Immunoblot analysis of antibodies obtained from rabbits immunized with a synthetic Binder of Sperm Protein-1 (BSP-1) epitope

This study aims to analyze whether antibodies produced through multiple vaccinations of a synthetic Binder of Sperm Protein-1 (BSP-1) epitope to local rabbits, can react against BSP-1 found in the ruminant sperm. BSP-1 is one of the biomarkers suggested to authenticate sire fertility. The method used was immunoblot assay against frozen semen of Bali cattle and local buffalo, also against the extracts of the caput-, and cauda-epididymis, as well as the testis of the buffalo. Analyses showed that the antibodies reacted to antigens found in the frozen semen of cattle and buffalo. The most notable result was that the antibody reacted specifically to buffalo frozen semen. No reactions occurred to the extracts of the caput-, and cauda-epididymis, as well as to the testis of the buffalo. Further research is needed to ascertain whether the antibody can be used to develop a sire fertility detection kit.


Introduction
In the livestock industry, especially regarding reproduction and breeding, more attention is generally paid to female fertility management than the sires fertility issues.This is because sire fertility is complex [1].Assessment of the sire's reproductive quality is more focused on their semen quality.In fact, assessment of semen quality is more appropriate for identifying a sire with reproductive problems, rather than for predicting the relative fertility of the sire and his offspring.For this reason, in the past decade research has been directed towards exploring the potential use of genomic and proteomic markers to predict the fertility performance of a sire [1].Recently, at least two candidate biomarkers have been proposed, namely the peptides Enolase-1 (ENO-1) and Binder of Sperm Protein-1 (BSP-1) [2].
It has been reported that Binder of Sperm Proteins (BSPs) are the most abundant group of seminal plasma proteins in rams and bulls and have been extensively studied in the last 30 years [3,4].BSPs account for more than 50% of bull seminal plasma proteins [5].In bulls, there are three BSP proteins involved in fertilization, namely BSP1 (originally known as PDC-109), BSP3 (BSP-A3), and BSP5 (BSP-30 kDa) [6].
Since BSP-1 has the capacity to stabilize the sperm membrane and mediate the sperm binding to the oviduct's epithelium [7,8], we recently focused on BSP-1 and tried to develop antibodies against it.For this purpose, we developed immunogenic epitopes of BSP-1 in silico [9].In this paper, we report the analyzing results of the antibodies by means of immunoblot assay.

Material and methods
In this study, the antibodies tested were developed in our laboratory (called BSP1-Ab), derived from local rabbit serum that had previously been multiple vaccinated using peptide synthetic from BSP-1 with the sequence of LPEDSVPDEERVFPFTYRNRKHF [9].
Before being used as a vaccine, the peptide was modified and conjugated with keyhole limpet hemocyanin (KLH) for the vaccine, or with bovine serum albumin (BSA) for positive control, which modified from [10].
Assays for the antibodies were performed using the immunoblot method, modified based on [10] and [11].Antigens used were derived from local buffalo testis extracts.The extracts included the whole testis extracts, extracts of parts of the caput-, and the cauda epididymis.Frozen semen from Bali cattle and local buffalo were also used.Each antigen, 10 μg each, was dripped onto a nitrocellulose membrane.After incubation at 4°C overnight, the membrane was blocked with 4% skimmed milk at 37°C, 60 min.After incubation and washing, the membrane was dripped with BSP1-Ab (10 ug/ml) and allowed to react for 60 minutes at 37°C.After washing and incubation, the secondary antibody, alkaline phosphatase (AP) conjugated goat-anti rabbit IgG (diluted 1/5000; Sigma Aldrich, USA) was added to the nitrocellulose membrane.The membrane was then incubated for 60 min at 37°C, then subjected to a color reaction by a substrate that reacts specifically with the enzyme to form a dot-like color that can be observed by the naked eye.The test was repeated three times (n=3), then scored for each dot-like color observed.Scoring consisted of 0, 1, 2, 3, and 4, for negative, weakly positive, -moderate, -strong, and -strongly positive, respectively.The observation results were analyzed descriptively.
All activities related to the use of experimental animals were handled in accordance with the procedures set out in the Faculty of Animal Science, University of Mataram

