Production of monoclonal antibodies targeting plasma membrane of porcine Y-chromosome-bearing sperm

The pig industry is interested in increasing the number of female piglets by using sexed semen. Immunological sperm sexing is a promising method. This study investigated and produced a monoclonal antibody (MAbs) targeted to plasma membrane epitopes on porcine Y-chromosome-bearing sperm. Two BALB/c mice were immunized with 92.08 % high-purity porcine Y-sperm, which was separated by a cell sorter flow cytometer. The hybridoma cells were a fusion of myeloma cells (P3X63Ag8.653) and splenocyte cells from immunized mice. Indirect ELISA screening for positive antibodies produced by a single clone well (C2B2) with a high titer specific to porcine Y-sperm. The C2B2 clone was used to produce and purify C2B2-MAbs, yielding 2.78 ± 0.78 µg/mL. The C2B2-MAbs was highly specific to Y-sperm (100.00 %) and had a low cross-reactivity with X-sperm (3.25 %). Therefore, the percentage cross-reactivity of C2B2-MAbs was low for conventional sperm from various livestock, including 0.34 % for Angus, 0.38 % for Holstein-Friesian, 0.20 % for goats, and 0.25 % for buffalo. The bright fluorescence of FITC displayed by the C2B2-MAbs bound to the plasma membrane of porcine Y-sperm provided evidence of affinity between them. However, the C2B2-MAbs bound to an X-sperm lacked fluorescence. C2B2-MAbs showed specificity for the plasma membrane of porcine Y-sperm, which can be used in porcine semen sexing in further studies.