Claims:I/We Claim:

1. Immunogenic peptides for detection of pregnancy in bovine wherein the immunogenic peptide is selected from the group comprising peptides of sequence ID No. 1 to 2, either alone or in any combination thereof; wherein the immunogenic peptide is capable of generating either polyclonal or monoclonal antibodies against pregnancy associated glycoproteins-7 (PAG-7) and pregnancy associated glycoproteins-18 (PAG-18).
2. The immunogenic peptides as claimed in claim 1, wherein peptide is selected from the PAG-7 protein comprising sequence ID No. 1 (GRLIDNIQKLIGAT), while the sequence ID No. 2 (KIPLRRVKTMRKILSEKN) selected from PAG-18 protein.
3. The immunogenic peptides as claimed in claim 1, wherein the peptides comprising any peptide sequence having 70 % or more identity with sequence ID No. 1 to 2.
4. The immunogenic peptides as claimed in claim 1, wherein immunogenic peptides generate polyclonal antibodies capable of binding with pregnancy associated glycoproteins 7 (PAG-7).
5. The immunogenic peptides as claimed in claim 1, wherein immunogenic peptides generate polyclonal antibodies capable of binding with pregnancy associated glycoproteins 18 (PAG-18).
6. Polyclonal antibodies generated against immunogenic peptides as claimed in claim 1 having sequence ID no. 1 to 2 wherein the polyclonal antibodies are capable of binding with pregnancy associated glycoproteins 7 (PAG-7) and pregnancy associated glycoproteins 18 (PAG-18).
7. The polyclonal antibodies generated against immunogenic peptides as claimed in claim 1, wherein the polyclonal antibodies having 70 % or more identity with any paratope regions of antibodies of claim 6.
8. Monoclonal antibodies generated against immunogenic peptides as claimed in claim 1 having sequence ID no. 1 to 2 wherein the monoclonal antibodies are capable of binding with pregnancy associated glycoproteins 7 (PAG-7) and pregnancy associated glycoproteins 18 (PAG-18).
9. The monoclonal antibodies generated against immunogenic peptides as claimed in claim 1, wherein the monoclonal antibodies having 70 % or more identity with any paratope regions of antibodies of claim 8.
10. The immunogenic peptides as claimed in claim 1,wherein the peptide and paratope region of antibody having any modifications or chemical modification or post translational modifications including amino acid substitutions in conserved regions, glycosylation, acetylation, phosphorylation, proteolytic cleavage, conjugation with repeated nucleotide sequences (CpGs) and/or peptides having identical sequences produced by chemical synthesis and recombinant DNA technology.
11. A method for detection of pregnancy associated glycoprotein in a biological test sample, wherein said method detect antigen/antibody complexes using an indicator reagent comprising a signal generating compound; wherein the detection of antigen/antibody complex is an indication that PAG-7 &/OR PAG-18 proteins are present in test sample.

, Description:FORM 2
THE PATENT ACT 1970
(39 OF 1970)
&
The Patents Rules, 2003
PROVISIONAL/COMPLETE SPECIFICATION
1. TITLE OF THE INVENTION
Immunogenic Peptides and Antibodies thereof for Detection of Pregnancy in Bovine and related species

2. APPLICANT(S)
(a) NAME: Protedge Biotech Solutions LLP
(b) NATIONALITY: Indian
(c) ADDRESS: 102 Pyramid Greenwoods Apts, Ayappa Temple Road, Bytarayanapura Sahakar Nagar, Bangalore, Karnataka-560092
3. PREAMBLE TO THE DESCRIPTION
PROVISIONAL
The following specification describes the invention.
COMPLETE
The following specification particularly describes the invention and the manner in which is to be performed.

4. Description
Field of the invention:
The present invention relates to the field of early pregnancy diagnostic assays (novel peptides and their antibodies). The immunogenic peptides were selected from predominantly expressed PAG-7 and PAG-18 isoforms and antibodies were generated against these peptides for pregnancy diagnosis in bovine.

