Generation and characterization of antibodies against synthetic peptides of porcine zona pellucida ZP3 alpha.
OBJECTIVE
Use of synthetic zona peptides as target immunogens is a promising approach to an anti-fertility vaccine. To elucidate contraceptive epitopes, we raised polyclonal antibodies against synthetic peptides of pig ZP3 alpha and evaluated for immunogenicity, ability to inhibit in vitro boar sperm-pig zona attachment, and cross-reactivity with human zonae.


METHODS
Five synthetic peptides were chosen based on hydropathicity analysis of ZP3 alpha, synthesized and coupled to keyhold limpet haemocyanin (KLH). Pairs of male rabbits were immunized with each peptide-KLH conjugate using a multi-site intradermal injection protocol. Resulting antisera were evaluated for immunoreactivity with cognate peptide and ZP3 alpha/EBGD (purified endo-beta-galactosidase-digested ZP3 alpha), contraceptive potential using an in vitro pig gamete bioassay, and cross-reactivity with human zonae using indirect immunofluorescence.


RESULTS
All anti-peptide-KLH sera demonstrated immunoreactivity with cognate peptide. Anti-peptide sera, except for anti-alpha-3-KLH serum, were less immunoreactive with ZP3 alpha/ EBGD. Antisera directed against alpha-1-KLH, alpha-2-KLH, and alpha-3-KLH demonstrated significant (P < .00001) inhibition of boar sperm-pig zona attachment. Only anti-alpha-2-KLH and anti-alpha-3-KLH sera cross-reacted with human zonae.


CONCLUSIONS
Synthetic peptides of pig ZP3 alpha are immunogenic, elicit antibodies cross-reactive with human zonae, and have in vitro contraceptive activity. These data support continued investigation of synthetic ZP3 alpha peptides as potential target immunogens for anti-fertility vaccine development.
Hall
L L
LL
Department of Obstetrics and Gynecology, C. S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, Detroit, Michigan, USA.
Yurewicz
E C
EC
Sacco
A G
AG
Moghissi
K S
KS
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D003429:Cross Reactions; D004527:Egg Proteins / Q000032:analysis* / Q000737:chemistry* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000737:chemistry* / Q000276:immunology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology*; D011817:Rabbits; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000502:physiology; D013552:Swine; D015044:Zona Pellucida / Q000502:physiology / Q000648:ultrastructure; D000072216:Zona Pellucida Glycoproteins
D000906:Antibodies; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; C082833:ZP4 protein, human; D000072216:Zona Pellucida Glycoproteins