UA66100A - IMMUNOENZYME ASSAY KIT FOR DETECTING IgM AGAINST TOXOPLASMA GONDII IN HUMAN BLOOD SERUM (DIA-TOXO-IgM) - Google Patents

Abstract

The immunoenzyme assay kit for detecting IgM against Toxoplasma gondii in human blood serum (DIA-TOXO-IgM) consists of immune sorbent and conjugate. The immune sorbent comprises the purified antigens of Toxoplasma gondii. The sera being assayed are used at a 1:10 dilution.

Description

The invention belongs to biotechnology, immunochemistry and medicine and can be used in the clinic to confirm the diagnosis of acute toxoplasmosis.

In Ukraine, there is no test system for detecting acute toxoplasmosis. In Russia, the test system "VektoToxo-IdM-strip" (ZAO "Vektor-Best", Novosibirsk) is produced, in which Tochoriazta dopayi antigens are sorbed on the solid phase; sera are studied at a dilution of 1:20, and monoclonal antibodies against human M 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 sorbed monoclonal antibodies against human DM. The studied sera are used in a dilution of 1:10. The composition of the conjugate includes purified antigens of Tochoriast dopai, labeled with peroxidase.

The enzymatic reaction is determined using a substrate solution with a chromogen. 91 serums can be analyzed in one setup (1.5 hours).

Example 1

Determination of antibodies of the IDM class against Tochoriazt dopaiy in the blood serum of patient A., born in 1978.

Determination is carried out according to the known method |2| solid-phase enzyme immunoassay. To do this, add 900 μl of serum dilution solution and 10 μl of the patient's blood serum to detect M-class antibodies in the wells of the immunosorbent tablet with fixed anti-human monoclonal antibodies on it, control samples (2 positive and 3 negative) are placed in separate wells. .

Cover the tablet with an adhesive film or a lid and incubate at a temperature of 37"C for 30 minutes.

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.

100 μl of the conjugate solution - purified toxoplasma antigens, labeled with peroxidase - are introduced into the wells of the tablet, and the tablet is incubated in a ЗОхв thermostat. at 3776.

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 results of the detection of antibodies of the IDM class against Tochoriazt dopaia in the patient's serum when using our test system were compared with the data 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 of the IDM class 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 of class I9M 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 found to be false-positive. The specificity of the comparative test system was 9895.

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

Used literature 1. Instructions for the use of test systems! "VektoToxo-IdDM-strip" produced by ZAO "Vektor-Best", p.

Koltsevo, Novosibirsk Region, 2003, Collection 2. A.T. Mikhailov, V.N. Simirskyi "Methods of immunochemical analysis in developmental biology". M. "Science",

Claims (1)

The immunoenzymatic test system for the detection of antibodies of the DM class against Tochoriazeta dopaia in human blood serum (PIA-TOHO-II9M), which uses an immunosorbent, Tochoriazeta dopaia antigens and monoclonal antibodies against IDM, which is characterized by the fact that the immunosorbent includes monoclonal antibodies against human immunoglobulins class M, sera are examined in a dilution of 1:10, and purified antigens of Tochoriazta dopai, labeled with peroxidase, are used as a conjugate.