Background of the invention:
Development of a farmer friendly pregnancy diagnostic kit in cattle and buffalo which can be practiced at the farmer’s door as early as possible is still an unmet need. Early pregnancy diagnosis has economic benefits to the farmers (Whitlock et al., 2008). To increase milk production in animals as per their genetic potential, artificial insemination and out of season breeding is required for optimal milk yield. Accurate as well as early pregnancy detection in dairy animals is of paramount importance for improving the reproductive efficiency and overall productivity (Lotfan et al., 2018 & Balhara et al., 2014). Methods of pregnancy diagnosis depend on visualization of the conceptus or determination of its secretory products in the maternal blood, milk or urine. Various methods have been used to diagnose pregnancy in cattle and buffalo like ultrasonography, rectal palpation and hormonal assays (Balhara et. al, 2014, Green et al., 2005, Ayad et. al., 2007). However, there is no method which a dairy farmer can himself practice for early diagnosis of pregnancy which otherwise causes economic loss to farmers due to longer unproductive phase of dairy animals in case of pregnancy loss. Generally, a dairy farmer has to wait for 50 to 60 days for pregnancy diagnosis by a trained veterinarian using per-rectal palpation method. At many occasions, this method is associated with undesirable infection and abortion (Franco et al., 1987). Moreover farmers are very much in need to have a door-step diagnostic kit for early detection of pregnancy. Over past few years a large number of studies carried in the direction of pregnancy diagnosis indicate that Pregnancy Associated Glycoproteins (PAGs) are important markers of pregnancy in bovine (Sausa et al., 2006, Lotfan et al., 2018). PAGs are secretory products of the mono-nucleate and binucleated trophoblastic cells in bovine placentomes. They are secreted in maternal blood when the conceptus attaches more closely to the uterine wall and placentome formation begins (Wooding, 1992). PAGs being the products of the trophoblastic cells, suggest that their determination in maternal blood can be useful for prediction of fetal well-being and to detect early placental abnormalities, embryonic mortality or abortion (Ectors et al.,1996).
The PAGs became important for the development of pregnancy diagnostic assays in ruminants similar to hCG tests in primates (Zoli et al., 1992, Gonzalez et al., 2004). PAGs as embryo specific pregnancy markers can be considered ideal candidate for developing pregnancy diagnostic kits (Zoli et al., 1992, El Amiri et al., 2006, Gonzalez et al., 2004, Jerome et al. 2011b). The level of PAGs rises to detectable levels in maternal blood and milk during days 22 to 28 after fertilization (Mialon et al., 1993, Sausa et al., 2006). As pregnancy failure occurs, PAG concentrations drop and disappear from milk and maternal blood (Klisch et al., 2005, Commun et al., 2016).PAG-7 and PAG-18 are dominantly expressed in early stages of pregnancy in buffalo (Lotfan et al., 2018).PAG proteins and their antibodies are commercially not available in the market, so design of immunogenic peptides and antibody generation is needed.
Present invention provides a user-friendly diagnostic assay for early and accurate detection of pregnancy in dairy animals. There are several benefits of using PAG based low cost, field applicable, rapid and accurate detection of pregnancy; experiment can be performed in in vitro condition, affinity and specificity to target is comparable to antibody etc. This invention is likely to be highly commercially viable because of its urgency of requirements by the dairy farmers and all the stake holders.

Existing methods and their limitations
Dairy farmer has to wait for 50 to 60 days for pregnancy diagnosis by trained veterinarian using per-rectal palpation method. This method is not advisable because of chances of the abortion of the fetus (Balhara et al., 2014). Moreover, farmers are very much in need to have a door-step diagnostic kit for early detection of pregnancy. Unfortunately, such kits are not available till date which can be used reliably in the field.PAG-1 based ELISA kit is available for detection of pregnancy in cattle (IDEXX & BIOPRYN). The cost per the test is more than Rs.500/ sample (prohibitive cost). Progesterone based ELISA kit is available for pregnancy detection in bovine (Gives false positive result in the case of embryo loss). None of these tests are cow-side or on-farm, so blood samples must be collected by farm personnel and sent by courier to a local or regional laboratory (Balhara et al., 2014, Commun et al., 2016).

