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.

Effect of Ethanol Extract of Whole Plant of Trichosanthes cucumerina var. cucumerina L. on Gonadotropins, Ovarian Follicular Kinetics and Estrous Cycle for Screening of Antifertility Activity in Albino Rats

Ethanol extract of whole plant of Trichosanthes cucumerina L. var. cucumerina was evaluated for antiovulatory activity in adult rats. The ethanol extract at the doses 200 and 400mg/kg body weight (orally) affected the normal estrous cycle showing a significant increase in estrus and metestrus phases and decrease in diestrus and proestrus phases. The extract also significantly reduced the number of healthy follicles (Class I-Class VI) and corpora lutea and increased the number of regressing follicles (Stage IA, Stage IB, Stage IIA, and Stage IIB). The protein and glycogen content in the ovaries were significantly reduced in treated rats. The cholesterol level was significantly increased, whereas, the enzyme activities like 3b-HSD and 17b-HSD were significantly inhibited in the ovary of treated rats. Serum FSH and LH levels were significantly reduced in the treated groups were measured by RIA. In acute toxicity test, neither mortality nor change in the behavior or any other physiological activities in mice were observed in the treated groups. In chronic toxicity studies, no mortality was recorded and there were no significant differences in the body and organ weights were observed between controls and treated rats. Hematological analysis showed no significant differences in any of the parameters examined (RBC, WBC count and Hemoglobin estimation). These observations showed the antiovulatory activity of ethanol extract of whole plant of Trichosanthes cucumerina L. var. cucumerina in female albino rats.

Dissociation of monoclonal antibody-antigen complexes: Implications for ELISA procedures

A single step solid phase radioimmunoassay (SS-SPRIA) has been developed for human chorionic,gonadotropin (hCG) using monoclonal antibodies (MAb) from culture media adsorbed immunochemically on plastic tubes. The assays have been found to be very simple in terms of operation and do not demand purification of MAbs. Several MAbs which do not show any displacement in liquid phase RIA and ELISA provide a satisfactory SS-SPRIA. Our investigations revealed that the assumption regarding the stability of the primary Mab-Ag complex during incubation and washing steps in ELISAs is not strictly valid for dissociable MAbs. A comparison of different assay systems suggests that the single step SPRIA offers additional advantages over conventionally used multistep ELISA procedures and provides a quantitative probe for the analysis of epitope-paratope interactions.

Establishment of the functional importance of thiamin carrier protein in pregnant rats by using monoclonal antibodies

Monoclonal antibodies (mAbs) to chicken thiamin carrier protein (TCP) have been produced by hybridoma technology to identify the crucial epitopes involved in bioneutralization of the vitamin carrier. The monoclonality of these mAbs (A4C4, F3H6, H8H3, C8C1 and G7H10) was sought to be confirmed by sub-class isotyping; they all belong to IgG1, k type. The epitopes recognized by all the five mAbs are conserved in TCP from the chicken to the rat as assessed by liquid phase RIA and immunoprecipitation of I-125-labelled proteins from pregnant rat serum. Among these mAbs, passive immunization of pregnant rats with the mAb C8C1 only on three consecutive days (day 10, 11 and 12) resulted in embryonic resorption. These results demonstrate the importance of epitopic structure specified by the mAb C8C1 on TCP during pregnancy in rats.

Activity of TiO2 in the System ZrO2-TiO2 and Gibbs Energy of Formation of ZrTiO4

The activity of Ti02 in single and two··phase regions of ihe system ZrOrTi02 has heen measured lIsing solid state cells based on yttria··doped tho ria (YDT) as the solid electrolyte at 1373 K. The cells used can be represented as: Pt, Tio.07PtO.Y3 + Zrj.,Tix0 2 / YDT / Ti02 + Tio.07Pto.93, Pt Pt, Tio.07Pto.93 + ZrJ.xTix02 + ZrTi04 / YDT / Ti02+ Tio.07PtO.93, Pt In each cell the composition of Pt-Ti alloy was identical at hoth electrodes. The emf of the cell is therefore directly related to the activity of Ti02 in oxide phase or oxide phase mixture: aTiO~ :;: cxp (-4FE/RT). The activity coefficient of Ti02 in th~ zirconia-rich solid solution with monoclinic structure (CUl2 2" XTi02 2" 0) can be expressed as:In the zirconia-rich solid solution with tetragonal structure (0.085 2" X ri02 2" 0.03), the activity coefficient is given by:In YTi02 (± 0.012) = 2.354 (1-XTiO? )2 +0.064 The standard Gibbs energy of formation of ZrTi04 is -5650 (± 200) J/mol at 1373 K .

Raman and infra-red microspectroscopy: towards quantitative evaluation for clinical research by ratiometric analysis

Biomolecular structure elucidation is one of the major techniques for studying the basic processes of life. These processes get modulated, hindered or altered due to various causes like diseases, which is why biomolecular analysis and imaging play an important role in diagnosis, treatment prognosis and monitoring. Vibrational spectroscopy (IR and Raman), which is a molecular bond specific technique, can assist the researcher in chemical structure interpretation. Based on the combination with microscopy, vibrational microspectroscopy is currently emerging as an important tool for biomedical research, with a spatial resolution at the cellular and sub-cellular level. These techniques offer various advantages, enabling label-free, biomolecular fingerprinting in the native state. However, the complexity involved in deciphering the required information from a spectrum hampered their entry into the clinic. Today with the advent of automated algorithms, vibrational microspectroscopy excels in the field of spectropathology. However, researchers should be aware of how quantification based on absolute band intensities may be affected by instrumental parameters, sample thickness, water content, substrate backgrounds and other possible artefacts. In this review these practical issues and their effects on the quantification of biomolecules will be discussed in detail. In many cases ratiometric analysis can help to circumvent these problems and enable the quantitative study of biological samples, including ratiometric imaging in 1D, 2D and 3D. We provide an extensive overview from the recent scientific literature on IR and Raman band ratios used for studying biological systems and for disease diagnosis and treatment prognosis.