Results and discussion
The main aim of this study was to analyze whether antibodies against BSP-1 could be obtained through repeated vaccination of BSP-1 peptide in local rabbits.These antibodies are needed to develop a fertility detection kit for male ruminants (cattle and buffalo), especially if the bulls are intended to be used as sires.Some of these detection kits include those based on antigen-antibody reactions, such as ELISA as well as lateral flow immunoassay, all of which require antibodies.One of the target analytes or biomarkers that has recently been suggested is BSP-1 [2].In our laboratory, we injected an in silico developed BSP-1 synthetic epitope vaccine into local rabbits.Two weeks after the third vaccination, blood was harvested, and immunoglobulins (IgG) were partially purified from the serum using 33% ammonium sulfate as modified from [12].The specificity of the IgG (called BSP1-Ab) was then analyzed by means of immunoblot, and a representation of the results is presented in Figure 1.
Figure 1 shows that based on the immunoblot results, the serum IgG obtained after repeated vaccination using synthetic epitope vaccines gave a positive response against some sampel antigens.It is quite interesting that of the antigen samples used, positive results that equivalent to the color intensity of the positive control (i.e. the peptide vaccine) were exhibited by antigen derived from frozen semen of buffalo (dot G).A similar reaction was not shown by the frozen semen derived from Bali cattle, i.e. its color intensity was relatively weaker (dot F).These results illustrate that the antibody (BSP1-Ab) obtained in this study is specific to buffalo semen.This specificity is likely because the vaccine used was derived from the peptide sequence of BSP-1 Bubalus bubalis [9].
From these immunoblot results there are some interesting points to note.Firstly, although the BSP1-Ab antibody reacted specifically to frozen buffalo semen, a similar reaction was not shown against antigens derived from the extracts of the caput-, and cauda-epididymis, as well as the testis of the buffalo used in this study.There are several possibilities to explain this phenomenon.Technically, there may have been a problem in the process of extracting testicular tissue and parts thereof.For example, the use of buffer containing phosphate to make the extracts, whereas phosphate is known to be an inhibitor of the alkaline phosphatase-labelled secondary reaction used in the immunoblot method in this study [13].Whether this is the case still needs to be investigated further.
Further consideration that needs to be taken into account is the results of previous research.It was revealed that the amount of BSP-1 was found to be highest in seminal plasma protein i.e. 31 mg/ml compared to BSP-3 and BSP-4 which were 3 mg/ml and 4 mg/ml respectively [14].While we understand that seminal plasma proteins are mostly derived from seminal vesicles and the prostate, which produce ~65-75% and ~20-30% of the volume of semen, respectively.Only a small proportion is sourced from the testes, epididymis, and bulbourethral and periurethral glands [15].It can be argued therefore that the presence of BSP-1, which is found only in small amounts in the testis and epididymis, could have been washed away during the tissue extraction process in this study.
It should be noticed here that the main point of this result is that the synthetic epitope vaccine available in our laboratory [9] has successfully triggered the local rabbit immune system to produce antibodies against BSP-1.Another study, such as the one that conducted by Ardon and Suarez [9], although using a residue sequence of the BSP-1 peptide different from us, i.e. "dqdeg vsteptqdg" as the vaccine, produced antibodies against BSP-1 with an immunoblot pattern that similar to ours.They revealed that the relative band intensity of BSP-1 in seminal plasma and frozen semen was significantly greater (p<0.05)than that in fresh sperm.However, they also mentioned that the results were strongly influenced by the semen diluent used [16].
Our study is not yet at the level of application of the antibodies against BSP-1 we obtained.Results with positive prospects that have been expressed by other researchers suggest that further research should be conducted, especially whether BSP-1 can be used as a biomarker to authenticate sire fertility, using BSP-1 antibody.

Conclusion
This study has successfully produced polyclonal antibodies against BSP-1 through repeated vaccination of BSP-1 peptide vaccine in local rabbits.The antibody reacted specifically against frozen semen of local high breed buffalo stud.Further research is needed, particularly whether BSP-1 can be used as a biomarker to authenticate sire fertility, utilizing antibodies against BSP-1.

Figure 1
Figure 1 Top panel: Representative immunoblot of BSP1-Ab reaction against antigen samples; bottom panel: average scoring of immunoblot results (n=3)