References
1. Ayad, A., Sousa, N.M., Sulon, J., Iguer-Ouada, M., Beckers, J.F. (2007). Comparison of five radioimmunoassay systems for PAG measurement: ability to detect early pregnancy in cows. Reproduction in Domestic Animals 42(4), 433-440.
2. Balhara AK, Gupta M, Singh S, Mohanty AK, Singh I (2014). Early pregnancy diagnosis in bovines: current status and future directions. Sci World J. (ArticleID 958540).
3. Barbato O, Sousa NM, Barile VL, Canali C, Beckers JF. Purification of pregnancy-associated glycoproteins from late-pregnancy Bubalus bubalis placentas and development of a radioimmunoassay for pregnancy diagnosis in water buffalo females. BMC Vet. Res. 2013;9: 89.
4. Commun L., K. Velek, J. B. Barbry, S. Pun, A. Rice, A. Mestek, C. Egli and S. Leterme. (2016). Detection of pregnancy-associated glycoproteins in milk and blood as a test for early pregnancy in dairy cows. J Vet Diagn Invest. 28 (3): 207- 13. DOI:10.1177/1040638716632815
5. El Amiri, B., Sulon, J. and Karen, A. (2006). Development and validation of two homologous radioimmunoassays for measuring pregnancy-associated glycoprotein (PAG) concentrations in ewe. Reprod. Domest. Anim.,48 :100.
6. Franco OJ, Drost M, Thatcher MJ, Shille VM, and Thatcher WW (1987). Fetal survival in the cow after pregnancy diagnosis by palpation per rectum.Theriogenology, vol. 27, no. 4, pp. 631– 644.
7. González, Fernando, Fernando Cabrera, Miguel Batista, Noemi´ Rodri´guez, DesiréÁlamo, JoseSulon, Jean-François Beckers, and AnselmoGracia. "A comparison of diagnosis of pregnancy in the goat via transrectal ultrasound scanning, progesterone, and pregnancy-associated glycoprotein assays." Theriogenology 62, no. 6 (2004): 1108-1115.
8. Green, Jonathan A., SancaiXie, Xiao Quan, BagnaBao, XinshengGan, Nagappan Mathialagan, Jean-François Beckers, and R. Michael Roberts. "Pregnancy-associated bovine and ovine glycoproteins exhibit spatially and temporally distinct expression patterns during pregnancy." Biology of reproduction 62, no. 6 (2000): 1624-1631.
9. Green, Jonathan A., Tina E. Parks, Mary PavloAvalle, Bhanu Prakash Telugu, April L.McLain, A. James Peterson, William McMillan et al. "The establishment of an ELISA for the detection of pregnancy-associated glycoproteins (PAGs) in the serum of pregnant cowsand heifers." Theriogenology 63, no. 5 (2005): 1481-1503.
10. Humblot, P., S. Camous, J. Martal, J. Charlery, N. Jeanguyot, M. Thibier, and G. Sasser."Diagnosis of pregnancy by radioimmunoassay of a pregnancy-specific protein in theplasma of dairy cows." Theriogenology 30, no. 2 (1988): 257-267.
11. Jerome, A. "An overview on Pregnancy Associated Glycoproteins in Cattle and Buffalo." Journal of Advanced Veterinary Research 2, no. 1 (2012): 50-58.
12. Karen A, Darwish S, Ramoun A, Tawfeek K, Hanh N, Sousa NM, Sulon J, Szenci O, Beckers JF. Accuracy of ultrasonography and pregnancy–associated glycoprotein test for pregnancy diagnosis in buffaloes. Theriogenology. 2007;68(8): 1150-55.
13. Klisch K, De Sousa N M, Beckers, J F, Leiser, R &Pich A. (2005). Pregnancy associated- glycoprotein- 1, -6, -7, and -17 are major products of bovine binucleate trophoblast giant cells at mid pregnancy. Mol Reprod Dev, 71: 453.
