Pramod V. Prasad

Extracellular calcium protects against verapamil-induced metaphase-II arrest and initiation of apoptosis in aged rat eggs.

Non‐specific L‐type calcium channel blockers, such as verapamil (≥50 μM), induce metaphase‐II (M‐II) arrest and apoptosis in aged rat eggs cultured in Ca2+‐deficient medium. However, the effects of extracellular Ca2+ on verapamil‐induced M‐II arrest and apoptosis have not yet been reported. We have demonstrated that postovulatory aging induced exit from M‐II arrest by extruding a second polar body, a morphological sign of spontaneous egg activation (SEA). Verapamil inhibited SEA and induced egg apoptosis in a dose‐dependent manner in Ca2+‐deficient medium. The initiation of apoptotic features was observed at 50 μM of verapamil. Extracellular Ca2+ (1.80 mM) reduced intracellular H2O2 level, bax protein expression, caspase‐3 activity, DNA fragmentation and protected against 50 μM, but not higher concentrations of ≥100 μM in verapamil‐induced egg apoptosis. These results suggest that extracellular Ca2+ ions have a role during SEA and protect against verapamil‐induced apoptosis in aged rat eggs.

Development of Colorimetric Enzyme‐Linked Immunosorbent Assay for Human Chorionic Gonadotropin

Abstract The present study demonstrated the development of a solid phase competitive enzyme linked immunosorbent assay (ELISA) for direct estimation of human chorionic gonadotropin (hCG) in serum and urine. Polyclonal antisera raised against the β‐ subunit of peak‐I hCG was used in the assay. The Peak‐IA hCG‐penicillinase was used as tracer. The performance of this antiserum and tracer was compared against hCG‐β antisera of NIH, USA and penicillinase conjugated to hCG‐β obtained from NIH, respectively. Almost parallel standard curves were obtained in both cases, suggesting that these antisera and enzyme label have much potential for developing ELISA system. To the anti‐rabbit gamma globulin (ARGG) coated polystyrene tubes, standard or serum or urine samples (50 µL), 100 µL of hCG‐β antiserum, 100 µL of peak‐I(A) hCG‐penicillinase conjugate and 350 µL of assay buffer were incubated at 37°C for 2 hours. Bound enzyme activity was measured using Penicilline V as substrate. In this new strategy, locally available polystyrene tubes were ground from inside and coated with ARGG. The sensitivity of the assay was 17 mIU/mL in urine and 18 mIU/mL in serum. The intra‐assay and inter‐assay coefficients of variation (CVs) appeared to be within acceptable limits of 10%. The serum and urinary hCG values, obtained by this method, correlated well with those obtained by radioimmunoassay (RIA) r=0.98 (n=100 for serum samples; n=250 for urinary samples).

Heterologous enzyme linked immunosorbent assay for measurement of testosterone in serum.

In steroid enzyme immunoassay (EIA), homologous and heterologous combinations of enzyme conjugate with immunogen influences labeled steroid recognition by antibodies that affect sensitivity of the assay. To develop testosterone enzyme linked immunosorbent assay (ELISA), antibodies were generated against testosterone-3-carboxymethyloxime-bovine serum albumin (T-3-CMO-BSA), testosterone-11-hemisuccinate-bovine serum albumin (T-11-HS-BSA), testosterone-17-hemisuccinate-bovine serum albumin (T-17-HS-BSA), testosterone-17-glucuronide-bovine serum albumin (T-17-G-BSA), and testosterone-19-carboxymethylether-bovine serum albumin (T-19-CME-BSA). Testosterone horseradish peroxidase (HRP) enzyme conjugate were prepared using carboxyl derivatives of 11-keto-testosterone (11-keto-T) and 1-dehydrotestosterone (1-Dehydro-T). Ten combinations of heterologous assays were evaluated. The data of the present study revealed that the use of the T-11-HS-BSA antibody in antigen plus site heterologous assay that employed 11-keto-testosterone-3-CMO-HRP as the label showed binding and displacement, and led to the development of sensitive and specific assay.

