30. Describe the indirect ELISA test that is used as a screening test for HIV AI

Question

30. Describe the indirect ELISA test that is used as a screening test for HIV AIDS) and include the following: a) sequence of procedures, b) reagents used, and c) detection and interpretation of a positive test. 31. Compare and contrast agglutination and precipitation reactions,and inciude a description of one test for each type of reaction. 32. Describe laboratories. 3 possible eampling sites and methods of collection for clinical 33. why do many physicians feel that stool cultu in certain circumstances, when diagnosing GI tract infections? res are not necessary, except

Explanation / Answer

30. Indirect ELISA:

ELISA is a test that detects and measures antibodies in your blood. This test can be used to determine if you have antibodies related to certain infectious conditions.

a) Reagents:

1. Coating buffer:

Anhydrous Na2CO3 = 1.5 g

Anhydrous NaHCO3 = 2.93 g

Distilled water = 1 liter, pH 9.6

2. Blocking buffer:

Phosphate buffer saline containing 1% w/v BSA.

3. Wash buffer:

Phosphate buffer saline containing 0.05 v/v Tween 20.

4. Substrates:

HRP conjugated antibodies or Alkaline phosphatase conjugated antibodies.

5. Stop Solution:

0.2 M H2SO4 or 1M NaOH.

b. Sequence of Procedure:

1. Coat microtitre plate wells with 100 ul of the antigen solution at a concentration of between 1-10 Ug/ml in coating buffer.

2. Cover the plate ancubate overnight at 4 degree celcius and wash the plate 3 times in wash buffer.

3. Add 150 ul of blocking solution to each well. Incubate for 1 hour at 37 degree celcius and wash 4 times in wash buffer.

4. Add 100 ul of unconjugated detection antibody which is diluted in wash buffer to each well.

5. Incubate it for 1 hour at 37 degree celcius and wash 3 times in the wash buffer.

6. Add 100 ul of enzyme conjugated secondary antibody to each well and again incubate for 1 hour at 37 degree celcius. Wash it 3 times in a wash buffer.

7. Add 100 ul of the appropriate substrate solution to each well. Incubate it at room temperature for 30 minutes or until the desired color changes is attained.

8. After sufficient color development, add 100 ul of stop solution to the wells.

9. Read the absorbance values immediately at the appropriate wavelength 405 nm with a plate reader.

c. Detection and Analysis of results:

1. Detection:

The HRP and alkaline phosphatase are the two widely used enzymes employed for the ELISa assay. They gives a specific color to the sample after addition of substrate. By observing the color, detection of positive or negative results can be done.

2. Analysis of results:

Prepare a standard curve from the data produced from the serial dilution with concentration on the X axis vs absorbance on the Y axis. Interprete the concentrationof the sample from this standard curve.

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