14. Lotfan M, Choudhary S, Y adav ML, Kumar S, Singh S, Kaushik JK, Mohanty AK (2018). Primary structures of different isoforms of buffalo pregnancy associated glycoproteins (BuPAGs) during early pregnancy and elucidation of 3-dimensional structure of the most abundant isoform BuPAG 7. PLoSOne. Nov 7;13(11):e0206143. doi: 10.1371/ journal.pone.0206143. eCollection 2018.
15. Lotfan, M., Ali, SA.,Yadav, ML., Chuadhary, S., Jena, M., Kumar, S., Mohanty, AK (2018). Genome-Wide Gene Expression Analysis of 45 days pregnant Fetal Cotyledons Vis-a-Vis non-pregnant crancules in buffalo (Bubalus bubalis). Gene, DOI:10.1016/j.gene. 2018.02.038.
16. Mialon, M. M., S. Camous, G. Renand, J. Martal, and F. Menissier. "Peripheral concentrations of a 60-kDa pregnancy serum protein during gestation and after calving andin relationship to embryonic mortality in cattle." Reproduction Nutrition Development 33,no. 3 (1993): 269-282.
17. Ranilla, María José, Joseph Sulon, M. D. Carro, A. R. Mantecon, and Jean-François Beckers. "Plasmatic profiles of pregnancy-associated glycoprotein and progesterone levelsduring gestation in Churra and Merino sheep." Theriogenology42, no. 3 (1994): 537-545.
18. Singh, S. K., S. K. Agarwal, U. Shankar, and Lavleen K. Gupta. "Purification and characterization of protein (s) from placental extracts in buffalo." INDIAN JOURNAL OFANIMAL SCIENCES 75, no. 7 (2005): 769.
19. Skinner, J. G., D. Gray, F. E. Gebbie, Jean-François Beckers, and Joseph Sulon. "Field evaluation of pregnancy diagnosis using bovine pregnancy-associated glycoprotein (bPAG)." Cattle Practice (United Kingdom) (1996).
20. Sousa, N.M., Ayad, A., Beckers, J.F. &Gajewski, Z. (2006). Prengancy-associated glycoproteins (PAG) as pregnancy markers in the ruminants. J. Physiol. Pharmacol. 57[Supp 8]:153-171.
21. Szenci, M. A. M., J. F. Beckers, J. Sulon, J. Varga, L. Borzsonyi, ChHanzen, and GySchekk. "Evaluation of false ultrasonographic diagnoses in cows by measuring plasma levels of bovine pregnancy-associated glycoprotein." The Veterinary Record 142 (1998):304-306.
22. Whitlock, B.K. & Maxwell, H.S. (2008). Pregnancy-associated glycoproteins and pregnancy wastage in cattle. Theriogenology 70:550-559.
23. Wong, Raphael C., and Y. Tse Harley. "Quantitative, false positive, and false negative issues for lateral flow immunoassays as exemplified by onsite drug screens." In Lateralflow immunoassay, pp. 1-19. Humana Press, 2009.
24. Wooding, F. B. P., R. M. Roberts, and J. A. Green. "Light and electron microscope immunocytochemical studies of the distribution of pregnancy associated glycoproteins (PAGs) throughout pregnancy in the cow: possible functional implications." Placenta 26,no. 10 (2005): 807-827.
25. Zoli, Andre P., Pierre Demez, Jean-François Beckers, Michel Reznik, and Albert Beckers."Light and electron microscopic immunolocalization of bovine pregnancy-associated glycoprotein in the bovine placentome." Biology of Reproduction 46, no. 4 (1992): 623-629.

