Production of antibodies against murine class II antigens using unconjugated synthetic peptides of the β chain region 63–78

Identification of residues in the skeletal muscle nicotinic acetylcholine receptor (AChR) that bind snake venom alpha-neurotoxin antagonists of acetylcholine [e.g., alpha-bungarotoxin (alpha-BTx)] provides structural information about the neurotransmitter binding region of the receptor. Using synthetic peptides of the human AChR alpha-subunit region 177-208, we previously localized a pharmacologically specific binding site for alpha-BTx in segment 185-199. To define in more detail the residues that influence the binding of alpha-BTx to this region, we prepared 16 peptide analogues of the alpha-subunit segment 185-200, with the amino acid L-alanine sequentially replacing each native amino acid. Circular dichroism spectroscopy did not reveal changes in the secondary structure of the peptides except for the analogue in which Pro194 was substituted with alanine. This implies that any change in alpha-BTx binding could be attributed to replacement of the native residue's side chain by alanine's methyl group, rather than to a change in the structure of the peptide. The influence of each substitution with alanine was determined by comparing the analogue to the parental sequence alpha 185-200 in solution-phase competition with native human AChR for binding of 125I-labeled alpha-BTx. The binding of alpha-BTx by analogue peptides with alanine substituted for Tyr190, Cys192, or Cys193 was greatly diminished. Binding of alpha-BTx to peptides containing alanine replacements at Val188, Thr189, Pro194, Asp195, or Tyr198 was also reduced significantly (p < 0.003). An unanticipated finding was that substitution of alanine for Ser191 significantly increased alpha-BTx binding (p < 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)

show abstract

Nine Residues Influence the Binding of α‐Bungarotoxin in α‐Subunit Region 185–200 of Human Muscle Acetylcholine Receptor

McCormick¹,

Liebenow²,

Griesmann

et al. 1993

Journal of Neurochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

Liposomes as Carriers of Peptide Antigens: Induction of Antibodies and Cytotoxic T Lymphocytes to Conjugated and Unconjugated Peptides

Alving

Koulchin

Glenn

et al. 1995

Immunological Reviews

135

View full text Add to dashboard Cite

In the quest for effective immunization against complex diseases such as cancer, parasitic diseases, AIDS, and other viral infections, numerous peptides and recombinant proteins have been synthesized, examined for the ability to induce antibodies and CTLs, and tested for binding capability and therapeutic or prophylactic efficacy against the original target cell or organism. A liposome formulation, consisting of alum-adsorbed liposomes containing both a potent adjuvant, lipid A, and encapsulated or surface bound antigen, has had a record of safety and strong effectiveness for induction of antibodies in human vaccine trials. These same liposomes can also serve as effective vehicles for delivering conjugated or unconjugated peptides and proteins to antigen presenting cells for presentation via MHC class I and class II pathways for induction of CTLs and antibodies in experimental animal models. Liposomal lipid A appears to be extremely important, and is often a requirement, as an adjuvant for induction of CTLs against liposomal peptide antigens. Computer-generated molecular modelling analysis of small unconjugated or lipid-conjugated peptides strongly suggests that the expression of peptide antigen on the surface of the liposomes can be an important factor both in the induction of antibodies and in determining antibody specificities to small peptides. However, antigenic surface expression of liposomal peptide is not required for induction of CTLs. The data suggest that small synthetic peptides, synthesized with or without a lipid tail, or chemically conjugated to the surface of liposomes, might serve as effective antigenic epitopes, in combination with liposomal lipid A for induction of antibodies and CTLs.

show abstract

Synthetic Peptide Segments of Inhibin α- and β-Subunits: Preparation and Characterization of Polyclonal Antibodies*

et al. 1989

View full text Add to dashboard Cite

The amino acid sequences of human inhibin alpha-, beta A- and beta B-subunits were analyzed for hydrophilicity and chain flexibility to predict regions that are on the surface of the subunits and, therefore, are potential antigenic sites. Based on these analyses, a total of nine peptides were synthesized, and rabbit antisera against the peptides were prepared. Peptides of the N-terminus (residues 1-16 and 13-24) and region 109-123 of the alpha-subunit produced high titer antibodies. Regions 69-79 and 93-105 of the beta A-subunit and region 93-104 of the beta B-subunit were also immunogenic. Immunoblotting of an inhibin preparation with anti-alpha-peptide antiserum revealed that a 32K band (inhibin) and an 18K band (alpha-subunit) were stained. Immunoblotting with anti-beta-peptide antiserum detected a 32K band (inhibin), a 24K band (activin), and a 14K band (beta-subunit). Injection (iv) of these antisera into rats induced dramatic elevation of serum FSH in 6-12 h and suggested immunoneutralization of endogenous inhibin. RIAs for each subunit were developed using radioiodinated peptides as tracers. Competition binding assays indicated crossreactivity with human follicular fluid, semen, serum, plasma, and crude inhibin preparations. Parallel dilution curves were obtained. Antisera against beta A- and beta B-subunit peptide cross-reacted with each other. In immunocytochemical studies, these antisera were used in conjunction with gold-labeled goat antirabbit immunoglobulin G to localize inhibin in cells of the rat testis. Specific staining of inhibin was localized within the Sertoli cells of some tubules in adult rat testis. Positive staining could be blocked by preadsorbing the sera with the appropriate synthetic peptide. These results suggest that antibodies against synthetic inhibin peptides are useful in elucidating the roles of inhibin and activin.

show abstract

scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.

Contact Info

customersupport@researchsolutions.com

10624 S. Eastern Ave., Ste. A-614

Henderson, NV 89052, USA

Blog Terms and Conditions API Terms Privacy Policy Contact Cookie Preferences Do Not Sell or Share My Personal Information

Made with 💙 for researchers

Part of the Research Solutions Family.

Production of antibodies against murine class II antigens using unconjugated synthetic peptides of the β chain region 63–78

Cited by 3 publications

References 30 publications

Nine Residues Influence the Binding of α‐Bungarotoxin in α‐Subunit Region 185–200 of Human Muscle Acetylcholine Receptor

Nine Residues Influence the Binding of α‐Bungarotoxin in α‐Subunit Region 185–200 of Human Muscle Acetylcholine Receptor

Liposomes as Carriers of Peptide Antigens: Induction of Antibodies and Cytotoxic T Lymphocytes to Conjugated and Unconjugated Peptides

Synthetic Peptide Segments of Inhibin α- and β-Subunits: Preparation and Characterization of Polyclonal Antibodies*

Contact Info

Product

Resources

About