Novel single-chain fragment variable antibody targeting plasma membrane epitopes on porcine Y-chromosome-bearing sperm.

The aim of this study was to construct and produce a single-chain fragment variable (scFv) antibody targeting the plasma membrane epitope on porcine Y-chromosome-bearing sperm (Y-sperm). The hybridoma cloned from 4D1-E8 exhibited the highest specificity, and Y-sperm was used to generate the scFv gene. The expected combination of 450 bp (VH) and 300 bp (VL) resulted in a 795 bp scFv gene. The scFv gene was inserted into the pET22b expression vector and expressed in E. coli BL21 (DE3). The resulting H4:L4 clone produced a highly specific scFv antibody to Y-sperm. The reactivity of the H4:L4 scFv antibody to porcine Y-sperm was confirmed via ELISA and flow cytometry. The H4:L4 scFv antibody exhibited low cross-reactivity with X-sperm (4.14%). The soluble H4:L4 scFv antibody exhibited significantly less cross-reactivity with X-sperm than did the 4D1-E8 mAb (4.14% vs. 15.5%). However, H4:L4 scFv and the 4D1-E8 mAbs had high cross-reactivity with other conventional livestock semen. The scFv antibodies and mAbs were detected on the Y-sperm surface via immunofluorescence, and the fluorescence intensities were particularly strong on the plasma membranes of Y-sperm. In this study, the production of a scFv antibody against porcine Y-sperm was successful and represents a novel achievement. This scFv antibody had a high affinity for porcine Y-sperm. The soluble scFv antibody and mAb can be used to sort Y- and X-sperm in treatments involving porcine semen bearing X or Y chromosomes.