INFLUENCE OF DIFFERENT LENGTH SPACERS CONTAINING ENZYME CONJUGATE ON FUNCTIONAL PARAMETERS OF PROGESTERONE ELISA

In steroid enzyme immunoassay (EIA), there is an increase or decrease of labeled steroid recognition by antibody due to homologous and heterologous combinations of enzyme conjugate with immunogen that affects sensitivity of the assay. We have introduced three to 18 atomic length linkers between enzyme and steroid moieties and studied their effects on functional parameters such as sensitivity, ED50, and specificity of progesterone enzyme immunoassays. Progesterone-3-carboxymethyloxime–bovine serum albumin (P-3-CMO-BSA) was used as an immunogen to raise the antiserum in New Zealand white rabbits. Five enzyme conjugates were prepared using 17-α-hydroxy-progesterone-3-carboxymethyloxime (17-α-OH-P-3-CMO) as carboxylic derivative of 17-α-hydroxy-progesterone and horseradish peroxidase (HRP) as label. These were 17-α-OH-P-3-CMO-HRP, 17-α-OH-P-3-CMO-urea-HRP (17-α-OH-P-3-CMO-U-HRP), 17-α-OH-P-3-CMO-ehylenediamine-HRP (17-α-OH-P-3-CMO-EDA-HRP), 17-α-OH-P-3-CMO-carbohydrazide-HRP (17-α-OH-P-3-CMO-CH-HRP), and 17-α-OH-P-3-CMO-adipic acid dihydrazide-6-aminocaproic acid-HRP (17-α-OH-P-3-CMO-ADH-6ACA-HRP). The influence of different atomic length linkers on sensitivity, ED50, and specificity were studied with reference to label without linker. The results of the present investigation revealed that the incorporation of ADH-6ACA spacer in 17-α-hydroxy-progesterone-enzyme conjugate improved the sensitivity in antigen plus bridge heterologous EIA system. The presence of spacer in enzyme conjugate improved the sensitivity and specificity (cross-reactivity) in some antigen plus bridge heterologous assay of progesterone.

Isolation of hCG and its Characterization by Radioimmunoassay, Enzyme‐Immunoassay, and Radio‐Receptor Assay

Abstract The nature of human chorionic gonadotropin (hCG) molecules present during early pregnancy of Indian women is poorly understood. Therefore, a study has been undertaken to isolate hCG and characterize different forms of hCG from urine. The hCG molecules from urine of pregnant women (45–75 days post LMP) were adsorbed onto kaolin, eluted with ammonium hydroxide, and precipitated using acetone and then lyophilized. The lyophilized extract was subjected to Sephadex G‐100 chromatography followed by ion‐exchange fractionation. Three major fractions of protein (i.e., Peaks I, II, and III) associated with carbohydrate activity were obtained. Peaks II and III eventually resolved into a single peak I following repeated ion exchange chromatography, which suggested the presence of aggregates of molecules. Further purification on an affinity column resolved all three peak fractions into one unadsorbed and two adsorbed (A and B) fractions. These adsorbed fractions were characterized by radioreceptor assay (RRA), radioimmunoassay (RIA), and enzyme linked immunosorbent assay (ELISA). The activity was standardized against WHO reference preparation 75/589. Peaks I (A and B) were found to have maximum at about 75% of immunologically potent hCG, followed by peaks II (40%) and III (5%). The molecular sizes of peaks I, II, and III on a Sephadex G‐200 column corresponded to 27,500D, 66,000D, and 84,000D, respectively. Relative mobilities of all adsorbed fractions in sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) showed the presence of hCG‐α (mol. wt. 19,539D) and hCG‐β (28,870D) subunits. The presence of both subunits of hCG were also revealed by Western blot analysis. For the first time, we report the low molecular weight hCG molecule, of 27,500D by size exclusion chromatography, which has immunological and biological activity as measured by RIA, ELISA, and RRA.

Development of Enzyme-Linked Immunosorbent Assay for Estimation of Urinary Albumin

Yearly estimation of urinary albumin is a prerequisite for predicting renal status in Diabetes Type II patients with negative dipstick results for overt proteinuria. A simple, sensitive, and cost-effective enzyme linked immunosorbent assay (ELISA) for urinary albumin has been developed using human serum albumin antiserum (HSA-antiserum), HSA-biotin, and streptavidin-horseradish peroxidase (SA-HRP) conjugates. To the antibody-coated wells, 100 μL of HSA standards followed by 1:100 diluted urine samples in duplicate were added and then 50 μL of HSA-biotin conjugates was added in all the wells. 100 μL of SA-HRP was added after washing. Bound enzyme activity was measured by adding 100 μL TMB/H2O2. The analytical sensitivity and ED50 of the developed method was found to be 0.01 μg/mL and 0.35 μg/mL, respectively. The percent recovery of the HSA from exogenously spiked urine pools were in the range of 98.13–100.29%. The intra- and inter-assay coefficient of variation (CVs) ranged from 3.38–10.32 % and 4.22–11.01%, respectively. The antibody showed 4.4% and 3.2% cross reactivity with monkey and horse serum albumin, respectively. There was no cross reaction with human β2-microglobulin, γ-globulin, and haemoglobulin.

