Modulatory Role for Substance P: MATERIALS AND METHODS(4)

EIA for pLH The assay used is similar to the EIA for porcine growth hormone developed recently in our laboratory, based on EIAs for rat prolactin and goldfish gonadotropin, with appropriate modifications. The assay involved 8 steps separated by washes with 200 |xl of 0.01 M PBS, pH 7.4, containing 0.05% Tween 20 (PT). Only the interior wells of a flat-bottomed, 96-well polystyrene plate were used (Bioreba AG, Reinach, Switzerland). All steps were carried out under gentle agitation. flovent inhaler
The following is a brief description of the pLH EIA procedure. 1) Coating. Each well received 200 ц.1 of PBS containing 80 ng of pLH (AFP-10714B, kindly provided by Dr. A.F. Parlow, Pituitary Hormones and Antisera Center, Harbor-University of Califomia-Los Angeles Medical Center). Wells used as blanks or 0% values received only PBS without pLH. Plates were incubated at 4°C for 20-24 h. 2) Preincubation of primary antiserum with antigen (concurrent, but separated from step 1). Porcine LH standard (250-2 ng/ml, serial dilutions in assay buffer, 0.5% BSA in PT) and samples to be measured were incubated in Eppendorf tubes with primary antiserum (anti-pLH; UCB Bioproducts, Brain L’ Alleud, Belgium) at a final dilution of 1:200 000. Tubes were incubated overnight (20-24 h) at 4°C. 3) Saturation. All wells were supplied with 200 (xl of saturation buffer (2% BSA in 0.01 M PBS) and incubated for 1 h at 37°C. 4) Primary antiserum. Two hundred microliters of the mixture prepared in step 2 was added to the corresponding wells.


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