Ultrasensitive, Specific, Two-Antibody

CLIN.CHEM.37/12, 2069-2075 (1991)

Ultrasensitive, Specific, Two-Antibody lmmunoradiometricAssay That Detects Free Alpha Subunits of Glycoprotein Hormones in Blood of Nonpregnant Humans SanJeev Grover, Jeanine Griffin,

and William D. OdelI’

This noncompetitive, sensitive, immunoradiometnc assay of the free alpha subunit of human pituitary glycoprotein hormones is based on two monoclonal antibodies and an avidin-biotin separation system. The affinity of the first antibody, mouse anti-alpha subunit covalently conjugated to biotin, is 3.8 x 1011 LJmol. The second antibody, radiolabeled with 125 has an affinity of 5.4 x 1011 LJmol. i A polystyrene ball coated with avidin serves as the separation system. Tests of “purified” immunochemicalgrade intact human glycoprotein hormones yielded crossreactions of -2% in the assay. Sephadex G-1 00 column chromatography showed that this “cross-reaction” was caused by contamination of the various hormone preparations with free alpha subunit. When the intact glycoprotein hormones were further purified with specific antialpha monoclonal antibody, their reaction in the alpha subunit assay was undetectable (

I-. C) 4

FRACTION NUMBER

4 I

Fig. 3. SephadexG-100 chromatograph of 40 zg of hLH (AFP 0642B)afterthestandard waspurified against an anti-alpha Immune column Fraction 0 representsthe voidvolume (V,), which Is 58 mL No alphaactivity was detected In the preparation. Intact hCG (CR125) and the alpha-subunitof hCG (CR123) werealsochromatographedonthesame column;their activities eluted near the predicted values

tein hormones in human serum. The IRMA showed 1-Z% reaction with “purified” preparations of bLH, hTSH, hFSH, and hCG. In each case, this “cross-reaction” was shown to be due to contamination of these intact hormone preparations with small amounts of free alpha subunit. When each preparation was further purified by immunopurification with specific anti-alpha monoclonal antibodies, the resulting intact hormones obtained showedno detectable reaction in the alpha subunit IRMA (