Deciphering a Conformation-Specific Epitope of hCG-β Through Immunokinetics

Abstract Proteins and peptides are comprised of both sequence-specific and conformation-specific epitopes. Sequence-specific epitopes are delineated by a peptide approach and other robust methods like competition assays, gene expression assays, synthetic peptide library based assays, etc. Available methods for deciphering conformation-specific epitopes are cumbersome (X-ray crystallography, etc.), time-consuming, and require expensive equipment. Therefore, it is indispensable to develop a simple method for identification and mapping of conformation-specific epitopes. In the present investigation, the radiolabeled human chorionic gonadotropin-β (125IhCGβ) was employed as a probe and nitrocellulose (NC) as a solid support to immobilize monoclonal antibody (MAb) G1G10.1. The NC-G1G10.1-125IhCGβ complex (NCcom) was prepared and the dissociation of radiolabeled hCGβ was carried out in the presence of excess unlabeled ligate. From the experimental dissociation data under varying ionic strength, dissociation constants (k−1), association constants (k+1), and affinity constants (ka) were calculated. The values obtained were utilized in exploring the amino acid residues constituting an epitopic region of hCGβ involved in interaction with the complementary paratope on MAb G1G10.1. Kinetic data of the present study supported our recently published findings [using single step-solid phase radioimmunoassay (SS-SPRIA)] that the core region of a conformation-specific epitope of hCGβconsists of Arg (94, 95) and Asp (99) while a Lys (104) and a His (106) are in proximity to the core epitopic region. Therefore, the results of the present investigation suggested that the dissociation kinetics coupled with SS-SPRIA unequivocally assists in deciphering amino acid residues constituting a conformation-specific epitope of hCGβ.

Kinetic analysis of a human chorionic gonadotropin-β epitope–paratope interaction

Kinetics of protein–protein or ligand–ligate interaction has predominantly been studied by optical spectroscopy (particularly fluorescence) and surface plasmon resonance biosensors. Almost all such studies are based on association kinetics between ligand–ligate and suffer from certain methodological and interpretational limitations. Therefore, kinetic analyses of dissociation data of such interactions become indispensable. In the present investigation, the radiolabeled human chorionic gonadotropin-β (125IhCGβ) was employed as a probe and nitrocellulose (NC) as a solid support to immobilize monoclonal antibody (MAb) G1G10.1. The NC–G1G10.1–125IhCGβ complex (NCcom) was prepared and the dissociation of radiolabeled hCGβ was carried out in the presence of excess unlabeled ligate. From the experimental dissociation data under varying ionic strength, dissociation constants (k− 1), association constants (k+1) and affinity constants (ka) were calculated. The values obtained were utilized in exploring the amino acid residues constituting an epitopic region of hCGβ involved in interaction with the complementary paratope on MAb G1G10.1. Kinetic data of the present study supported our recently published findings [using single step-solid phase radioimmunoassay (SS-SPRIA)] that the core region of hCGβ epitope consists of Arg (94,95) and Asp (99) while a Lys (104) and a His (106) are in proximity to the core epitopic region. Based on the results of present investigation, we conclude that dissociation kinetics coupled with SS-SPRIA unequivocally provides considerable insight into the study of ligand–ligate interactions and epitope analysis.

Ergonomical Evaluation of Manual and Power Operated Weeders in Dry Land Condition

Weeding operation is an important among field operations, which affects the yield 30 to 60 per cent due to delay and negligence in operation. Drudgery involved in weeding operation increases stress on the worker causing increase in heart rate and oxygen consumption. The main focus of the study was to evaluate ergonomical and mechanical parameters of power weeder and wheel hoe. The estimation of oxygen consumption rate (OCR) by measuring the energy expenditure rate (EER) is a fairly accurate and acceptable method. The heart rate of workers varied from 109.47 to 130.66 beats/min by using power weeder and 130.33 to 147.52 beats/min by using wheel hoe. The oxygen consumption rate of workers from 0.873 to 1.302 L/min with power weeder and 1.389 to 1.738 L/min with wheel hoe. The actual field capacity of 114 and 208 man-h/ha were observed for power weeder and wheel hoe, respectively. The weeding efficiency of power weeder and wheel hoe were observed to be 8 and 75 per cent, respectively. The maximum value of weeding efficiency (8%) was observed in case of power weeder.

Isolation of α‐ and β‐Subunits of Peak‐I hCG and Generation of Highly Specific Polyclonal Antisera

Abstract Development of polyclonal antisera is still a choice in some hard‐pressed budget laboratories. In the present study, an attempt was made to isolate α‐ and β‐subunits from peak‐I hCG, generation of polyclonal antisera and their characterization. The anti‐hCG‐α antisera showed titres of 1∶8000 and anti‐hCG‐β antisera 1∶16,000 at 50% binding to radiolabelled hCG in RIA. Studies on specificity using anti hCG‐β antiserum demonstrated no cross‐reaction with several hormones tested in the present study, except for hCG‐β and hCG, thus eliciting a highly specific hCG‐β antiserum.