ENZYME IMMUNOASSAY

Enzyme Immunoassay of the steroid hormone cortisol

Enzyme Immunoassay of the steroid hormone cortisol

Introduction

Saliva, as a biological fluid for alternative to measurement of steroid hormones in plasma, has been gaining popularity.1, 2 Salivary steroids assay has many advantages over plasma as a biological sample.3 Frequent, easy collection by noninvasive, stress-free techniques makes it highly acceptable. Moreover, saliva collection for F assay rules out the false increase in needle-fearing patients, thus providing an ideal technique, especially for clinical assay of F hormone profile. Saliva providers find little difficulty in salivating directly into disposable tubes, providing adequate volumes for determining steroid profile, which can be done in a short time. The other advantage is the ease of storage, as saliva can be stored at -20°C for 6-9 months, at 4°C for 7 days, or at room temperature for 48 hours without any significant change in steroid amount.

 

Antibody Production

The process for production of F polyclonal antibody was modified from that mentioned in our earlier work.19 Rabbits were immunized with a mixture of Cortisol-11--ol-dione hemisuccinate: BSA and Freund adjuvants were administered once a week for a month, and then, once every month. The first immunization was with complete adjuvant, while later, incomplete adjuvant was used. The antibody titer was checked routinely after a month, and when an appropriate titer level was detected, the animals were humanely slaughtered to obtain the antibody, which was diluted and stored for later use.

Enzyme Immunoassay Procedure The EIA plates were prepared by coating the 96-well plates (Coster). Briefly, 200 µL of coating buffer (prepared in one liter of double distilled water (DDW)) containing 2.93 g of NaHCO3 (Merck Art.6329), 1.59 g of Na2CO3 (Osaka TNO 461), Thimerosal 0.1 g (Sigma, T-5125) at a final pH of 9.6, containing polyclonal cortisol antibody at a dilution of 40,000x, was provided per well to the EIA plate. The antibody coated plates were stored overnight at 4°C following which they were washed with washing buffer (containing 10.86 g of Na2HPO4 · 2H2O, 5.38 gm of NaH2PO4 · H2O, 1 g of Thimerosal, and 10 mL of Tween-20 in ten liters of DDW at pH 7.0) before blocking with a blocking buffer. The blocking buffer (containing 5.0 g of gelatin, which was placed in 500 mL of DDW, subjected to continuous stirring and heating to 50°C for 15 minutes, after which the volume was raised to 2 liters by addition of DDW and added with 17.5 g of NaCl, 12.1 g of Tris-base (Sigma T-1503), 3.6 g of EDTA (Sigma), 1.0 mL of Tween-20 and the final pH of the buffer was maintained at 8.0); EIA plates were then sealed with a plastic plate sealer and stored at 4°C until further used.

At the time of assay, the saliva samples were taken from the refrigerator, thawed, and centrifuged. They, as well as the standards and quality control samples, were then diluted with assay buffer in a recycled EIA plate. The diluted samples, standards, and QCs were then transferred to the cortisol antibody coated EIA plate in duplicate, 50 µL in each well. Ten times diluted F-HRP from original stock was ...