Limitations

Estriol was assayed using ELISA kit by Delta Biologicals S.R.L, Pomezia Rome; a subsidiary of Erba Diagnostic, Inc.

1. Principle of the assay

This assay employs the quantitative competitive enzyme immunoassay technique. A monoclonal antibody specific for estriol (anti- estriol) has been pre-coated onto a microplate (solid phase).

Standards and samples are pipetted into the wells and any estriol present is bound by the immobilized antibody. Estriol in the sample (antigen) competes with horseradish-peroxidase (HRP)-estriol (enzyme-labeled antigen) for binding onto the limited number of anti- (HRP)-estriol coated on the microplates. After incubation, a simple washing is performed to wash away any unbound substances.

This is followed by the addition of enzyme substrate solution to the wells and color develops. The color development is stopped and the intensity of the color is measured. The intensity of colour measured is inversely proportional to the estriol in the sample.

84 2. Reagent preparation

Wash solution was prepared by diluting content of vail of the buffered wash solution concentrate with deionized water to final volume of 500 mL. The diluted wash solution is stable for 30 days at 2-8 Ċ.

3. Test procedure

All reagents and specimen were allowed to attend room temperature (20-28Ċ) before use. All reagent were mixed gently without foaming. All standards, samples, and controls were run concurrently. Standards were run in duplicates

Appropriate number of microtitre wells were secured in the frame holder. Twenty five microliters (µL) of each standard, control and samples were dispensed with new disposable tips into appropriate wells. One hundred microliters of enzyme conjugate was dispensed into each well and mixed thoroughly for 10 seconds. It is important to have a complete mixing in this step. This was incubated for 60 minutes at 37 Ċ. The microplates was washed properly 3 times with 300µL of diluted wash solution. Absorbent paper was used to remove residual water droplets. The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure.

This was followed by the addition of 100µL of enzyme substrate solution to each well and incubated for 15 minutes at room temperature (20-28Ċ) in the dark. The reaction was ended by adding 50µL of stop solution. Absorbance reading was at 450nm.

The concentration of the controls and samples were generated automatically by the automatic microplate reader from standard curve obtained from the average absorbance values of each set of standards plotted against their concentrations.

85 3.2.4 Anti-β2-Glycoprotein 1 antibodies (a-β2-GP1)

Anti-β2-Glycoprotein 1 antibodies was assayed using ELISA kit by Delta Biologicals S.R.L, Pomezia Rome; a subsidiary of IVAX Diagnostics, Inc

1. Principle of the assay

This assay employs the quantitative indirect solid phase enzyme immunometric assay (ELISA) technique. This method is designed for quantitative measurement of IgA class outoantibodies directed against Glycoprotein 1. The assay is based on micoplate coated with highly purified β2-Glycoprotein 1. Standards, controls and prediluted samples are pipetted into the wells. Any present antibody binds to the immobilized antigen on the inner surface of the microplate wells. After 30 minutes incubation the micoplate is washed with wash buffer to remove non-reactive serum components. An antihuman-IgA horseradish-peroxidase (HRP) conjugate solution is pipetted into the wells to bind to the autoantibodies bound to the immobilized antigens. After a 15 minutes incubation a second washing is done to wash off excess enzyme conjugate.

This is followed by the addition of enzyme substrate solution to the wells and color develops. The color development is stopped and the intensity of the color is measured. The intensity of colour measured is directly proportional to the concentration of antibodies present in the sample.

Reagent preparation

Wash solution was prepared by diluting content of vail of the buffered wash solution concentrate with deionized water to final volume of 1L. The diluted wash solution is stable for 30 days at 2-8 Ċ.

86

Sample diluent was prepared by diluting content of vail of the diluent concentrate with deionized water to final volume of 100mL. The diluted diluent solution is stable for 30 days at 2-8 Ċ.

Test procedure

All reagents and specimen were allowed to attend room temperature (20-28Ċ) before use. All reagent were mixed gently without foaming. All standards, samples, and controls were run concurrently. Standards were run in duplicates

Appropriate number of microtitre wells were secured in the frame holder. Samples were diluted 1:100 with sample diluent before assay (10 µL of sample to 1000 µL of sample diluent). One hundred microliters (100µL) of each calibrators, control and prediluted samples were dispensed with new disposable tips into appropriate wells. This was incubated for 30 minutes at room temperature (20-28Ċ). The microplates was washed properly 3 times with 300µL of diluted wash solution. Absorbent paper was used to remove residual water droplets. The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure.

One hundred microliters (100µL) of enzyme conjugate was added into each well, mixed thoroughly for 10 seconds and incubated for 15 minutes at room temperature (20-28Ċ).

The microplates was washed properly 3 times with 300µL of diluted wash solution. Absorbent paper was used to remove residual water droplets. The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure.

This was followed by the addition of 100µL of enzyme substrate solution to each well and incubated for 15 minutes at room temperature (20-28Ċ) in the dark. The reaction was ended by adding 50µL of stop solution. Absorbance reading was at 450nm.

87

The concentration of the controls and samples were generated automatically by the automatic microplate reader from standard curve obtained from the average absorbance values of each set of standards plotted against their concentrations.