UA66096A - IMMUNOENZYME ASSAY KIT FOR DETECTING IgG AGAINST TOXOPLASMA GONDII IN HUMAN BLOOD SERUM (DIA-TOXO-IgG) - Google Patents

Abstract

The immunoenzyme assay kit for detecting IgG against Toxoplasma gondii in human blood serum (DIA-TOXO-IgG) consists of immune sorbent and conjugate. The immune sorbent comprises the purified surface antigens of Toxoplasma gondii. The conjugate comprises peroxidase labeled anti-human IgG monoclonal antibodies. The undiluted sera are used for the assay. In addition, the kit contains the reference samples for calibration purposes.

Description

The invention belongs to biotechnology, immunochemistry and medicine and can be used to assess the immune status of women before or at the beginning of pregnancy, as well as to conduct sero-epidemic studies.

In Ukraine, there is no test system for detecting infection of people, especially pregnant women and babies, with toxoplasma. In Russia, the test system "VectoToxo-antibodies" (CJSC "Vector-Best",

Novosibirsk), in the composition of which recombinant polypeptides-protein analogues are used on the solid phase

Tochoriazta dopaiy; sera are examined after a preliminary dilution of 1:100, and anti-species antibodies against human C-class immunoglobulins labeled with peroxidase are taken as a conjugate (11.

The invention is based on the task of creating an immunoenzymatic test system based on an immunosorbent with purified Tochoriazt dopai antigen surface proteins. Antigens are adsorbed on the surface of polystyrene tablets. The studied sera are used in a dilution of 1:10. Conjugates include monoclonal antibodies against human immunoglobulins of class C, labeled with a peroxidase enzyme. The enzymatic reaction is determined using a substrate solution with a chromogen. 88 sera can be analyzed in one setup (2 hours).

Example 1

Determination of antibodies against Tochoriazt dopaia in blood serum of patient L. born in 1977.

Determination is carried out according to the known method |2| solid-phase enzyme immunoassay. To do this, add 9 µl of serum dilution solution and 100 µl of the patient's blood serum to detect antibodies of class c (jus) into the wells of the immunosorbent tablet with the Tochoriaet antigen proteins fixed on it.

90 μl of serum dilution solution and control samples are placed in four wells: in three wells - 10 μl of positive control serum samples with determined antibody titers (for building a calibration curve), and in one - TOmcl of negative control.

Cover the tablet with an adhesive film or a lid and incubate at a temperature of 37"C for 2 hours.

At the end of the incubation, remove the contents of the wells using a washer or an 8-channel pipette and wash the wells four times with a solution for washing tablets.

Add 100 μl of a solution of a monoclonal conjugate against human immunoglobulins labeled with peroxidase to the wells of the tablet, and incubate the tablet at 37"C in a ZOhv thermostat.

After incubation, the wells are washed six times and 100 μl of the developer solution (substrate solution with chromogen) are added and incubated at 18-22"C in the dark at 3°C.

The color reaction is stopped by adding 100 μl of stop reagent to all samples.

No more than because of them. after stopping the color reaction, determine the optical density (OD) in the wells in the two-wave mode using a spectrophotometer. The OG value of the sample is directly proportional to the amount of antibodies in the serum.

The value of the number of antibodies against Tochoriazt dopaia in the patient's serum, determined when using our test system, was compared with the values obtained when using the test system produced by ZAO "Vektor-

Best" (Russia).

The studied sera of the patient were identified as positive for the presence of antibodies against antigens

Tochoriazta dopaii.

Example 2

Determination of sensitivity and specificity of the proposed test system.

Blood sera were examined using the named test systems (20 definitely positive and 100 definitely negative sera). All sera were tested in duplicate with each test system according to the described method. All 20 positive sera showed the presence of antibodies against Tochoriazt dopaia, which is 10095 sensitivity. Out of 100 negative sera, all 100 turned out to be truly negative (specificity - 10095) in the proposed test system. In the compared test system from Russia, out of 20 definitely positive sera, 19 were positive. One low titer serum was not detected. The sensitivity of the test system was 9595. Out of 100 negative sera, 2 sera were detected as false positives. The specificity of the comparative test system was 9895.

Thus, the proposed test system provides the detection of class da antibodies against Tochoriast dopaia antigens in human sera. Simple and reliable in operation, shows high sensitivity and specificity.

Used literature 1. Instructions for the use of test systems! "VectoToxo-antibodies" produced by CJSC "Vector-Best", p.

Koltsevo, Novosibirsk region, 2003, Zs. 2. A.T. Mikhailov, V.N. Simirsky "Methods of immunochemical analysis in developmental biology". M, "Science",

Claims (1)

Immunoenzymatic test system for the quantitative determination of antibodies of the class ds against Tochoriast dopaia in human blood serum (OIA-TOHO-Ida), in which Tochoriast dopaia antigens are used as an immunosorbent, and peroxidase-labeled antispecies antibodies are used as a conjugate , that the immunosorbent includes purified surface proteins-antigens Tochoriazta dopai), the tested sera are used without prior dilution, control calibration samples with a determined antibody titer are added to the set, and the conjugate contains enzyme-labeled monoclonal antibodies against human immunoglobulins of class C.