UA66095A - IMMUNOENZYME ASSAY KIT FOR DETECTING IgG AGAINST HERPES VIRUSES TYPES I AND II IN HUMAN BLOOD SERUM (DIA-HSV 1/2-IgG) - Google Patents

Abstract

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

Description

The invention belongs to biotechnology, immunochemistry and medicine and can be used to detect immunoglobulins of class I against herpes virus 1 and 2 in human blood serum; the invention can be applied in the clinic to establish and confirm the diagnosis of primary or recurrent herpes infection, as well as to conduct seroepidemic studies.

In Ukraine, test systems are not produced to detect human infection with the herpes virus. In Russia, the test system "VektoVPG-Ida-strip" (CJSC "Vektor-Best", Novosibirsk) is produced, which contains proteins-antigens of the herpes virus on the solid phase; sera are examined after a preliminary dilution of 1:100, and as a conjugate, anti-species antibodies against immunoglobulins of the human class, labeled with peroxidase (11.

The invention is based on the task of creating an immunoenzymatic test system based on an immunosorbent with a mixture of purified membrane proteins-antigens of herpes virus types 1 and 2. Antigens are adsorbed on the surface of polystyrene tablets. The researched sera are used in a 1:50 dilution. Monoclonal antibodies against human immunoglobulins of class C, labeled with an enzyme, are taken as a conjugate. 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 against the herpes virus in the blood serum of patient O., born in 1975.

Determination is carried out according to the known method |2| solid-phase enzyme immunoassay. For this, 100 μl of diluted (1:50) sera for the detection of class C antibodies (IDS) are introduced into the wells of an immunosorbent tablet with a mixture of antigen proteins of herpes virus types 1 and 2 fixed on it.

Add 100 μl of the positive control sample to two wells, and 100 μl of the negative control sample to three wells.

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.

100 μl of a solution of a monoclonal conjugate against human immunoglobulins, labeled with an enzyme, is introduced into the wells of the tablet, and the tablet is incubated at 37"C in a ZOkhv 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 the herpes virus in the patient's serum, determined when using our test system, was 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 class dO against antigens of the herpes 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 against the herpes virus, which is 10095 sensitivity. Out of 100 negative sera in the proposed test system, all 100 turned out to be truly negative (specificity - 10090). In the compared test system from Russia, out of 20 definitely positive sera, 20 were positive (sensitivity 10095), but out of 100 negative sera, 2 sera were identified as false-positive.

The specificity of the compared test system was 98905.

Thus, the proposed test system provides detection of antibodies of the DS class; against herpes virus antigens in human sera. Simple and reliable in operation, shows high sensitivity and specificity.

Used literature 1. instruction on the use of test systems! "VektoVPG-Id(-strip" produced by ZAO "Vektor-Best", p.

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

Claims (1)

The immunoenzymatic test system for the detection of antibodies of the yes class against herpes viruses of the 1st and 2nd types in human blood serum (OIA-H5M 1/2-IdS), in which antigens of the herpes virus are used as an immunosorbent, and as a conjugate - anti-species antibodies labeled with peroxidase, which is characterized by the fact that the immunosorbent includes a mixture of purified membrane proteins-antigens of herpes virus types 1 and 2, the tested sera are used in a 1:50 dilution, and the conjugate contains monoclonal antibodies against human immunoglobulins of class C, labeled with an enzyme.