RU2282191C2 - Method of determining functional activity of antichlamydial antibodies - Google Patents

Abstract

FIELD: laboratory diagnostics.

SUBSTANCE: gist of invention resides in that functional activity of antichlamydial IgG antibodies in blood serum is determined from value of relative affinity (RHAV) obtained by studying intensity of antigen-antibody complex bonding after treatment of ammonium thiocyanate solution with different concentrations. When RHAV value is below 500, low functional activity of antichlamydial antibodies is stated and , when RHAV value is above 500, high functional activity of antichlamydial antibodies is stated.

EFFECT: extended choice of means of diagnosing chlamidiosis utilizing blood serum procedures and estimating patient treatment quality.

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Description

Scope: The present invention relates to medicine, namely to laboratory diagnostics.

State of the art

Analogues of this invention are methods for assessing the functional activity of specific antibodies to infectious agents described in domestic and foreign literature (Korovina N.A. et al., 1999; Kulakov A.V. et al., 1997; Meringova L.F. et al. , 1997; Blackburn NK et al., 1991; Bodeus M. et al., 1998; Luxton RW et al., 1990; Klimova S.V. et al., 1997), which, according to a number of common features, can be divided into two groups:

1. Evaluation of the functional activity of antibodies using the dissociation constant (Kulakov A.V. et al., 1997; Meringova LF et al., 1997).

2. Determination of the avidity (functional affinity) of antibodies based on the avidity index (Korovina N.A. et al., 1999; Blackburn NK et al., 1991; Bodeus M. et al., 1998) and using the formula for determining the relative value functional affinity (Luxton RW et al., 1990; Klimova S.V. et al., 1997).

A common feature of the methods of the first group is the use of the equilibrium constant as a measure of the affinity of the studied interaction. The availability of information on the exact concentration of antigen, antigenic determinants allows us to determine the value of the constant with high accuracy by the slope of the straight line constructed in the coordinate axes. The disadvantages of these research methods are the complexity and duration of their implementation, the use of additional materials (antigen solutions).

The most widespread in practical immunology is the second group of research methods, characterized by the speed of execution, minimal costs for additional reagents and low cost of research. The basis of these research methods is the use of solutions of detergents with a known concentration, which have the ability to break the formed bonds in the antigen-antibody complex. The results are taken into account using the avidity index, which reflects the percentage of affinity antibodies, or using the formula by which the relative affinity value is calculated. The avidity index of antibodies, in particular, to cytomegalovirus, allows to differentiate between acute and chronic infection (Blackburn NK et al., 1991), pregnancy outcome (Bodeus M. et al., 1998), intrauterine and postnatal infection (Korovina N.A. et al. ., 1999). However, the above methods do not provide information on the functional activity of affinity antibodies to weak immunogens, in particular to chlamydia.

A prototype of the proposed method is the method of Matikainen M.T., Lehtonen O.P. (1984). The essence of the method is to determine the affinity of anti-Chlamydia antibodies in the supernatant of cell culture and ascidic fluid using solutions of known concentration of antigen under conditions of excess of specific antibodies using enzyme-linked immunosorbent assay. The affinity of anti-Chlamydia antibodies is evaluated using the extrapolated curve fitting procedure plotted in the coordinate axes. The disadvantages of this research method are the complexity and duration, the use of additional materials (antigen solutions).

SUMMARY OF THE INVENTION

The purpose of the invention is to develop a method for determining the functional activity of specific anti-Chlamydia IgG antibodies by assessing the intensity of their interaction with the antigen (affinity) after treatment with solutions of ammonium thiocyanate.