Objectives of invention:
? The main objective of invention is to design, synthesize and evaluate immunogenic peptides deduced from buffalo (Bubalus bubalis) PAG-7 & PAG-18.
? Another objective of the invention is to generate antibodies (polyclonal) against PAG-7 &PAG-18 peptides (SEQ ID No- 1& SEQ ID No- 2).
? Yet another objective of the present invention is to provide antibodies capable of binding to PAG proteins.
? Yet another objective of the present invention is to provide a method for detection of PAG-7 & PAG-18 proteins in bovine blood and milk by using the selected peptides and their antibodies.
Summary
In view of the foregoing, the invention herein provides immunogenic peptides for detection of pregnancy in bovine.
An aspect of the present invention provides the immunogenic peptides i. e. GRLIDNIQKLIGAT (seq. ID 1) and KIPLRRVKTMRKILSEKN (seq. ID 2) selected from the PAG-7 and PAG-18 genes respectively.
An aspect of the present invention provides polyclonal antibodies against pregnancy associated glycoproteins-7 (PAG-7) and pregnancy associated glycoproteins-18 (PAG-18).
Another aspect of the present invention provides a method for detection of pregnancy associated glycoprotein in a biological test sample.

Brief description of the drawings:
Figure 1. Designing and evaluation of PAG-7 & PAG-18 immunogenic peptides for their epitope exposure on its protein surface.
Figure 2: Antibody purification by Protein G/L column and SDS-PAGE analysis.
Figure 3: Assessment of immunogenicity of PAG-7 & PAG-18 antibodies. (A) Dot blot assay using PAG-7 antibody; (B) Dot blot assay using PAG-18 antibody, interacted very well with respective PAG antigens and does not show cross reactivity with other non-PAG proteins.
Figure 4: Screening and validation of pregnant and non-pregnant serum samples in dot blot using PAG-7 antibody.
Figure 5: Screening and validation of pregnant and non-pregnant serum samples in dot blot using PAG-18 antibody.

List of Abbreviations used
PAG= Pregnancy associated glycoproteins
LFA= Lateral flow assay
hCG= human chorionic gonadotropin
ECL= Enhanced chemiluminescence
IFN-?= Interferon tau
BSA= Bovine serum albumin

Detailed Description
While the present invention is described herein by way of example using embodiments and illustrative drawings, those skilled in the art will recognize that the invention is not limited to the embodiments of drawing or drawings described, and are not intended to represent the scale of the various components. Further, some components that may form a part of the invention may not be illustrated in certain figures, for ease of illustration, and such omissions do not limit the embodiments outlined in any way. It should be understood that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the scope of the present invention as defined by the appended claim.

This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art.

The present invention relates to novel peptide sequences and their antibodies specific for PAG-7 and PAG-18 respectively. These peptide sequences and their antibodies have shown their detection capability of PAG-7 and PAG-18 proteins in serum and milk samples of bovine (cattle & buffalo) and related species.
Design of immunogenic peptide and its synthesis
In an embodiment of present invention whole PAG protein sequences were used for prediction of antigenic epitopes. Signal sequences were analyzed and removed from the sequence.
In an embodiment of present invention different software were used for antigenic epitope prediction like SVMTriP, EMBOSS Antigenic tool. The epitopes with high score were selected for peptide synthesis and antibody generation.
In an embodiment of present invention, the length of peptides selected for antibody generation was 14-18 amino acids. Multiple alignments were done for predicted epitopes and unique epitopes were chosen for peptide synthesis. The protein BLAST search was done to make sure that peptide does not match with other proteins reported in bovine database.
In an embodiment of present invention, epitope surface exposure was checked by using 3D structure predicted by SWISS-MODEL (swissmodel.expasy.org). Peptide solubility was checked by using an online peptide calculator software“PepCalc.com”. The possible post-translational modifications were checked by Expassy tools i. e. phosphorylation by NetPhos and glycosylation by NetNGlyc, NetOGlyc.
In another embodiment of present invention PAG peptides were synthesized and conjugated with carrier molecule KLH (Keyhole Limpet Hemocyanin) and used for antibody generation.
Table 1. List of buffalo PAG-7 & PAG-18 peptides used for antibody generation
S.No. Protein Epitope Sequence Epitope Name
1 PAG-7 GRLIDNIQKLIGAT Seq. ID No.1
2 PAG-18 KIPLRRVKTMRKILSEKN Seq. ID No.2

Raising polyclonal antibodies against peptides of PAG-7 (GRLIDNIQKLIGAT) and PAG-18 (KIPLRRVKTMRKILSEKN)
In an embodiment of present invention, polyclonal antibodies against buffalo PAG-7 and PAG-18 peptides were raised in rabbit (New Zealand white) as well as in rats (Wistar). The PAG peptides were coupled with KLH by EDC and NHS method (Bremus et. al., 2012). Equal amount of antigen and adjuvant were mixed till emulsion formation. PAG peptide antigens of 500 ug were injected subcutaneously at least at four sites per rabbit. First immunization was done with FCA (Freund's complete adjuvant) and subsequent immunizations were done with FIA (Freund's incomplete adjuvant). Pre-immune sera were collected from animal prior to immunization. Immunization details are given below in details in tables for rabbit models (Table 2). All anti-PAG-7 and Anti-PAG-18 antibodies were purified by using Protein G/L column and were used in western blot and dot blot analysis for antigen-antibody interaction study (Table 3).
Table 2. Immunization details of rabbits (New Zealand white)
S.No Protocol day Description
1 Day 0 Pre-immune serum
2 Day 1 Immunize 500 ug with FCA
3 Day 15 Booster 1: 300 ug with FIA
4 Day 25 Booster 2: 300 ug with FIA
5 Day 35 Booster 3: 300 ug with FIA
6 Day 45 Booster 4: 200 ug with FIA
7 Day 55 Booster 5: 200 ug with FIA
8 Day 65 Bleed 1
9 Day 75 Bleed 2

