Method of serum diagnostics of pertussis

FIELD: medicine.

SUBSTANCE: analysed human blood serums are brought in wells of 96-well polystyrene plates with an immobilised complex of pertussis microbe antigens, added with a human IgG3 antibody peroxidase conjugate, added with a substrate mixture that is followed with recording the optical density of the mixture to derive a titre of immunosorbent attached human IgG3 antibodies. The substrate mixture is a ready form of tetramethylbenzidine and hydrogen peroxide. The human antibody titre 1:40 is accepted as pertussis related.

EFFECT: higher sensitivity and specificity of pertussis diagnostics.

1 ex, 2 tbl

 

The invention relates to medicine, namely to laboratory diagnosis, and can be used for serological diagnosis of pertussis enzyme-linked immunosorbent assay (ELISA).

Despite the high coverage of the population in Russia and abroad vaccination against pertussis incidence of pertussis remains at a high level, and the level of asymptomatic disease is much higher than the manifest forms of the infection. Therefore, timely diagnosis of the disease plays an important role in the fight against whooping cough, with great importance are methods for the serological diagnosis of pertussis, from which the optimal method is an enzyme-linked immunosorbent assay (ELISA)based on detection of antibodies to Bordetella pertussis antigens in sera of patients with whooping cough.

A known method for the diagnosis of whooping cough by immobilization on a 96-well flat-bottomed polystyrene tablets complex antigens pertussis germ, added dilutions analyzed sera, prilipanie to them peroxidase conjugates monospecific antisera to total IgG, IgM and IgA person, then add substrate mixture (hydrogen peroxide and 5-aminosalicylic acid), measuring the optical density of the substrate mixture in a vertical photometer at a wavelength of 450 nm, characterized in that for detecting prisoedinivshiisya to the immunosorbent antibody using peroxidase conjugates of antibodies to IgG-General, IgM and IgA as well as antibodies in the composition of the conjugate using monospecific antisera (Emizet, Ikimashou, Masterova, Lignanotrain, Nassarawa. Diagnostic value of IgG, IgA and IgM antibodies to Bordetella pertussis antigens in patients with pertussis. Journe. microbiol., 2008, No. 6, p.23-26).

The known method is based on the determination of increased levels of whooping cough booster total IgG, IgM and IgA antibodies to pertussis antigens microbe in the blood sera of patients with whooping cough. Elevated levels of whooping cough booster antibodies are serological marker for pertussis, but whooping cough booster IgM antibodies in the sera of patients are defined only in some patients and, mainly, in a short time from the onset of the disease, and elevated levels of total IgG and IgA antibodies also can be found in healthy people. The younger children are relatively high levels of total IgG antibodies may be the result of vaccination, and in older children and adults high titers of total IgG and IgA antibodies are the result of previously deferred pertussis in asymptomatic or erased form. Elevated levels of total IgG and IgA antibodies can persist for a long time after recovery, making it difficult for the serological diagnosis of pertussis. Thus, the problem of increasing the specificity of diagnosis kokosavaya up to date.

The present invention is to develop a method for the diagnosis of whooping cough by ELISA with high specificity and sensitivity.

The technical result of the proposed method is to increase the specificity of the assay for the serological diagnosis of pertussis, allowing to differentiate between immunological indicators current pertussis infection from parameters of the immune system of healthy individuals and recover from whooping cough.

To achieve the technical result in the method for the serological diagnosis of pertussis by adding to the wells of 96-well polystyrene tablets with immobilized complex antigens pertussis germ investigated sera of human blood, the addition of peroxidase conjugate antibodies to human IgG3, addition of substrate mixture, check the optical density of the mixture and on the basis of the optical density of the mixture of revealing titer joined the immunosorbent whooping cough booster IgG3 antibodies person, according to the invention as a substrate mixture use a ready-made form with tetramethylbenzidine hydrogen peroxide revealed titers of IgG3 antibodies person 1:40 accept as relevant to the disease whooping cough.

The sensitivity of the method is assessed according to the frequency of detection of increased levels of IgG3 antibodies in patients with whooping cough.

ISOE is isawanya peroxidase conjugate monoclonal mouse antibodies to human IgG3 for the serological diagnosis of pertussis based on the features of catabolism and short half-life of IgG3 antibodies, resulting in elevated levels of IgG3 antibodies are determined only in patients with pertussis and absent from healthy individuals and suffered whooping cough.

The invention is illustrated by the following example.

Example.