The method is as follows:

In pre-washed (once) phosphate buffer wells of a polystyrene plate with immobilized chlamydophilic antigens of the main protein of the outer membrane of chlamydia (MOMP Chlamydia), 80 μl of serum dilution solution are added to all wells. Then, 20 μl of a negative control sample (K ^- ) are added to any two wells, 20 μl of a positive control sample (K ⁺ ) to the next two wells, and 20 μl of previously diluted test serum containing A1-A5 Chlamydia antibodies. Chlamydia positive serum of patients is taken in a dilution that gives an optical density of 1000-1500 units at a wavelength of 492 nm. The tablet is closed with a lid and incubated in an incubator at 37 ° C for 30 minutes. As a result of incubation, the antibodies are fixed on the antigen (formation of the antigen-antibody complex). After incubation in a thermostat and subsequent washing with phosphate buffer to remove antibodies not bound to the antigen, 100 μl of phosphate buffer is added to the wells with control samples and one well with test serum (A5). In the remaining four wells containing the test serum (A1-A4), add 100 μl of ammonium thiocyanate (CH ₄ N ₂ S - r.h.s.) ("MERCK", Germany) in various concentrations (1.0; 1, 5; 2.0; 2.5 M) and incubated for 10 minutes at room temperature. Under the influence of the detergent, weak bonds in the antigen-antibody complex are broken and antibodies that have become free are removed as a result of five-fold washing of the plate wells with phosphate buffer. Then, 100 μl of conjugate solution (anti-human IgG antibodies conjugated to horseradish peroxidase) was added to all wells, the plate was closed and incubated in an incubator at 37 ° C for 30 minutes. During the incubation process, a new antibody-antigen complex is formed that contains an enzyme label (horseradish peroxidase). Antigen in this complex are antibodies against human IgG. After incubation, the washing procedure is repeated to remove unbound antibodies. Then add 100 μl of a chromogen solution and incubate at room temperature in a dark place for 20 minutes. As a result of a chemical reaction during the interaction of horseradish peroxidase and chromogen, the solution becomes colored. The reaction is stopped by adding 50 μl of stop reagent. The staining intensity is estimated by the value of optical density using a photometer at a wavelength of 492 nm.

The relative value of the affinity of antibodies is determined by the formula: RHAV = A1 × 7 + A2 × 6 + A3 × 5 + A4 × 4, where A1, A2, A3, A4 is the percentage of the initial amount of antibodies remaining in the well after they are treated with ammonium thiocyanate in concentrations of 2.5; 2.0; 1.5; 1.0 M respectively. For 100% (initial amount), the absorbance value reflecting the content of antichlamydial antibodies in the test serum, which was not exposed to ammonium thiocyanate (A5), is taken.

It was found that the value of RHAV <500.0 srvc characterizes a decrease in the activity of the B-cell immunity link and promotes the formation of humoral reactions of a pathological type (pathology).

RHAV value> 500.0 srvc indicates a full anti-chlamydial humoral response (normal).

A value of 500.0 conventional units reflects the borderline state. Upon receipt of this result, it is recommended to re-examine after 2 weeks.

Example 1. Sergeeva O.A. (history of childbirth No. 6438) was hospitalized at a gestational age of 14 weeks due to the threat of miscarriage. When tested for infection by PCR and ELISA, antibodies (IgG to Chlamydia MOMP) and Ch. trachomatis. The titer of specific anti-Chlamydia IgG was 1/80, RHAV = 51.5 srvc. Based on the obstetric history and laboratory tests, a diagnosis of urogenital chlamydial infection is made. After antibiotic therapy, the patient was examined at 27 and 35 weeks of gestation.

Chlamydia antigens were not detected at 27 weeks. The titer of anti-chlamydial IgG was 1/320, RHAV = 97.2 srvc.

At 35 weeks, the titer of specific IgG was 1/160, RHAV = 925.6 srvc. The result of PCR (on chlamydia) is negative. The perinatal pregnancy outcome, in accordance with our data, was expected to be favorable.

Clinical diagnosis of the newborn Sergeyeva (developmental history of the newborn No. 56438): Mild chronic hypoxia. Almost healthy.

Example 2

Dubrovina O.A. (history of childbirth No. 1072) was examined and treated for urogenital chlamydia. During an initial examination at a gestational age of 7 weeks, Ch.trachomatis antigens were detected by PCR. In a period of 10 weeks - IgM (titer 1/200) and IgG (titer 1/640), the value of RHAV (IgG to Chlamydia MOMP) was 285.0 srvc. The diagnosis of acute chlamydial urogenital infection was verified and appropriate treatment was carried out. In pregnancy at 13 and 18 weeks, Ch.trachomatis antigens were not detected. After childbirth, a repeated serological examination was performed: titer of anti-Chlamydia IgG -1/2560, RHAV = 221.0 conventional units

Based on the results of a laboratory examination, an adverse perinatal pregnancy outcome was assumed.

