Immunochemical approach to the mapping of an assembled epitope of human chorionic gonadotropin: Proximity of CTP-alpha to the receptor binding region of the beta-subunit

A single-step solid-phase RIA (SS-SPRIA) developed in our laboratory using hybridoma culture supernatants has been utilised for the quantitation of epitope-paratope interactions. Using SS-SPRIA as a quantitative tool for the assessment of epitope stability, it was found that several assembled epitopes of human chorionic gonadotropin (hCG) are differentially stable to proteolysis and chemical modification. Based on these observations an approach has been developed for identifying the amino acid residues constituting an epitopic region. This approach has now been used to map an assembled epitope at/near the receptor binding region of the hormone. The mapped site forms a part of the seat belt region and the cystine knot region (C34-C38-C88-C90-H106). The carboxy terminal region of the alpha-subunit forms a part of the epitope indicating its proximity to the receptor binding region. These results are in agreement with the reported receptor binding region identified through other approaches and the X-ray crystal structure of hCG.

Structural basis for the function of anti-idiotypic antibody in immune memory

We had earlier proposed a hypothesis to explain the mechanism of perpetuation of immunological memory based on the operation of idiotypic network in the complete absence of antigen. Experimental evidences were provided for memory maintenance through anti-idiotypic antibody (Ab2) carrying the internal image of the antigen. In the present work, we describe a structural basis for such memory perpetuation by molecular modeling and structural analysis studies. A three-dimensional model of Ab2 was generated and the structure of the antigenic site on the hemagglutinin protein H of Rinderpest virus was modeled using the structural template of hemagglutinin protein of Measles virus. Our results show that a large portion of heavy chain containing the CDR regions of Ab2 resembles the domain of the hemagglutinin housing the epitope regions. The similarity demonstrates that an internal image of the H antigen is formed in Ab2, which provides a structural basis for functional mimicry demonstrated earlier. This work brings out the importance of the structural similarity between a domain of hemagglutinin protein to that of its corresponding Ab2. It provides evidence that Ab2 is indeed capable of functioning as surrogate antigen and provides support to earlier proposed relay hypothesis which has provided a mechanism for the maintenance of immunological memory.

Structural basis for the function of anti-idiotypic antibody in immune memory

We had earlier proposed a hypothesis to explain the mechanism of perpetuation of immunological memory based on the operation of idiotypic network in the complete absence of antigen. Experimental evidences were provided for memory maintenance through anti-idiotypic antibody (Ab(2)) carrying the internal image of the antigen. In the present work, we describe a structural basis for such memory perpetuation by molecular modeling and structural analysis studies. A three-dimensional model of Ab(2) was generated and the structure of the antigenic site on the hemagglutinin protein H of Rinderpest virus was modeled using the structural template of hemagglutinin protein of Measles virus. Our results show that a large portion of heavy chain containing the CDR regions of Ab(2) resembles the domain of the hemagglutinin housing the epitope regions. The similarity demonstrates that an internal image of the H antigen is formed in Ab(2), which provides a structural basis for functional mimicry demonstrated earlier. This work brings out the importance of the structural similarity between a domain of hemagglutinin protein to that of its corresponding Ab(2). It provides evidence that Ab(2) is indeed capable of functioning as surrogate antigen and provides support to earlier proposed relay hypothesis which has provided a mechanism for the maintenance of immunological memory.

An avidin-biotin microELISA for rapid measurement of total and allergen-specific human IgE

his paper describes an improved microtiter solid-phase enzyme immunoassay for the determination of total and allergen-specific human IgE. This assay technique is unique in its use of the avidin-biotin interaction to increase sensitivity. The avidin-biotin microtiter enzyme-linked immunosorbant assay (AB-microELISA) was performed in polyvinyl chloride microtiter plates using biotinylated anti-IgE and horseradish peroxidase (HRP)-avidin conjugate. This AB-microELISA technique enabled the quantitation of human serum IgE in the range of 0.1–5 ng/ml (10–500 pg/test) in less than 3 h. Total serum IgE, whether measured by the AB-microELISA or the paper radioimmunosorbant test (PRIST) was similar (correlation coefficient, r = 0.92). Further, the presence or absence of positive skin tests to 7 specific allergens determined in serum donors generally agreed with the presence or absence of allergen-specific IgE in their sera as measured by the AB-microELISA. The quantity of short ragweed allergen-specific IgE as determined by the AB-microELISA agreed with values obtained by the radioimmunosorbant test (RAST) (correlation coefficient, r = 0.89). No significant interference by ragweed-specific IgG (blocking antibody) was observed in the quantitation of allergen-specific IgE. The AB-microELISA is not only rapid and inexpensive, but also more sensitive than other published ELISA procedures and comparable to solid-phase radioimmunoassays in the quantitation of total and allergen-specific IgE.

