Thesis (Ph.D.)--Boston University. / The inhibition technique was used in an attempt to determine the chemical structure of Rh antigens. A saline agglutinating Rh antibody was mixed with a chemical compound of known structure, containing carbohydrate, and believed to be similar in configuration to the Rh antigen in question. This mixture was first incubated, then the appropriate erythrocytes were added. Following a second incubation, the degree of agglutination was read in the usual manner for Rh testing. A saline control was carried out for each experiment. If the unknown showed less agglutination than the control, then the chemical compound was said to have an inhibitory effect. In all these experiments the inhibitor was diluted and not the antibody. The three Rh antibodies, anti-C, anti-D, and anti-E, were tested with the same carbohydrates, D- and L-glucose, D- and L-mannose, D-ribose, and L-lyxose. The antibodies were also tested with antibiotics which included streptomycin, stylomycin, carbomycin, and two degradation products of the first two, streptobiosamine and 6-dimethyl-amino-9-(3'amino-31deoxy-beta-D-ribofuranosyl)-purine. Some of the above carbohydrates and streptobiosamine were coupled with aniline and p-aminophenol, and then tested as inhibitors. 5'adenylic acid and 2', 3'adenylic acid were also tested as inhibitors. Their reactions suggested the use of indol acetic acid and indol butyric acid. Two amino acids, tyrosine and phenylalanine, were tested. In addition, paminophenyl acetic acid and salicin were also used as inhibitors. [TRUNCATED]

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The antral hormone gastrin has generally been regarded as a non antigenic molecule necessitating its conjugation with a large "carrier” to be an effective inducer of antibody. Such conjugation has normally taken the form of covalent binding to a highly antigenic molecule or ionic binding to an inert particle. Alternatively, some success has been achieved by the injection of impure antral extracts. This report describes four approaches to the problem of the induction of antibodies specific for gastrin. Initially, natural hog gastrin was obtained after the procedure of Gregory and Tracy (1964). The pure active hormone was modified by alkaline hydrolysis to liberate an N-terminal amino group for covalent conjugation. The modified gastrin, however, was not specifically identified or isolated in quantity. Consequently, pure and impure antral extracts, hemocyanin-bound synthetic human gastrin and latex-bound gastrin were applied to a variety of animals. Passive hemagglutination tests in ducks revealed titres of 400 to 1600 to both the pure and impure extracts but a series of four injections of pure gastrin into an antrectomized rabbit failed to raise detectable antibody. Injections of 3 mg. of SHG bound to hemocyanin was unsuccessful; antibody titre to the carrier molecule was high but no specificity appeared to be directed to the synthetic hapten. The immunization of chickens with latex-bound natural gastrin was more fruitful. Although a number of precipitin tests established the presence of antibodies to gastrin, it was apparent that the assay was inadequate as a sensitive test for the bivalent antigen. Furthermore, the antibody titre was not sufficiently high to be successfully applied to a radioimmunoassay. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

Modifications to the standard techniques of somatic cell fusion are presented which have resulted in the derivation of hybrid cell lines secreting monoclonal antibody to the low molecular weight antigen ferredoxin. Antibody secreted by one of these lines has been purified using the technique of protein. A sepharose chromatography. Using standard immunochemical methods it has been determined that this monoclonal antibody has an approximate isoelectric pH of 8.5 - 8.6 and belongs to the IgG₂[sub b] subclass of immunoglobulin. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Thesis (M.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Outer membrane proteins from unencapsulated(nontypable) Haemophilus influenza were prepared by incubation with EDTA and shearing followed by molecular sieve chromatography in a lipooligosaccharide (LOS) disaggregating deoxycholate buffer. Outer membranes proteins collected were determined to be free of LOS by polyacrylamide gel electrophoresis and then covalently attached to cyanogen bromide activated Sepharose. Antibodies to outer membrane proteins were then isolated by affinity chromatography and characterized by enzyme-linked immunosorbant assay. These antibodies will prove useful for passive immunization experiments to establish the role of outer membrane proteins in host protection from infection with nontypable Haemophilus influenza. / 2031-01-01

Antisperm antibodies (ASA) are thought to be a predominate cause of immune infertility by interfering with various aspects of sperm function in both the male and the female reproductive tracts. The precise mechanism by which these antibodies contribute to infertility, as well as their etiology, remains to be established. ASA are present in a variety of biological substrates, such as genital tract secretions, and the blood sera of both males and females. Although not all ASA underly infertility, a substantial body of research suggests that certain ASA, referred to as sperm immobilizing antibodies (SI-Abs) and sperm agglutinating antibodies, significantly impair sperm transportation in the female reproductive tract. High titers of sperm agglutinating or sperm immobilizing antibodies have been associated with reproductive failure. CD52g is a GPI anchored glycoprotein found on mature sperm and in seminal plasma (SP). Antibodies against a male reproductive tract-specific epitope of CD52g are known to readily agglutinate sperm. The current study sought to develop an ELISA to quantify the prevalence of CD52g antibodies in the sera of male and female patients with infertility, and to determine if there was a correlation between the prevalence of CD52g antibodies and the prevalence of sperm agglutinating antibodies in the sera of these patients. Ultimately, CD52g antibodies were only detected in the sera of patients (21%) with sperm agglutinating antibodies. While detecting CD52g antibodies in sera via an ELISA proved challenging, the results of this study corroborate research demonstrating that CD52g antibodies have a remarkable capacity to agglutinate sperm. Elucidation of the mechanisms underlying this immune response would advance our understanding of immune modulation in human reproductive tracts, further the diagnosis of immune infertility, and are currently providing the basis for the development of a potent dual purpose immunocontraceptive, that both prevents unintended pregnancy, and prevents the transmission of sexually transmitted infections (STIs).

Phage libraries are a versatile source of recombinant antibody fragments directed against a wide variety of antigens. Recombinant antibodies have the advantage that they can be engineered to improve their binding or other characteristics. A chicken single chain variable fragment (scFv) phage library was panned against the 16 kDa antigen of Mycobacterium tuberculosis. Three phage displayed antibodies were obtained which bound specifically to the antigen. In soluble scFv format, however, they produced low ELISA signals. For this reason they were able to be used as models for antibody engineering. Three mutant sub-libraries were created by random mutagenesis. High stringency panning of the mutant sub-libraries against the target antigen yielded stronger binders which produced ELISA signals of up to eleven times higher than the parent scFvs. An increase in affinity was confirmed by surface plasmon resonance. One mutant scFv with a single amino acid exchange also showed an increase in the yield of scFvs it produced. Upon shortening the linker sequence between the heavy and light chains, size exclusion chromatography showed that multimerisation had occurred. Dimers, trimers and tetramers were formed thus increasing the avidity of the scFvs. Tetramers derived from the unmutated scFv showed the greatest improvement in ELISA binding. To improve expression and purification, an alternate bacterial expression vector with a histidine tag was investigated. For this series of experiments the coding region for a chicken scFv directed against VP7 of bluetongue virus was transferred from the pHEN1 display vector to the pSANG 14-3F vector, which fuses the scFv gene to a bacterial alkaline phosphatase gene. A bi-functional chicken scFv-alkaline phosphatase fusion protein that exhibits both alkaline phosphatase activity and specific antigen binding was expressed in Escherichia coli and purified in a single step via metal affinity chromatography. Furthermore the scFv-AP fusion protein was directly detected in ELISA without the use of secondary detection reagents. This study confirms that the strategies used can efficiently enhance the characteristics of chicken scFvs. / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted

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