The formulation of the ELISA is carried out in a 96-well polystyrene tablets with a flat bottom. In the wells of tablets make a 0.1 ml solution of complex antigens pertussis bacteria at a concentration of 40 g/ml in 0.05 M carbonate-bicarbonate buffer solution with a pH of 9.6 and incubated the plates for 2 h at a temperature of 37 deg. With and then for 16-18 h at a temperature of 4-6 degrees. C. then the wells tablets thrice washed with 0.3 ml of 0.1 M phosphate buffer solution pH to 7.4 with 0.005% tween-20. Later in the wells of tablets make a serial dilution of the investigated sera 1:10, 1:20, 1:40, 1:80, 1:160 in 0.1 M phosphate buffer solution pH to 7.4 with 0.005% tween-20 and 1% bovine serum albumin. Tablets incubated 1 h at 37 deg. C and washed three times wells 0.3 ml of 0.1 M phosphate buffer solution pH to 7.4 with 0.005% tween-20. Then in the wells of tablets contribute to 0.1 ml of the working dilution of peroxidase conjugate monoclonal mouse antibodies to human IgG3 in 0.1 M phosphate buffer solution pH to 7.4 with 0.005% tween-20 and 1% bovine serum albumin. Tablets incubated for 1 h at 37 deg. S, after which the wells tablets thrice washed with 0.3 ml of 0.1 M phosphate buffer what about the solution pH to 7.4 with 0.005% tween-20 and added to 0.1 ml of the prepared substrate mixture with tetramethylbenzidine hydrogen peroxide. Tablets incubated for 15 min at room temperature, out of reach for the light and add to the wells, 0.1 ml of 2 M sulfuric acid. When interacting peroxidase from peroxidase conjugate with the substrate mixture and sulfuric acid appears yellow staining mixture, which is recorded as absorbance values on the vertical scanning photometer at a wavelength of 450 nm.

Titers of IgG3 antibodies 1:40 take appropriate disease whooping cough. The titer of the sera take the values back to the maximum dilution at which the absorbance values not less than 50 per cent higher than the values of optical density in the wells with control reagents (adsorbed antigen - peroxidase conjugate - substrate mixture).

Using the proposed method in comparison with known were investigated 184 serum of patients with whooping cough, 140 sera of healthy children 3-5 years and 115 sera of healthy children 9-11 years with the purpose of revealing of whooping cough and evaluation of the specificity of the claimed method. The results are presented in Table 1.

Table 1
The sensitivity study claimed and known methods for the serological diagnosis of pertussis
ExaminedThe surveyed gave a positive reaction to the total number of surveyed
IgAIgG totalIgG3
Patients with pertussis n=184Abs.%Abs.%Abs.%
155/18484,2159/18486,4168/184for 91.3

As follows from the data presented in table 1, the study of the sensitivity of the inventive method showed the presence of the disease in 168 patients with pertussis from 184 patients studied, which accounted for 91.3%, while using a known method with the determination of IgA and IgG total antibody the presence of the disease was identified in 155 of 184 patients and 159 of 184 patients, respectively, which was 84,2% and 86.4%, respectively. Thus, the sensitivity of the claimed method was high, was not inferior and even slightly exceeded the known sensitivity of the method.

Comparative study specificn the STI claimed and known methods (table 2) showed an advantage of the inventive method, since the examination of sera 140 healthy children 3-5 years by the claimed method only 2 children gave nonspecific response that amounted to 1.4%, in the study of sera 115 healthy children 9-11 years - 2 children gave nonspecific reaction, which corresponded to 1.7% and testified to the high specificity of the proposed method, while in the study of sera from these same groups of children with determination of IgA antibodies non-specific reaction gave 8 children in the first group and 14 children in the second group, which corresponded to 5.7% and 12.2%, and total IgG antibodies 23 children in the first group and 13 children in the second group, which accounted for 16.4% and 11.3%, respectively. Thus, the inventive method of diagnosis is highly specific, as it reveals elevated titers of IgG3 antibodies only in patients with pertussis (% nonspecific reactions was low and did not exceed 1.7 percent), while when diagnosing a known manner % nonspecific reactions was significantly higher and reached 16.4 percent. The differences between the claimed and known method statistically significant (at p<0,05).

Table 2
Study of the specificity of the claimed and known methods for the serological diagnosis of pertussis
About ladouanie The surveyed gave a positive reaction to the total number of surveyed
IgAIgG totalIgG3
Abs.%Abs.%Abs.%
Healthy children 3-5 years (group 1) N=1408/140the 5.723/14016,42/1401,4
Healthy children 9-11 years (group 2) n=11514/11512,213/11511,32/1151,7

The advantage of the invention lies in the fact that the developed method for the serological diagnosis of pertussis, with high sensitivity and specificity superior to the known method and allowing for immunological indicators to identify the current pertussis infection in patients. The method can be used for the diagnosis of pertussis infection.

With the royals for the serological diagnosis of pertussis by adding to the wells of 96-well polystyrene tablets with immobilized complex antigens pertussis germ investigated sera of human blood, the addition of peroxidase conjugate antibodies to human IgG3, addition of substrate mixture, check the optical density of the mixture and on the basis of the optical density of the mixture identifying titer joined the immunosorbent whooping cough booster IgG3 antibodies person, characterized in that the substrate mixture use a ready-made form with tetramethylbenzidine hydrogen peroxide revealed titers of IgG3 antibodies person 1:40 accept as relevant to the disease whooping cough.



 

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