A delphi study on health in future India

PIP: A delphi study was conducted to identify or envision health scenarios in India by the year 2000. Questionnaires consisting of 48 questions on 5 areas (diagnosis and therapy; family planning; pharmaceuticals and drugs; biochemical and biomedical research; health services) were mailed to 250 experts in India. 36 responded. Results were compiled and mailed back to the respondents for changes and comments. 17 people responded. Results of the delphi study shows that policy decisions with respect to compulsory family planning as well as health education at secondary school level will precede further breakthroughs in birth control technology. Non operation reversible sterilization procedures, immunological birth control, Ayurvedic medicines for contraception and abortion, and selection of baby's sex are all possible by 2000 thereafter. Complete eradication of infectious diseases, malnutrition and associated diseases is considered unlikely before 2000, as are advances in biomedical research. Changes in health services (e.g., significant increases in hospital beds and doctors, cheap bulk drugs), particularly in rural areas, are imminent, leading to prolonging of life expectancy to 70 years. Genetic engineering may provide significant breakthroughs in the prevention of malignancies and cardiac disorders. The India delphi study is patterned after a similar delphi study conducted in the U.S. by Smith, Kline and French (SKF) Laboratories in 1968. The SKF study was able to predict some breakthroughs with basic research which have been realized.

Purification of Antibodies Specific to a Dinucleotide Using the Hapten Bound to Deae Cellulose as an Affinity Column

The dinucleotide dpTpA held electrostatically on DEAE cellulose was used as an affinity column for the purification of dpTpA specific antibodies. Chromatography of the y-globulin fraction from dpTpA specific antisera on this column resulted in the retention of dpTpA specific antibodies which were later eluted along with the bound dpTpA using 1M NaC1. Dextran coated charcoal was the method of choice for the dissociation and removal of dpTpA bound to the antibodies. This method may extend itself to the purification of antibodies specific for other oligonucleotides.

Chemical Modification Studies On Ricinus-Communis (Castor Bean) Agglutinin

Ricinus communis agglutinin was subjected to various chemical treatments and the effect on its hemagglutinating and saccharide-binding properties was studied. Acetylation, succinylation and citraconylation led to a complete loss in the activity of the agglutinin, whereas reductive methylation had no effect on the activity, showing that charged amino groups were involved in the hemagglutinating and saccharide-binding activity of Ricinus agglutinin. Modification of tryptophyl, arginyl and carboxyl-group-containing residues did not lead to any loss in the activity of the agglutinin. Acetylation of tyrosyl groups with N-acetylimidazole strongly reduced the hemagglutinating and saccharide-binding property of Ricinus agglutinin. The loss in activity was restored on deacetylation of the tyrosyl groups. Modification of tyrosyl residues also led to a change in the immunological properties of the agglutinin. The initial rate of modification of tyrosyl and amino groups and the concomitant loss of activity was reduced in the presence of lactose.

Arginine Decarboxylase from Lathyrus sativus Seedlings Purification and Properties

Arginine decarboxylase which makes its appearance in Lathyrus sativus seedlings after 24 h of seed germination reaches its highest level around 5–7 days, the cotyledons containing about 60% of the total activity in the seedlings at day 5. The cytosol enzyme was purified 977-fold from whole seedlings by steps involving manganese chloride treatment, ammonium sulphate and acetone fractionations, positive adsorption on alumina C-γ gel, DEAE-Sephadex chromatography followed by preparative disc gel electrophoresis. The enzyme was shown to be homogeneous by electrophoretic and immunological criteria, had a molecular weight of 220000 and appears to be a hexamer with identical subunits. The optimal pH and temperature for the enzyme activity were 8.5 and 45 °C respectively. The enzyme follows typical Michaelis-Menten kinetics with a Km value of 1.73 mM for arginine. Though Mn2+ at lower concentrations stimulated the enzyme activity, there was no dependence of the enzyme on any metal for the activity. The arginine decarboxylase of L. sativus is a sulfhydryl enzyme. The data on co-factor requirement, inhibition by carbonyl reagents, reducing agents and pyridoxal phosphate inhibitors, and a partial reversal by pyridoxal phosphate of inhibition by pyridoxal · HCl suggests that pyridoxal 5'-phosphate is involved as a co-factor for the enzyme. The enzyme activity was inhibited competitively by various amines including the product agmatine. Highest inhibition was obtained with spermine and arcain. The substrate analogue, l-canavanine, homologue l-homoarginine and other basic amino acids like l-lysine and l-ornithine inhibited the enzyme activity competitively, homoarginine being the most effective in this respect.

