same manner as at the stage of selection of initial sera. At least 2 samples are taken from the screening so that in the first sample, antibodies to both antigens are detected in a titer of not less than 1/20, and in the second one in a titer of not more than 1:10 with an optical signal 1.5 times higher OPcrit.

Correction of the optical signal of the samples, if necessary, is done by adding the initial positive serum or diluting solution.

To ensure the reliability of the control, the panel includes two or more positive samples of each type obtained from different initial sera from different stages of the disease.

One negative sample is prepared from each donor serum.

All selected positive and negative serum samples are further purified by centrifugation or filtration and stabilized by the addition of known preservatives or stabilizing mixtures, poured into vials and lyophilized.

Example

According to the method described above, a panel is made comprising 12 positive and 6 negative sera. When evaluating the initial sera and diluted samples, solutions for washing, diluting sera and conjugate, as well as substrates based on RPD and TMB from the diagnostic sets “SIF-DSM-IgG” (CJSC “Biomedical Union”, Berdsk) are used. The characteristics of the initial sera and panel samples are given in table 1.

Table 1
Characteristics of the original sera and panel samples Panel No. Source Serum Panel Samples Floor Syphilis stage Antibody titer Breeding ratio OP ₄₉₂ on tablets with antigens p17 p41 p17 p41 p17 + p41 one m hidden 1:80 1:20 2 2,242 0.885 2,050 2 5 1,224 0.124 0.934 3 well hidden 1: 640 1: 160 16 1,932 0.424 1,546 four 40 0.731 0,095 0.634 5 m secondary 1: 320 1: 640 thirty 1,483 1,996 1,802 6 45 0.215 0.968 0.845 7 well primary 1:20 1:80 2 0.652 1,654 1,477 8 5 0.253 0,903 0.763 9 m secondary 1: 640 1: 640 25 1,412 1,218 1,762 10 fifty 0.611 0.765 0.801 eleven well primary 1: 320 1: 320 10 1,284 1,168 1,414 12 25 0.586 0.832 0.710 13 well Negative Donor Sera 0 0 0 0.124 0.142 0.154 fourteen well 0 0 0 0.123 0.132 0.152 fifteen m 0 0 0 0.119 0.125 0.108 16 well 0 0 0 0.136 0.118 0.146 17 m 0 0 0 0.128 0,112 0,112 eighteen m 0 0 0 0.115 0.137 0.125

The panel is used to evaluate plates sensitized with sorption mixtures containing different ratios of p17 and p41 antigens, as well as conjugates for determining total antibodies containing different ratios of p17 and p41 antigens conjugated with peroxidase.

Example 1 - The tablets are sensitized with a sorption mixture with a predominance of p17 antigen, the conjugate is a / IgG-Hum-HRP.

Example 2 - The tablets are sensitized with a sorption mixture with a predominance of p41 antigen, the conjugate is a / IgG-Hum-HRP.

Example 3 - The tablets are sensitized with a sorption mixture with the optimal ratio of antigens p17 and p41, the conjugate is a / IgG-Hum-HRP.

Example 4 - The tablet is the same as in example 3, an antigenic conjugate with a predominance of protein p17.

Example 5 - The tablet is the same as in example 3, antigenic conjugate with a predominance of protein p41.

Example 6 - The tablet is the same as in example 3, an antigenic conjugate with an optimal ratio of proteins p17 and p41.

The evaluation results are given in table.2.

table 2
Examples of application of the panel to assess the balance of the sorption mixture for sensitization of tablets and antigenic conjugate No. Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 one 2,162 1,182 2,050 0.963 0.657 1,026 2 1,145 0.228 0.934 0.421 0.220 0.439 3 1,835 0.634 1,546 0.740 0.446 0.875 four 0.712 0.236 0.634 0.419 0.216 0.334 5 1,238 1,874 1,802 0.654 0.763 0.989 6 0.267 0.768 0.845 0.233 0.432 0.554 7 0.716 1,439 1,477 0.441 0.541 0.767 8 0.271 0.812 0.763 0.230 0.430 0.581 9 1,324 1,118 1,762 0.655 0.657 0.816 10 0.587 0.645 0.801 0.438 0.434 0.534 eleven 1,196 1,234 1,414 0.561 0.762 0.923 12 0.674 0.712 0.710 0.432 0.429 0.577 13 0.118 0.136 0.154 0,078 0,074 0,092 fourteen 0.121 0.128 0.152 0,084 0,078 0.105 fifteen 0.129 0.123 0.108 0,092 0,083 0.103 16 0.114 0.125 0.146 0,089 0,076 0.133 17 0.118 0.118 0,112 0,086 0,083 0,092 eighteen 0.135 0.129 0.125 0,095 0.102 0.106 Opcrit 0,300 0,300 0,300 0.280 0.280 0.280

