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

IF 2.5 3区生物学 Q3 REPRODUCTIVE BIOLOGY

Reproductive biology Pub Date : 2025-03-15 DOI:10.1016/j.repbio.2025.101009

Onpreeya Chot , Marninphan Thongkham , Apinya Satsook , Chaiwat Arjin , Supamit Mekchay , Surat Hongsibsong , Korawan Sringarm

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Abstract

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.

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养猪业希望通过使用性别化精液来增加雌性仔猪的数量。免疫学精子性别鉴定是一种很有前景的方法。本研究调查并制备了一种针对猪 Y 染色体精子质膜表位的单克隆抗体（MAbs）。用 92.08% 的高纯度猪 Y-精子对两只 BALB/c 小鼠进行免疫，并用细胞分拣流式细胞仪对其进行分离。杂交瘤细胞是骨髓瘤细胞（P3X63Ag8.653）与免疫小鼠脾细胞的融合。间接 ELISA 筛选单个克隆孔（C2B2）产生的阳性抗体，该抗体对猪 Y-精子具有高滴度特异性。C2B2 克隆用于生产和纯化 C2B2-MAbs，产量为 2.78 ± 0.78 µg/mL。C2B2-MAbs 对 Y-精子的特异性很高（100.00%），与 X-精子的交叉反应性很低（3.25%）。因此，C2B2-MAbs 与各种家畜的常规精子的交叉反应率较低，其中安格斯为 0.34%，荷斯坦-弗里斯兰为 0.38%，山羊为 0.20%，水牛为 0.25%。与猪 Y 型精子质膜结合的 C2B2-MAbs 显示出明亮的 FITC 荧光，这证明了它们之间的亲和性。然而，与 X 精子结合的 C2B2-MAbs 却没有荧光。C2B2-MAbs 对猪 Y 型精子的质膜具有特异性，可用于猪精液性别鉴定的进一步研究。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Reproductive biology 生物-生殖生物学

CiteScore

3.90

自引率

0.00%

发文量

审稿时长

29 days

期刊介绍： An official journal of the Society for Biology of Reproduction and the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn, Poland. Reproductive Biology is an international, peer-reviewed journal covering all aspects of reproduction in vertebrates. The journal invites original research papers, short communications, review articles and commentaries dealing with reproductive physiology, endocrinology, immunology, molecular and cellular biology, receptor studies, animal breeding as well as andrology, embryology, infertility, assisted reproduction and contraception. Papers from both basic and clinical research will be considered.