IMMUNOENZYMETIC TEST-SYSTEM FOR DETERMINATION OF ANTIBODIES TO THE HEPATITIS C VIRUS
A. M. Abdukavumov, P.G.Chistvakov. G.R.Garayshina
«Radi opr eparat» enterprise o f the Institute o f Nuclear Physics, Uzbekistan Academy o f Sciences, Tashkent
Interest to the study of different aspects of Hepetitis C is defined first of all by its wide spreadness. According to rated data there are infected HCV not less than 5 hundred million persons in the world. It reaches 10,1% of the whole human population I I I . Annually about
8 - 1 0 thousand lethal cases are connected with chronic HCV infection. Exactly HCV infection is one of the main reasons for the whole group of the chronic liver problems - a chronic hepatitis, cirrhosis and anothers.
In spite of the significant progress in the study of hepatitis C, Hepetitis C virus is mysterious virus. It was not understood the HC pathogenes completely wtat would explained a possibility of lethal cases after 25 years after infection.
HCV recombinant protein NS5 is encoded by the putative NS5 region of the HCV genome. Comparisons of amino acid and nucleotide sequences of flaviviruses and pestiviruses with HCV suggest that NS5 is derived from a non-structural region of the genome that encodes the viral polymerase: the enzyme involved into the replication process of HCV. Studies have indicated that a significant proportion of persons infected with HCV develop antibodies to NS5
12, 3/.
The use of HCV recombinant proteins derived from the core, NS3, NS4 and NS5 regions of the HCV genome has shown to be effective for determination a greater number of diagnoses for acute and chronic non-A, non-B hepatitis patients than single antigen (c l00-3) assays /4, 51. In
addition, the use of these additional proteins allows the earlier detection of seroconversion following HCV infection.
The primary purpose of this assay is to screen blood donations so that units containing HCV antibody can be identified and eliminated from the blood supply. Although the presence of anti- HCV does not constitute a diagnosis of HCV infection, the determination of anti-HCV may be used as an aid in the diagnosis of hepatitis C and in the differential diagnosis of non-A, non-B hepatitis in conjunction with determination of liver enzymes, additional serological markers and clinical evaluation.
The method for determination of anti - HCV was developed at "Radiopreparat" enterprise. It is ensuring high characteristics of the test and safety of functioning. Antibodies are formed to
each of virus proteins located at structural or unstructured region of HCV. The offered test is the system of the third generation. It is intended for the specific diagnostics of the hepatitis C virus infection and can be used in the service of the blood transfusion, in AIDS preventive maintenance centers, infectious hospitals, centers epidemiological sanitary control and other medical institutions.
The offered test - system "ELISA - anti - HCV" represents the diagnostics kit based on the principle of immunoassay test by means of recombinant structural and unstructural antigens of the hepatitis C virus (core, E2NS2, NS3, NS4A, NS4B, NS5A, NS5B) immobilized on the
stable phase that allows to increase a percentage of determination of the specific antibodies to the hepatitis C virus antigens in serum or plasma of the human blood. Duration of the test is about 1,5 hours. Specificity is more than 98%.
The solution of the HCV fusion antigens was prepared to effect the sensibilisation of the polystirol plate wells. 0,1 ml of the fusion antigens solution was put into the every well.
The plate was sealed by film and kept during the night in a household refrigerator at 4°C. After termination of the incubation the contents of a plate was removed by shaking. 0,2 ml of the protein buffer (0,1 M of a diethylamine, pH 7,5; 1% - BSA) was introduced into each well and kept 1 hour at of 37°C for the suppressing of the sorbence activity of a plates. The contents of a plate were removed by shaking after the termination of the incubation.
Marking the wells of the plate and entering of the samples were carried out according to the following scheme which is indicated below (where 1-12 - number of wells, A - H - numbers of plate rows).
The scheme of the entering of analyzed samples into the wells.
A CS X X X X X X X X X X X B CC X X X X X X X X X X X C PS X X X X X X X X X X X D PS X X X X X X X X X X X E PS X X X X X X X X X X X F NS X X X X X X X X X X X G NS X X X X X X X X X X X H NS X X X X X X X X X X X
Note: CS - the supervision of the substrate; CC - the supervision of the conjugate; PS - the positive control sample; NS - the negative control sample.
Time of the analyzed samples entering into the wells should not exceed 15 minutes.