Technological-forecasting and research inputs in area of birth-control technology

Technological forecasting, defined as quantified probabilistic prediction of timings and degree of change in the technological parameters, capabilities desirability or needs at different times in the future, is applied to birth control technology (BCT) as a means of revealing the paths of most promising research through identifying the necessary points for breakthroughs. The present status of BCT in the areas of pills and the IUD, male contraceptives, immumological approaches, post-coital pills, abortion, sterilization, luteolytic agents, laser technologies, and control of the sex of the child, are each summarized and evaluated in turn. Fine mapping is done to identify the most potentially promising areas of BCT. These include efforts to make oral contraception easier, improvement of the design of the IUD, clinical evaluation of the male contraceptive danazol, the effecting of biochemical changes in the seminal fluid, and researching of immunological approaches and the effects of other new drugs such as prostaglandins. The areas that require immediate and large research inputs are oral contraception and the IUD. On the basis of population and technological forecasts, it is deduced that research efforts could most effectively aid countries like India through the immediate production of an oral contraceptive pill or IUD with long-lasting effects. Development of a pill for males or an immunization against pre gnancy would also have a significant impact. However, the major impediment to birth control programs to date is attitudes, which must be changed through education.

Studies on the variants of the protein toxins ricin and abrin

his study elucidates some structural and biological features of galactose-binding variants of the cytotoxic proteins ricin and abrin. An isolation procedure is reported for ricin variants from Ricinus communis seeds by using lactamyl-Sepharose affinity matrix, similar to that reported previously for variants of abrin from Abrus precatorius seeds [Hegde, R., Maiti, T. K. & Podder, S. K. (1991) Anal. Biochem. 194, 101–109]. Ricin variants, subfractionated on carboxymethyl-Sepharose CL-6B ion-exchange chromatography, were characterized further by SDS/PAGE, IEF and a binding assay. Based on the immunological cross-reactivity of antibody raised against a single variant of each of ricin and abrin, it was established that all the variants of the corresponding type are immunologically indistinguishable. Analysis of protein titration curves on an immobilized pH gradient indicated that variants of abrin I differ from other abrin variants, mainly in their acidic groups and that variance in ricin is a cause of charge substitution. Detection of subunit variants of proteins by two-dimensional gel electrophoresis showed that there are twice as many subunit variants as there are variants of holoproteins, suggesting that each variant has a set of subunit variants, which, although homologous, are not identical to the subunits of any other variant with respect to pI. Seeds obtained from polymorphic species of R. communis showed no difference in the profile of toxin variants, as analyzed by isoelectric focussing. Toxin variants obtained from red and white varieties of A. precatorius, however, showed some difference in the number of variants as well as in their relative intensities. Furthermore, variants analyzed from several single seeds of A. precatorius red type revealed a controlled distribution of lectin variants in three specific groups, indicating an involvement of at least three genes in the production of Abrus lectins. The complete absence or presence of variants in each group suggested a post-translational differential proteolytic processing, a secondary event in the production of abrin variants.

Prospects of riboflavin carrier protein (RCP) as an antifertility vaccine in male and female mammals

Riboflavin carrier protein (RCP) is obligatorily involved in yolk deposition of the vitamin, riboflavin, in the developing oocyte of the hen. The production of this protein is inducible by oestrogen. It is evolutionarily conserved in terms of its physicochemical, immunological and functional characteristics. It is the prime mediator of vitamin supply to the developing fetus in mammals, including primates. Passive immunoneutralization of the protein terminates pregnancy in rats. Active immunization of rats and bonnet monkeys with avian RCP prevents pregnancy without causing any adverse physiological effects of the mother in terms of her vitamin status, reproductive cycles or reproductive-endocrine profile. Denatured, linearized RCP is more effective in eliciting neutralizing antibodies capable of interfering with embryonic viability either before or during peri-implantation stages. Two defined stretches of sequential epitopes, one located at the N-terminus and the other at the C-terminus of the protein have been identified. Active immunization with either of these epitopes conjugated with diptheria toxoid curtails pregnancy in rats and monkeys. Immunohistochemical localization of RCP on ovulated oocytes and early embryos shows that the antibodies cause degeneration only of early embryos. RCP is produced intra-testicularly and becomes localized on acrosomal surface of mammalian spermatozoa. Active immunization of male rats and monkeys with denatured RCP markedly reduces fertility by impairing the fertilizing potential of spermatozoa. These findings suggest that RCP, or its defined fragments, could be a novel, first generation vaccine for regulating fertility in both the sexes.

