Mapping of assembled epitopic regions of human chorionic gonadotropin reveals proximity of CTPα to the determinant loop ß93-100.

Three overlapping assembled epitopes of ßhCG have been mapped using MAb probes and a single step solid phase radioimmunoassay. These epitopes have been shown to be at receptor binding region comprising of the loop region ß Cys93-Cys100. Importance of disulphide bonds in maintaining integrity of these epitopes is assessed. Two MAbs (INN 58 and INN 22) interact with the ß region as well as the α C-terminal peptide, while the other MAb INN 24 interacts with only the ß region. Cross-reactivity pattern with ßhCG and hLH as well as the reported crystal structure of hCG substantiates the epitope identification. The results demonstrate utility of MAbs as probes in investigations on three-dimensional structure of gonadotropins.

Determination of kinetic parameters of epitope-paratope interaction based on solid phase binding: An inexpensive alternate to biospecific interaction analysis.

A method has been developed for biospecific interaction analysis between antigen and antibody using solid phase binding approach. Real time kinetics between monoclonal antibody and human chorionic gonadotropin have been studied. Kinetic constants of the bimolecular reaction are determined. Affinity constants measured by several independent methods have been found to be relatively consistent. Convenient and simple procedures to determine affinity constant, Kon and Koff of monoclonal antibody-human chorionic gonadotropin interaction using binding of [125I]hCG to immobilized monoclonal antibody are presented. Values obtained compare well with those obtained using surface plasmon resonance technology,making this method a viable alternative.

Ventricular tachycardia with left bundle branch block morphology in the absence of ischaemic heart disease--clinical and electrophysiological observations.

Clinical and electrophysiological features of 20 patients presenting with ventricular tachycardia (VT) of left bundle branch block (LBBB) morphology without evidence of coronary artery disease were studied. The mean age of the patients was 35.2 +/- 12 (range 15-57 years). The rate of VT varied between 140-240/min (182 +/- 80). Six (30%) patients experienced giddiness or syncope during palpitations. Structural heart disease was found in 10 (50%) of these patients, which included arrhythmogenic right ventricular dysplasia in five, submitral left ventricular (LV) aneurysm in one, anterolateral LV dyskinesis in one, dilated cardiomyopathy in one, endomyocardial fibrosis in one and nonobstructive hypertrophic cardiomyopathy in one case. Ten patients were free of structural heart disease. Electrophysiological study was done in all patients. VT with same morphology as spontaneous VT was inducible in only 14 patients. Seventeen patients were treated medically with total or partial amelioration of symptoms. In three patients, two with arrhythmogenic right ventricular dysplasia and one with structurally normal heart, who were unresponsive to drug therapy, the VT focus could be mapped in right ventricular outflow tract and successful electrical ablation was done. Thus in patients who present with VT with LBBB morphology, the heart is often structurally normal but organic disease is not uncommon, and should be carefully searched.

Radioimmunoassay of polypeptide hormones using immunochemically coated plastic tubes.

A method has been developed for immobilisation of antisera on fresh plastic tubes through an immunochemical bridge. This type of immobilisation has been shown to be more consistent than direct adsorption on plastic. Such immunochemically coated antisera on plastic tube has been used in the development of a noncentrifugation radioimmunoassay. This assay system has been found to be technically as sound as the conventional method.

Immunological crossreactivity of the antisera to purified hormones.

Antisera to ovine follicle stimulating hormone free of ovine lutinising hormone contamination has been obtained in monkeys. These antisera have been shown to be able to crossreact with ovine lutinising hormone. Quantitation of the binding data for ovine follicle stimulating hormone and ovine lutinising hormone show that 10–40% of the total antibody population to ovine follicle stimulating hormone can bind to ovine lutinising hormone and the affinity constant for ovine lutinising hormone is about 2–20 times lesser than for ovine follicle stimulating hormone. These binding data indicate that there are common epitopes exposed in ovine follicle stimulating hormone and ovine lutinising hormone through the α- subunit. Results are obtained which match with the above conclusions when ovine lutinising hormone antisera is analysed for ovine follicle stimulating hormone binding. These results show that the α-subunit when combined with different ß-subunits will have common epitopes exposed, but would be sterically disposed differently in the two hormones.

Use of epoxysepharose for protein immobilisation.

Epoxy Sepharose, an activated affinity matrix which has been used for immobilisation of carbohydrates has been tried for immobilisation of proteins. Under normal conditions of coupling at neutral or alkaline pH proteins do not couple to epoxy Sepharose. However, a very high salt concentration during coupling allows the binding of proteins to epoxy Sepharose at a pH as low as 8·5. Increasing ionic strength and/or pH facilitates the binding. The bioactivity of the proteins is not destroyed by the immobilisation. This matrix, unlike cyanogen bromide-Sepharose, retains its ability to bind albumin by 80–90% even after 60 days of storage in aqueous suspension at 4°C. Its capacity to bind proteins is comparable to that of cyanogen bromide-Sepharose.

A solid phase radioimmunoassay for detection of early pregnancy in the South Indian bonnet monkey Macaca radiata.

Using an antibody raised in the rabbit to ovine leutenizing hormone β subunit coupled to activated cellulose, a solid phase radioimmunoassay to detect early pregnancy in the South Indian bonnet monkey has been developed. Non-specific inhibition due to serum was eliminated by inclusion of new born calf serum in the assay tubes. The assay is simple, needs only one centrifugation and can be completed in 6 h at room temperature with no false positive results.

Practical considerations in analyzing radioreceptor assays by Scatchard analysis and capacity determination in irreversible hormone receptor interactions.

The Scatchard plot in a radioreceptor assay depends upon the definition of specific binding and the quality of the iodinated hormone used. Iodination of protein hormones may alter it so that it no longer binds to the receptor and methods are available to measure the extent of this inactivation. When appropriate corrections are made for specific binding and the amount of inactive iodinated hormone in an assay, both qualitative and quantitative differences were observed in estimates of binding capacity and affinity in some well characterised hormone receptor systems. Theoretical predictions derived from Scatchard analysis of irreversible unimolecular hormone-receptor interactions were applicable, both qualitatively and quantitatively to two irreversible hormone-receptor systems. A method described permits a more accurate estimate of capacity from radioreceptor assay data.

Irreversibility of the interaction of human growth hormone with its receptor and analysis of irreversible reactions in radioreceptor assays—Theoretical considerations.

Kinetic studies of the binding and dissociation of [125I]-human growth hormone to rabbit liver and mammary gland membrane receptors have showed that the binding of [125I]- human growth hormone was largely irreversible to liver membrane receptors and completely to the solubilised mammary gland receptor. As Scatchard analysis assumes complete reversibility of the hormone-receptor interaction the validity of estimates of affinity and capacity of receptors derived by this analysis may be questionable. Theoretical considerations show that in unimolecular irreversible interactions of hormone and receptor, a nonlinear (concave) or a linear Scatchard plot can be obtained. In linear Scatchard plots the capacity of the receptor obtained by extrapolation represents an overestimation of true capacity. This overestimation correlates with the value of the intercept in the Scatchard plot.