At the first stage 0,1 ml of the solution of the 0,1 M phosphate buffer, pH 7,5 are introduced into the every well (for exception A1 and B1 wells). 0,01 ml of the positive control sample solutions are introduced into the wells #1, rows C, D, E and the negative checking sample are introduced into the wells #1, rows F, G, H. Into the rest of wells are introduced in the duplicates 0,1 ml the analyzed blood serum or plasma. The plate is sealed by the tape and is incubated during 30 minutes at the temperature 37C . After the termination of the incubation the wells of the plate are washed out by 0,2 M phosphate buffer containing 0,05% of the Twin - 20. Then the solution of the conjugate is prepared. 0,1 ml of the conjugate solution is introduced into the all wells of the plate. The plate is sealed by the tape and incubated during 30 minutes at the temperature 37°C. The substrate mixture is prepared 10 minutes before the termination of the incubation. After the termination of the incubation the wells of the plate are washed out by 0,1 M phosphate buffer containing 0,05% of the Twin - 20. 0,1 ml of the substrate mixture is introduced to all wells of the plate. The plate is sealed by the tape and is incubated during 10 - 20 minutes at the room temperature in dark place. 0,1 ml of the stop - reagent (7% - sulfuric acid) is introduced into all wells of the plate after termination of the incubation. Optical density of the wells is detected by the photometer of vertical scanning at the wave length 492 nm. The results are estimated just in that case if the optical density of the conjugate control is not more then 0,15 optical units. Positive control well should be not more then 0,3 optical units and the data of the optical density of the positive control should exceed optical density negative control not lees them in 3 time. Test serum is estimated as the positive if the measured optical density of the tested sample higher then average value of the optical density of the negative control samples multiplied in 2,1 times (statistically calculated coefficient), if lower or equal - then it is negative. The offered method ensures high specificity of the analysis. In the Table 1 are represented the comparative data for the test-systems different origin.
REFERENCES
1. Sherlock Sh. Antiviral Therapy for chronic hepatitis C viral infection . - J. Hepatol., 1995, V.23, Suppl. 2, P. 3 - 7.
2. Pontisso P., Gerotto M., Chemello L. et al. Hepatitis C virus genotypes HCV - 1a and HCV - 1b. The clinical point of view. - J. Infect. Dis., 1995, V. 171, N° 3, P. 760 - 761. 3. Enomoto N., Takada A., Nakao T., Date T. There are two mayor types of hepatitis C
virus in Japan. - Biochem. Biophys. Res. Commun., 1990, V. 170, P. 1021 - 1025.
4. Simmonds P. Variability of hepatitis C virus. - Hepatology, 1995, V. 21, N° 2, P. 570 - 583.
5. Zein N.N., Persing D.H. Hepatitis C genotypes: Current trends and future implication. -Mayo Clin. Proc., 1996, V. 71, 5, P. 458 - 463.
Table 1. Absorbances of serum panel samples containing and not containing anti - HCV.
N° Serum and Rekombi - ORTO AQVA - Radiopreparat Conclusion panel samples Best anti -VGC PAST enterprise
1 0,072 0,299 0,043 0,102 negative 2 0,075 0,449 0,058 0,286 negative 3 0,092 0,251 0,037 0,128 negative 4 0,096 0,399 0,115 0,173 negative 5 0,101 0,218 0,130 0,215 negative 6 0,170 0,539 0,250 0,194 negatiive 7 0,632 1,321 0,504 2,304 positive 8 0,720 1,561 0,519 2,587 positive 9 1,423 1,636 0,839 3,010 positive 10 1,243 1,631 0,735 2,345 positive 11 0,697 2,199 0,870 2,987 positive 12 1,537 2,339 1,501 2,725 positive 13 1,681 2,421 0,972 3,001 positive 14 1,692 2,438 1,580 2,436 positive 15 1,603 1,820 1,566 2,228 positive 16 1,563 2,145 2,197 2,147 positive 17 1,641 2,145 1,207 2,038 positive 18 1,607 2,117 1,458 2,303 positive 19 0,520 2,488 0,590 2,126 positive 20 1,636 1,880 1,193 2,758 positive 21 1,476 2,312 1,046 2,148 positive 22 1,657 2,474 0,916 2,313 positive 23 2,065 1,823 1,905 2,056 positive 24 2,154 2,462 2,107 2,338 positive OD critical 0,304 0,590 0,295 0,384 519
