Comparative evaluation of various solid phases for the development of coated tube assays for the estimation of progesterone in human serum, bovine serum and bovine milk.

Immobilization of progesterone antibody using three polystyrene surfaces and two progesterone radiotracers for use in the development of a coated tube assay for the evaluation of progesterone levels in human serum, bovine serum and bovine milk was studied. The selection of the solid phase and the tracers were based on the maximum binding, non-specific binding, sensitivity and percentage recovery. Amongst the polystyrene tubes studied, streptavidin coated tubes showed the acceptable assay features such as low non-specific binding (0.5-1.0%), adequate sensitivity (0.13-0.16 ng/ml) and recovery (85-115%) for all the three sample matrices, human serum, bovine serum and bovine milk.

Surface modification of polystyrene using polyaniline nanostructures for biomolecule adhesion in radioimmunoassays.

The selection of an appropriate surface as a solid phase for coupling antibodies is a critical step in the development of solid-phase immunoassays. Availability of a new method of preactivating the surface of polystyrene tubes with a layer of another polymer for enhanced immobilization of antibodies seems to be promising. In this paper, we report the activation of a polystyrene surface using a layer of polyaniline and its effect on immobilizing antibodies for use as a solid phase in a T3 immunoassay. The modified surface on the polystyrene was characterized by optical absorption, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The modified tubes were coated with antibody and evaluated for their performance in the assay and validated for radioimmunoassay of T3. AFM images of the modified surface showed an enhancement in the surface roughness (Ra of 20.2 nm), as compared to an unmodified surface (Ra of 6 nm), allowing more adsorption of antibodies to the surface. XPS revealed the presence of N (binding energy approximately 400 eV) on the modified surface, which could help the antibody molecules to bind to these preactivated (modified) tubes. The modified tubes, when coated with antibody, not only showed an increase in the binding with the radioiodinated tracer but also improved the precision of coating the antibody. The present method of activating polystyrene surfaces is simple, does not involve severe chemical treatment, and may have wide applicability to functionalize other supports for immobilizing biomolecules.

Studies on the influence of the structural modifications in the tracer on the immunoassay of progesterone.

The main objective of the present study was to examine the influence of different bridges in radioiodinated tracers on the assay performance of progesterone using antibodies. Three homologous and two heterologous immunoassay systems for the measurement of progesterone in human serum are described. Using an antiserum raised against progesterone-11alpha-hemisuccinate-bovine serum albumin (BSA), assays with homologous radioligands, namely progesterone-11alpha-hemisuccinate-125I-tyrosine methyl ester (TME) and progesterone-11alpha-hemisuccinate-125I-histamine, heterologous bridge radioligand, namely progesterone-11alpha-hemiphthalate-125I-TME, and a heterologous site radioligand namely progesterone-3-(O-carboxymethyl) oxime (CMO)-125I-histamine were optimized. A homologous assay system, using antiserum raised against progesterone-3-carboxymethyl oxime-BSA and progesterone-3-CMO-125I-histamine as the radioligand was also optimized to develop a radio-immunoassay (RIA) for serum progesterone. Amongst the two homologous radioligands, viz., progesterone-11alpha-hemisuccinate-125I-histamine and the corresponding TME conjugate tracer, the former yielded a standard curve with a higher slope (-0.6) as compared to the latter (-0.5). The heterologous bridge system with progesterone-11alpha-hemiphthalate-125I-TME resulted in a more sensitive assay (slope of -0.8) than the homologous tracers, whilst the heterologous site radioligand, viz., progesterone-3-CMO-125I-histamine gave the most sensitive assay (slope of -1.2). The homologous assay with antiserum against progesterone-3-CMO-BSA and progesterone-3-CMO-125I-histamine tracer gave a standard curve having a slope of -0.97. The two antibodies developed against progesterone, viz., progesterone-11alpha-hemisuccinate-BSA and progesterone-3-CMO-BSA were characterized for their titre, sensitivity, and specificity. Considering the slope, sensitivity, cross-reactivity, and the quality of tracer, the assay system using antiserum against progesterone-11alpha-hemisuccinate-BSA and progesterone-3-CMO-125I-histamine was found to be suitable for the development of RIA for serum progesterone. The bridges used in an immunogen for production of antibodies, as well as in the preparation of tracer, have a great influence on the assay characteristics.