The clinical diagnosis of the newborn Dubrovin (history of the development of the newborn No. 51072): infection of the perinatal period (acute infectious enterocolitis, hypotoxic disorders of the central nervous system).

Example 3 Muzofarova A.V. (history of childbirth No. 540) was admitted to the obstetric department during the period of 40-41 weeks of pregnancy. According to obstetric history in the second trimester of pregnancy, the patient received treatment for urogenital chlamydia. Before birth, a serological examination was performed to identify antibodies to chlamydia. Detected IgG to Ch.trachomatis in titer 1/40, RHAV = 588.8 conventional units A sufficiently high activity of affinity antibodies made it possible to predict a minimal risk of chlamydial infection in a newborn.

The clinical diagnosis of the child Muzofarov (source. Development of the newborn No. 50540): healthy.

Thus, the above examples demonstrate the possibility of using the proposed method for assessing the functional activity of antichlamydial antibodies as an additional criterion for the diagnosis and assessment of the quality of treatment of a patient.

It has been reliably established that the value of the RHAV value of anti-Chlamydia IgG antibodies, greater than 500.0 conventional units, corresponds to a “full-fledged" humoral response (normal).

A decrease in the RHAV value of specific IgG antibodies to less than 500.0 conv.units of antibodies characterizes the immaturity of the humoral immune response (pathology). The patient requires immunocorrection and appropriate etiotropic treatment.

A value of 500.0 conventional units reflects the borderline state. Upon receipt of this result, it is recommended to re-examine after 2 weeks.

Information sources

1. Klimova S.V., Pinegin B.V., Kulakov A.V. and others // Immunology. -1997. - No. 3 - S.50-52.

2. Korovina N.A., Zaplatnikov A.L., Cheburkin A.V., Zakharova I.N. Cytomegalovirus infection in young children (clinic, diagnosis, modern treatment options) // Rukov. For doctors. -M .: Posad, 1999.-56p.

3. Kulakov A.V., Klimova S.V., Yarilin D.A., Rubtsova M.V., Pinegin B.V. The study of the affinity of natural antibodies of human blood serum to a component of the bacterial cell wall - glucosaminylmuramyl dipeptide with adjuvant activity // Immunology. - 1997.-№1.-S.21-24.

6. Bodeus M., Feyder S., Goubau P. Avidity of antibodies distinguishes primary from non-primary cytomegalovirus infection in pregnant women // Clin. Diagn. Virol. - 1998. - Vol. 9. - (1). - R. 9 - 16.

7. Matikainen M.T., Lehtonen O.P. Relation between avidity and specificity of monoclonal anti-chlamydial antibodies in culture supernatants and ascitic fluids determined by enzyme immunoassay. // J. Immunol. Methods - 1984.-Vol. 72 .- (2) .- P. 341-347.

8. Luxton R.W., Thompson E.J. Affinity distributions of antigen-specific in patients with multiple sclerosis and patients with viral encephalitis // Journal of Immunological Methods. - 1990. - Vol. 131. - P. 277 - 282.

Claims (1)

A method for determining the functional activity of antichlamydia antibodies by the relative affinity value, characterized in that control and experimental serum samples are introduced into the wells of the immobilized Chlamidia MOMP antigen, incubated, ammonium thiocyanate is added to concentrations of 1.0, 1.5, 2.0, 2.5 M, incubated, washed with phosphate buffer, determine the number of antibodies by optical density, and the functional activity of antichlamydia antibodies (RHAV) is calculated by the formula RHAV = A1 · 7 + A2 · 6 + A3 · 5 + A4 · 4, where A1, A2, A3, A4 is the percentage of antibodies remaining in the well after their treatment with ammonium thiocyanate at concentrations of 2.5, 2.0, 1.5, 1.0 M , respectively, with a RHAV value of less than 500, a low functional activity of antichlamydial antibodies is determined, and with a RHAV value of more than 500, a high functional Chlamydia antibody activity.