Specific Fab fragments recovered by phage display technique recognizing human spermatozoa.
Human hybridoma cell lines are often unstable and loose ability for antibody production. Sometimes, they show low and varying levels of heavy and light chains synthesis. Therefore it is reasonable to preserve generated specificities of light and heavy chains by cloning them to phagemid vector and creating phage display library. The aim of this study was to construct phage display library of Fab fragments recognizing sperm surface antigens. The source of mRNA constituted seven hybridoma cell lines producing antisperm antibodies which was proved by ELISA, and agglutination test as well as by inhibition of sperm to penetrate hamster oocytes. Fragments of cDNA encoding kappa/lambda and gamma chains were cloned into pComb3HSS phagemid vector and amplified in XL-1Blue. The library was panned against whole unfixed sperm cells. Three positive clones selected after fourth round of panning showed heavy chain belonging to VH4 family, two of them (G28, K61) possessed lambda chain from VL2 family and one (H43) kappa chain from VK1 family. As these Fabs revealed similarities to antibodies against some proteins involved in sperm motility and cell fusion it can be suggested that these Fabs may be a cause of infertility. Finally, we proved that it is feasible to preserve specificities produced by human hybridomas using phage display technique and we recovered some Fabs which may be of diagnostic and research value, and may also have some value for contraceptive vaccine.
Fiszer
Dorota
D
Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
Pupecka
Małgorzata
M
Schmidt
Katarzyna
K
Rozwadowska
Natalia
N
Kamieniczna
Marzena
M
Grygielska
Beata
B
Kurpisz
Maciej
M
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D007150:Immunohistochemistry; D008297:Male; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013094:Spermatozoa / Q000276:immunology*
D017931:DNA Primers; D018076:DNA, Complementary; D007140:Immunoglobulin Fab Fragments