UA66099A - IMMUNOENZYME ASSAY KIT FOR DETECTING IgM AGAINST CYTOMEGALOVIRUS IN HUMAN BLOOD SERUM (DIA-CMV-IgM) - Google Patents

Abstract

The immunoenzyme assay kit for detecting IgM against cytomegalovirus in human blood serum (DIA-CMV-IgM) consists of immune sorbent and conjugate. The immune sorbent comprises anti-human IgM monoclonal antibodies. The conjugate comprises peroxidase labeled mixture of recombinant polypeptides p28 and p150 - analogues of CMV antigens. 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 cytomegalovirus infection.

Test systems for detecting acute cytomegalovirus infection are not produced in Ukraine. In Russia, the test system "VektoCMV-IdM-strip" (CJSC "Vektor-Best", Novosibirsk) is produced, which contains cytomegalovirus antigens sorbed on the solid phase; sera are studied at a dilution of 1:100, 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 conjugates includes a mixture of purified recombinant polypeptides p28 and p150 - analogs of peroxidase-labeled cytomegalovirus antigens. The enzymatic reaction is determined using a substrate solution with a chromogen. 91 serums can be analyzed in one setup (2 hours).

Example 1

Determination of class IM antibodies against cytomegalovirus in blood serum of patient K., born in 1977.

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 studied serum of the patient to detect class M antibodies and control samples (2 positive and 3 negative) into the wells of the immunosorbent tablet with monoclonal antibodies against human DM fixed on it.

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 - a mixture of recombinant polypeptides p28 and p150 - analogs of peroxidase-labeled cytomegalovirus antigens - are introduced into the wells of the tablet, and the tablet is incubated in a ЗОхв thermostat. at 3770.

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 determination of antibodies of the DM class against cytomegalovirus in the patient's serum, determined when using our test system, were compared with the data obtained when using the test system manufactured by Vector-Best CJSC (Russia).

The studied sera of the patient were identified as positive for the presence of antibodies of the class (DM against cytomegalovirus antigens.

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 the IDM class against cytomegalovirus, 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, 20 were also positive. The sensitivity of the test system was 10095. 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 DM class antibodies against cytomegalovirus antigens in human sera. Simple and reliable in operation, shows high sensitivity and specificity.

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

Koltsevo, Novosibirsk Region, 2003, 3 p. 2. A.T. Mykhaylov, V.K. Simirsky "Methods of immunochemical analysis in developmental biology. "Nauka", 1981.

Claims (1)

The immunoenzymatic test system for the detection of antibodies of the DM class against cytomegalovirus in human blood serum (0IA-SMU-IDM), which uses an immunosorbent, cytomegalovirus antigens and monoclonal antibodies against IDM, which is characterized by the fact that the immunosorbent includes monoclonal antibodies against human immunoglobulins of the M class , sera are examined in a dilution of 1:10, and a mixture of recombinant polypeptides p2g8 and p150 - analogs of peroxidase-labeled cytomegalovirus antigens is used as a conjugate.