Epitope-based in silico peptide design yields peptide-directed antibodies that recognize the buffalo luteinizing hormone

What is it about?

We present a novel peptide sequence identified through in silico epitope design and the later generation of peptide-directed antibodies recognizing the buffalo luteinizing hormone. Peptides and antibodies, specific to reproductive hormones, are valuable tools for developing point-of-care immunodiagnostic tools. The study predicted an epitope peptide in silico from buffalo luteinizing hormone and the generation of polyclonal antibodies against this peptide sequence. In this quest, we identified a novel epitope peptide sequence (luteinizing hormone peptide, LHP) through bioinformatics tools. The peptide was further synthesized and characterized. The polyclonal antibodies (anti-LHP) were raised against the peptide in the rabbit. Thereafter, we explored a strategy for detecting buffalo luteinizing hormone (LH) using the anti-peptide antibodies developed. The affinity of the peptide, bovine lutropin beta, and crude LH (prepared from buffalo pituitary) towards the raised antibodies was established by dot blot and ELISA. Specific recognition of the luteinizing hormone by the raised polyclonal antibodies highlights the ability of the identified peptide (LHP) and developed polyclonal antibodies (anti-LHP) as suitable diagnostic reagents for sensing the buffalo luteinizing hormone. Through this work, we analyzed and translated the “-omics” information in the LH gene sequence for the development of a novel peptide and antibodies as valuable immuno-reagents.

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Why is it important?

The polyclonal antibodies specific for the antigens or bio-analytes are highly desirable as immunodiagnostics. The oligopeptides and antibodies find their use in veterinary-medical diagnostics, therapeutics, and sensors. Similarly, modern bioinformatics and computational tools are now very important and essential for mapping immunogenic regions in proteins. We were interested to develop peptides and antibodies as immunodiagnostic reagents for the detection of LH. Our results were indicative of the successful recognition of LHP and LH by the raised antibodies. Since the alpha subunit of LH is species-specific and homologous within a species (same in LH, FSH, CG & TSH), we focused on the beta subunit as it confers hormone specificity. Here, we presented that an epitope peptide sequence “CGGPRTQPLTCDHPPL” (luteinizing hormone peptide, LHP) unique to the beta (β) subunit of LH (responsible for biological specificity) could lead to the generation of polyclonal antibodies. These antibodies showed specific immune recognition to the corresponding peptide, bovine LH beta subunit, bovine, and buffalo LH. The anti-peptide antibodies enabled the differential identification of bovine/bubaline LH from FSH. The developed anti-LHP polyclonal antibodies were capable of immune recognition towards buffalo and bovine luteinizing hormone. The affinity of LHP, lutropin beta, and crude LH (prepared from buffalo pituitary) was also established towards raised anti-LHP by dot blot and ELISA techniques. The ELISA and Western blot experiments confirmed that the antibodies against LHP recognize both LHP as well as the LH. The work thus relates to the design, synthesis, and evaluation of a synthetic oligopeptide, which has not been described, deduced, and designed earlier from the buffalo LH and which can be used in the production of polyclonal antibodies by immunization in a suitable laboratory animal. In our view, the peptide and the developed antibodies will be applicable in strategies for efficient identification of luteinizing hormone in biological samples. These results also matched with the buffalo estrus characteristics. A recent study has already established that LH is increased in the urine of buffaloes during the estrus phase and this can be effectively used in predicting ovulation for timely artificial insemination of buffaloes in the estrus phase [50]. Together, the experiments conducted in this study have provided valuable diagnostic reagents with implications in the development of future sensors and probable use in controlling fertility and assistance in assisted reproductive technologies (graphical abstract). In one of the earlier studies, it was also demonstrated that the ovulation rate is increased in ewes treated with LH antiserum [51]. Therefore, if the anti-LHP antibodies mimic the action of anti-LH antibodies, the anti-LHP antibodies may probably be used as an anti-LH vaccine ahead having implications in buffalo fertility. Similarly, LHP mimicking the action of LH will also have implications in assisted reproductive technologies. However, these hypotheses need to be tested and validated ahead of.