Bio-elisa HCV

Principle:

Bio-ELISA HCV is an immunoenzymatic method in which the wells of a micro plate are coated with recombinant antigens representing HCV. Serum or plasma samples are added to these wells. If antibodies specific fir HCV represent in sample, they will form stable complexes with HCV antigens on well. Excess sample is removed by a wash step and a rabbit anti-human IgG conjugated with peroxidaes is then added and allowed to incubate. The conjugate will bind to any antigen-antibody complexes formed. After a second wash, a solution of enzyme substrate and chromogen is added. This solution will develop a blue color if sample is positive. The blue color changes to yellow after vlocking the reaction with sulphuric acid. The intensity of color is propotional to anti-HCV antibodies concentration in sample. Well containing negative sample remains colorless.

PROCEDURE:

Allow all reagents to reach room temperature (20-25c) before running assay.

Gently mix all liquid reagents before use.

Dilute the concentrate washing solution 1/10 with distilled water. For one plate, mix 50ml of the concentrate solution with 450 ml of water.

Dilute the concentrated conjugate 1/51 with conjugate diluent. For one plate, add 300 μl of concentrate conjugate to 15 ml of conjugate diluent. Mix gently.

ASSAY PROCEDURE:

Transfer 100μl of each control positive and negative, to the assigned wells. Use at least three wells for negative control. 1 well for the positive control and one well for substrate blank, in all microplates.Leave the blank well empty.

Transfer 100 μl of each sample to be analysed to the corresponding well.

3- Cover the plate with an adhesive seal and incubate for 60 min at 37c.

4-Remove and discard the adhesive seal. Aspirate the contents of the well and fill them completely with diluted washing solution. Repeat the process of aspiration and wshing 3 more time.

5- Transfer 100μl of diluted conjugate into each well of the microplate expects the blank well.

6-Cover the plate with an adhesive seals and incubate for 30 minutes at 37c.

7- During the last 5-10min of this incubation prepare the substrate-chromogen solution. If the entire plate is used add 280μl of chromogen(TMB) to the bottle containing the substrate buffer and mix well..Working solution has a pink color,discard if it became blue.

8-Remove and discard the adhesive seal. Aspirate and wash the plate.

9- Add 100μl of substrate TMB solution to each well,including blank.

10-Incubate for 30 min t room temperature.

11-Stop the reaction by adding 100ul of stopping solution in same sequence and time interval as for the substrate TMB.

12- Blank the reader at 450nm with blank well and read the absorbance of each well, within 30 minutes.

RESULT:

The presence or absence of HbsAg in sample analysed is determined by relating the absorbance value of each sample to the cut-off value .

1-Calculate the cut-off value by adding 0.040 to the mean absorbance of negative control.

2-Divide the sample absorbance by the cut-off value.

                   Positive: ratio absorbance/cut-off≥1.0

                   Negative: ratio absorbance/cut-off<0.9