Effect of Nucleic Acid Reactive Antibodies on Tumor Cells Grown in Vivo

Nucleic acid reactive antibodies have been reported to inhibit various nucleio acid mediated functions in cell free systems. These antibodies were also shown to inhibit the growth of transformed cells in culture due to the high rate of endocytosis in transformed cells as compared to normal cells. In this report, we have tested the possibility of nucleic acid reactive antibodies inhibiting the growth of tumor cells in vivo. The life span of mice bearing Dalton's lymphoma ascites tumor cells was increased, when they were immunized with conjugates of guanosine-BSA, GMP-BSA and tRNA-MBSA complex before transplanting the tumor cells. A similar effect was also observed when mice were injected intraperitoneally with antibodies to guanosine oi GMP along with the tumor cells. The specificity was ascertained, as immunization with non-specific antigens did not show any significant effect on tumor bearing mice. The results shows that nucleic acid. reactive antibodies inhibit the growth of tumor cells in vivo.

Hormonal modulation of riboflavin carrier protein secretion by immature rat Sertoli cells in culture

We report here that a protein species with biochemical and immunological similarity with chicken egg riboflavin carrier protein (RCP) is synthesized and secreted by immature rat Sertoli cells in culture. When quantitated by a specific heterologous radioimmunoassay, optimal concentrations of FSH (25 ng/ml) brought about 3-fold stimulation of RCP secretion. FSH, in the presence of testosterone (10−6 M) brought about 6-fold stimulation of secretion of RCP over the control cultures which were maintained in the absence of these two factors. The aromatase inhibitor (1,4,6-androstatrien-3,17-dione) curtailed 85% of the enhanced secretion of RCP, suggesting that the hormonal stimulation is mediated through in situ synthesized estrogen and this could be confirmed with exogenous estradiol-17 β which brought about 3 — fold enhancement of secretion of RCP at a concentration of 10−6 M. When tamoxifen (10 μM) was added along with FSH and testosterone, there was 75% decrease in the enhanced secretion of RCP. Addition of this anti-estrogen together with exogenous estradiol resulted in 55% decrease in elevated levels of RCP. Cholera toxin (1 μg/ml) and 8-bromo-cyclic AMP (0.5 mM) mimicked the action of FSH on the secretion of RCP thus suggesting that FSH stimulation of RCP production may be mediated through cyclic AMP. These findings suggest that estrogen mediates RCP induction in hormonally stimulated sertoli cells presumably to function as the carrier of riboflavin to the developing germ cells through blood-testis barrier in rodents.

Expression of Human Growth Hormone in Silkworm Larvae through RecombinantBombyx moriNuclear Polyhedrosis Virus

We have generated a recombinantBombyx morinuclear polyhedrosis virus, vBmhGH, harboring the full-length human growth hormone gene (2.4-kb genomic DNA, with four introns and the signal peptide sequences) under the control of the polyhedrin promoter. BmN cells in culture infected with the recombinant virus showed the presence of RNA corresponding to the authentic growth hormone mRNA as well as its incompletly processed precusor. Electrophoretic analysis and immunoprecipitation of proteins of recombinant virus-infected BmN cells revealed the presence of the growth hormone protein. Infection of silkworm larvae with vBmhGH led to the synthesis and efficient secretion of the protein into hemolymph. The recombinant human growth hormone was biologically active in a radioreceptor competition binding assay. The secreted protein was isolated and purified to homogeneity by a single step immunoaffinity chromatography, to a specific activity of 2.4 × 104U/mg. The recombinant hGH retained the immunological and biolological properties of the native peptide. We conclude that BmNPV vectors can be used successfully for expressing chromosomal genes harboring multiple introns.

Prediction of Candidate Primary Immunodeficiency Disease Genes Using a Support Vector Machine Learning Approach

Screening and early identification of primary immunodeficiency disease (PID) genes is a major challenge for physicians. Many resources have catalogued molecular alterations in known PID genes along with their associated clinical and immunological phenotypes. However, these resources do not assist in identifying candidate PID genes. We have recently developed a platform designated Resource of Asian PDIs, which hosts information pertaining to molecular alterations, protein-protein interaction networks, mouse studies and microarray gene expression profiling of all known PID genes. Using this resource as a discovery tool, we describe the development of an algorithm for prediction of candidate PID genes. Using a support vector machine learning approach, we have predicted 1442 candidate PID genes using 69 binary features of 148 known PID genes and 3162 non-PID genes as a training data set. The power of this approach is illustrated by the fact that six of the predicted genes have recently been experimentally confirmed to be PID genes. The remaining genes in this predicted data set represent attractive candidates for testing in patients where the etiology cannot be ascribed to any of the known PID genes.