Table 3. Antibodies generated against PAG peptides
S.No. Antibody Name Antibody Type Antibody Seq. ID Antibodies generated against given peptides
1 PAG-7_PA Polyclonal Seq. ID No.3 Seq. ID No.1
2 PAG-18_PA Polyclonal Seq. ID No. 4 Seq. ID No.2

In an embodiment of present invention Quality assessment of antibodies raised against PAG peptides (Seq. ID No.1 & Seq. ID No.2) was performed.
In an embodiment of present invention, purified polyclonal antibodies were quantified and used for antigen-antibody interaction studies (Table 4). Antibodies reactivity and specificity were checked in dot blot and western blot. Antibodies generated against PAG peptides interacted very well with PAG proteins and did not show any cross reactivity with other non PAG proteins (BSA and Interferon Tau:IFN-?) (Fig. 3A & 3B).
In an embodiment of present invention, screening of pregnant and non-pregnant bovine serum samples were done by dot blot.
In an embodiment of present invention, pregnant and non-pregnant bovine serum samples were collected and screened by using PAG-7 peptide based generated polyclonal antibodies in dot blot assay (Fig. 4). Pregnant animal samples of different days were checked in dot blot with enhanced chemiluminescence (ECL). PAG-7 based polyclonal antibodies reacted very well with natural positive pregnant samples. Serum samples of pregnant and non-pregnant spotted on nitrocellulose membrane (NCM) for dot blot assay (Fig. 4 & 5). Anti-PAG-7 antibody interacted very well with PAG antigens in positive serum samples (Fig. 3). Pregnant and non-pregnant serum samples can easily be differenced in dot blot assay.
In an embodiment of present invention, pregnant and non-pregnant serum samples were collected and screened by using PAG-18 peptide based generated polyclonal antibodies in dot blot assay (Fig. 5). Pregnant animal samples of different days (35 day, 42 day) were checked in dot blot with enhanced chemiluminescence (ECL) system. The reactivity of the anti-PAG-18 raised antibody (PAG-18_PA) showed positive results with pregnant samples while no reactivity was observed in non-pregnant serum samples in dot blot assay (Fig. 5). Anti-PAG-18 based polyclonal antibody reacted specifically with natural positive pregnant samples. Pregnant and non-pregnant serum samples are easily differentiable in dot blot assay using these antibodies.
Analysis of result:
In an embodiment, the polyclonal antibodies against PAG-7 peptide (Seq. ID No. 1) confirmed the specificity, sensitivity and quality with antigens. The immunogenicity assessment of the raised antibodies were done against bovine PAGs and positive pregnant serum samples in dot blot assay. These novel immunogenic peptides and their antibodies are more specific and reactive with native PAGs present in pregnant samples. Finally inventors have successfully developed a proof of concept for early detection of pregnancy in bovine using these peptides and antibodies unique to PAG-7 and PAG-18. Pregnant and non-pregnant animals can be screened using these antibodies. This invention may be helpful to evolve user-friendly diagnostic assay for early and accurate detection of pregnancy in dairy animals.
Commercial application:
The developed polyclonal antibody (PAG-7_PA & PAG-18_PA) have commercial application especially in development of PAG based detection of early pregnancy in bovine using serum and milk samples which is beneficial to farmers and dairy industry. PAG based LFA device can be developed using these antibodies. Development of low cost and field applicable rapid test will help the farmers for early detection of pregnancy in bovine and increase farmer’s economy. The potential of PAG based cheaper and accurate diagnostic kit has a large application in India as well as worldwide.
Novelty of the invention:
PAG-7 and PAG-18 isoforms are the most dominant protein isoforms which are expressed preferentially in the fetal cotyledons during early pregnancy in buffalo and equally applicable for cattle (Lotfan et al., 2018). Inventors have developed polyclonal antibodies against these PAG-7 and PAG-18 and assessed its suitability as a diagnostic marker for early pregnancy. The identified immunogenic peptides of PAG-7 and PAG-18 are novel sequences and antibodies generated against these peptides are unique in nature. The PAG specific antibodies showed their potential to recognize PAG antigens in pregnant bovine serum as early as 35th day of pregnancy. Pregnant and non-pregnant animals can be easily screened by using these PAG generated antibodies. This work may be helpful to evolve user-friendly diagnostic assay for early and accurate detection of pregnancy in dairy animals.

