UA66097A - IMMUNOENZYME ASSAY KIT FOR DETECTING IgM AGAINST HERPES SIMPLEX VIRUSES TYPES I AND II IN HUMAN BLOOD SERUM (DIA-HSV 1/2-IgM) - Google Patents

Abstract

The immunoenzyme assay kit for detecting IgM against herpes simplex viruses types I and II in human blood serum (DIA-HSV 1/2-IgM) consists of immune sorbent and conjugate. The immune sorbent comprises anti-human IgM monoclonal antibodies. The conjugate comprises peroxidase labeled mixture of the purified envelope protein antigens of herpes viruses types I and II. The sera being assayed are used at a 1:50 dilution.

Description

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

In Ukraine, there is no test system for detecting acute herpes infection. In Russia, the test system "VektoVPG-IdM-strip" (JSC "Vektor-Best", Novosibirsk) is produced, which contains antigens of the herpes simplex virus (HSV) 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 researched sera are used in a 1:50 dilution. The composition of the conjugate includes a mixture of purified antigens of herpes simplex virus types 1 and 2, labeled with peroxidase. 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 antibodies of the IDM class against herpes simplex virus types 1 and 2 in the blood serum of patient K., born in 1977.

Determination is carried out according to the known method |2| solid-phase enzyme immunoassay. For this purpose, 100 μl of the patient's blood serum diluted 1:50 to detect M-class antibodies, as well as control samples (2 positive and 3 negative) are introduced 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 purified antigens of herpes simplex virus types 1 and 2, labeled with peroxidase - are introduced into the wells of the tablet, and the tablet is incubated in a ЗОхв thermostat. at 370.

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 IDM class against the herpes simplex virus in the patient's serum, determined when using our test system, were compared with the values obtained when using the test system manufactured by Vector-Best CJSC (Russia).

The examined sera of the patient were identified as positive for the presence of antibodies of the class (DM against antigens of the herpes simplex virus.

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 class (DM against the herpes simplex virus, which has a sensitivity of 10095. 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, it was definitely of the positive sera, 20 were also positive. The sensitivity of the test system was 10095. Out of 100 negative sera, 2 sera were detected as false positives. The specificity of the comparative test system was 989.

Thus, the proposed test system provides detection of antibodies of the DM class against antigens of the herpes simplex virus in human sera. It is simple and reliable in operation, shows high sensitivity and specificity.

Used literature 1. Instructions for use of test systems "VektovpG-IdOM-strip" produced by ZAO "Vektor-Best", p.

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

Claims (1)

Immunoenzymatic test system for the detection of IM class antibodies against herpes simplex virus types 1 and 2 in human blood serum (0О1А-Н5М 1/2- IM), which uses an immunosorbent, herpes simplex virus antigens and monoclonal antibodies against IM , which differs in that the immunosorbent includes monoclonal antibodies against human immunoglobulins of the M class, sera are examined in a dilution of 1:50, and a mixture of purified antigens of herpes simplex virus types 1 and 2, labeled with peroxidase, is used as a conjugate.