The Development of a Radioimmunoassay Procedure for the Estimation of Tamm—Horsfall Glycoprotein in Human Serum

1. A quantitative radioimmunoassay was developed for the measurement of nanogram amounts of Tamm—Horsfall (TH) glycoprotein in the presence of serum proteins at concentrations above 30 mg/ml. 2. Specific anti-(TH-glycoprotein) antibodies were labelled with 125I and these were usable for a period of 8 weeks. 3. Agarose beads (Sepharose 4B), to which TH-glycoprotein had been coupled via cyanogen bromide activation of the Sepharose, were used as the solid phase in the assay. This proved stable upon storage at 4°C for periods in excess of 4 months. 4. The dissociation of the glycoprotein in the presence of serum proteins that was necessary for quantification was achieved by subjecting the sample to ultrasonication. 5. Assays conducted on a small sample of normal serum produced evidence that normal serum contained amounts (50–180 ng/ml) of a substance that reacted similarly to TH-glycoprotein in the assay procedure and in a series of experiments conducted to confirm the presence of this substance in human serum.

An Alternative Coupling Procedure for Preparing Activated Sepharose for Affinity Chromatography of Penicillinase

Most applications of affinity chromatography employ the cyanogen bromide activation scheme first devised by Axtm et al. (1967). Porath and Sundberg (1972) reported an alternative procedure in which phloroglucinol and divinylsulphone are used in activating reactions. The advantages of this scheme and parameters relevant to the activating reactions are reported here. Conditions for the attachment of various ligand molecules to sepharose using a divinylsulphone activation method are defined, and a comparison with cyanogen bromide activating and coupling techniques is drawn. a-Chymotrypsin is immobilized by covalent attachment to activated sepharose. The optimum coupling pH is 8� 0-8� 6 and the reaction is virtually complete after 20 h at room temperature. Conjugates containing as much as 2 g of enzyme per gram dry weight of polymer were obtained. The immobilized enzyme retained 41 % of the free enzymic activity. An affinity column of divinylsulphone-activated methicillin-sepharose was used to demonstrate the reversible adsorption of penicillinase.

SECOND ANTIBODY CHEMICALLY LINKED TO CELLULOSE FOR THE SEPARATION OF BOUND AND FREE HORMONE: AN IMPROVEMENT OVER SOLUBLE SECOND ANTIBODY IN GONADOTROPHIN RADIOIMMUNOASSAY

ABSTRACT Coupling the second antibody to a solid pase (DASP) was found to be a definite improvement over the classical technique of soluble second antibody (DA) separation of bound and free hormone in the radioimmunoassays of gonadotrophins in plasma or in serum. The duration (2.5 to 7.5 min) and the pH (10.5 to 12.5) of the cyanogen bromide activation of microcristalline cellulose did not affect the coupling capacity or the immunoreactivity of the second antibody, nor did it augment aspecific adsorption of free gonadotrophin on the cellulose matrix. Precision of duplicates was comparable for both methods but the accuracy and the detection limit of the solid phase system were higher due to a better separation of bound and free hormone, to the lower blank value and to the absence of aspecific interference of protein concentration. Cost was lower since much less second antibody was needed in the solid phase system than in the soluble system and since the experimental procedures were simplified by the absence of prozoning effect and the solid nature of the cellulose matrix.