Using the panel described above allows you to evaluate:

1. the health of the test system,

2. sensitivity of the test system,

3. the specificity of the test system,

4. optimality of the ratio of antigens on sensitized tablets,

5. the optimal ratio of antigens in the conjugate to detect total antibodies,

6. stability of the test system during storage, stability of individual proteins on the tablets and in the antigenic conjugate during storage.

BIBLIOGRAPHY

1. Japanese application No. 9304391 "Immunological Treponema pallidum diagnostic reagent and its manufacture" G 01 N 33/571.

2. US patent 6479248 "Process and agent for detecting antibodies against Treponema pallidum" G 01 N 33/53.

3. RF patent “Kit for the detection of antibodies to pale spirochete Treponema pallidum, recombinant polypeptide antigen Treponema pallidum 15 kDa, recombinant polypeptide antigen Treponema pallidum 17 kDa and recombinant polypeptide antigen Treponema pallidum TMP A31, C 12 N N.

4. An enzyme-linked immunosorbent assay system for detecting class G antibodies to Treponema pallidum using recombinant proteins FSP 42-0117-0281-00 “Recombest Anti-Pallidum”.

5. RF patent No. 2094807 "Method for producing a standard positive panel of sera for quality control of enzyme-linked immunosorbent assay systems and immunoblots", G 01 N 33/96, 33/53.

Claims (7)

1. The panel of serums for quality control of test systems for serodiagnosis of syphilis, characterized in that it contains a set of samples with normalized content of IgG antibodies to p17 and p41 antigens T. pallidum: a) samples with a predominance of antibodies to p17 antigen; b) samples containing antibodies only to p17 antigen; c) samples with a predominance of antibodies to the p41 antigen; d) samples containing only antibodies to the p41 antigen; e) samples containing a mixture of antibodies to p17 and p41 antigens in equal concentrations. 2. The serum panel according to claim 1, characterized in that it further includes positive samples made from two or more source sera having similar ratios of antibody titers. 3. The serum panel according to claims 1 and 2, characterized in that it additionally includes several negative samples made from sera that do not contain antibodies to T. pallidum. 4. A method of obtaining a panel of sera according to claims 1 to 3, characterized in that the selection of positive sera is carried out by determining the ratio (K) of the reverse titers of antibodies to T. pallidum antigens with mol. mass of 17 and 41 kDa (K = T ^-1 p ₁₇ / T ^-1 _p41 ), while the initial samples of sera having the ratios K≥2, K≤1 / 2 and K = 1 are taken and diluted with non-immune donor serum to obtaining the necessary concentrations of specific antibodies. 5. The method according to claim 4, characterized in that the determination of concentrations (titers) of antibodies to p17 and p41 antigens is carried out on ELISA test systems using tablets sensitized with individual recombinant p17 or p41 antigens. 6. The method according to claim 4, characterized in that when diluting sera with ratios of antibody titers K> 2 and K <1/2, at least 2 samples are obtained from each initial serum, and dilutions are made so that antibodies to are detected in the first sample both antigens, and in the second sample, only the most represented antibodies (antibodies to only one antigen) with an optical signal level exceeding 1.5 times the level of OPcrit were determined. 7. The method according to claim 4, characterized in that when diluting sera with a ratio of K = 1, at least 2 samples are obtained from each initial serum, and dilutions are made so that antibodies to both antigens in the first dilution are determined in a titer of at least 1:20, and in the second - in the title no more than 1:10 with the level of the optical signal exceeding the level of OPcrit by 1.5 times.