I/We Claim:

1. Immunogenic peptides for detection of pregnancy in bovine wherein the immunogenic peptide is selected from the group comprising peptides of sequence ID No. 1 to 2, either alone or in any combination thereof; wherein the immunogenic peptide is capable of generating either polyclonal or monoclonal antibodies against pregnancy associated glycoproteins-7 (PAG-7) and pregnancy associated glycoproteins-18 (PAG-18).
2. The immunogenic peptides as claimed in claim 1, wherein peptide is selected from the PAG-7 protein comprising sequence ID No. 1 (GRLIDNIQKLIGAT), while the sequence ID No. 2 (KIPLRRVKTMRKILSEKN) selected from PAG-18 protein.
3. The immunogenic peptides as claimed in claim 1, wherein the peptides comprising any peptide sequence having 70 % or more identity with sequence ID No. 1 to 2.
4. The immunogenic peptides as claimed in claim 1, wherein immunogenic peptides generate polyclonal antibodies capable of binding with pregnancy associated glycoproteins 7 (PAG-7).
5. The immunogenic peptides as claimed in claim 1, wherein immunogenic peptides generate polyclonal antibodies capable of binding with pregnancy associated glycoproteins 18 (PAG-18).
6. Polyclonal antibodies generated against immunogenic peptides as claimed in claim 1 having sequence ID no. 1 to 2 wherein the polyclonal antibodies are capable of binding with pregnancy associated glycoproteins 7 (PAG-7) and pregnancy associated glycoproteins 18 (PAG-18).
7. The polyclonal antibodies generated against immunogenic peptides as claimed in claim 1, wherein the polyclonal antibodies having 70 % or more identity with any paratope regions of antibodies of claim 6.
8. Monoclonal antibodies generated against immunogenic peptides as claimed in claim 1 having sequence ID no. 1 to 2 wherein the monoclonal antibodies are capable of binding with pregnancy associated glycoproteins 7 (PAG-7) and pregnancy associated glycoproteins 18 (PAG-18).
9. The monoclonal antibodies generated against immunogenic peptides as claimed in claim 1, wherein the monoclonal antibodies having 70 % or more identity with any paratope regions of antibodies of claim 8.
10. The immunogenic peptides as claimed in claim 1,wherein the peptide and paratope region of antibody having any modifications or chemical modification or post translational modifications including amino acid substitutions in conserved regions, glycosylation, acetylation, phosphorylation, proteolytic cleavage, conjugation with repeated nucleotide sequences (CpGs) and/or peptides having identical sequences produced by chemical synthesis and recombinant DNA technology.
11. A method for detection of pregnancy associated glycoprotein in a biological test sample, wherein said method detect antigen/antibody complexes using an indicator reagent comprising a signal generating compound; wherein the detection of antigen/antibody complex is an indication that PAG-7 &/OR PAG-18 proteins are present in test sample.

Dated 05th day of October, 2020

Garima Singhal
APPLICANT’S AGENT
Regd. Patent Agent [IN/PA-3332]

Immunogenic Peptides and Antibodies Thereof for Detection of Pregnancy in Bovine and Related Species

ABSTRACT
The present invention refers to the design, synthesis and evaluation of immunogenic peptides from pregnancy associated glycoproteins (PAG-7 & PAG-18), isoform of PAGs and use of Polyclonal AND/OR monoclonal antibodies thereof. The sequences for the immunogenic peptides were generated and derived upon using different bioinformatics approaches. The identified peptides were used for the generation of antibodies in rabbit. The selected peptides from PAG-7 and PAG-18 were screened for its immune-reactivity in dot blot and western blot using specific antibodies raised against them. The peptide specific antibodies showed its potential to recognize PAG antigens in pregnant bovine serum samples as early as day 35th after artificial insemination. This invention may help to evolve user-friendly immune-diagnostic assays for early and accurate detection of pregnancy in animals using PAGs.