Noelita Melo de Sousa, Aline Tchimbou Jean-François Beckers Project D31-1284 Assay of Pregnancy-Associated Glycoproteins in milk: Integration of the assay in periodic milk control to follow pregnancy in dairy cows Noelita Melo de Sousa, Aline Tchimbou Jean-François Beckers Physiology of Reproduction, Faculty of Veterinary Medicine, ULg

Pregnancy diagnosis and follow-up in dairy cows Sensitivity  identification of pregnant females Specificity  identification of non pregnant females Simplicity  test in farm, transfer to the lab, direct examination by ultrasonography… or assay in milk samples (periodic milk control) Low cost ! Registration of result and integration in farm computer No additional manipulation of the cows and minimal work for farmer.

Approaches to establish pregnancy diagnosis and follow-up in cows In practice… One single early diagnosis is not enough ! Due to late embryonic and early fœtal mortality ! Frequency of fetal losses : minimum 5% Necessity of control after day 60th (Fricke, 2010 ; http://www.extension.org/pages/When_to_Pregnancy_ Check_Dairy_Cattle_ and_Why)

Frequency of discrepancy between the reality and the log book of the farmer In a field study, 510 dairy cows were blood sampled between day 100 and day 120 after the « last » insemination and PAG was determined by RIA. Results : 5.1% of the cows presented levels lower than 0.8 ng/ml 1.4 % presented levels between 1 and 5 ng/mL indicating a later fertilization (not recorded by farmer) 93.5% gave values corresponding to pregnancies evolving for more than hundred days.

The present research project Assay of PAG concentration in milk samples in order to assist farmers through the periodic milk control organized in many countries for dairy cows !

Pregnancy Associated Glycoproteins (PAGs) First purified in the 80’s in USA by Butler et al. (1982), in Belgium by Beckers et al. (1988), and in France by Mialon et al. (1993). fœto-placental units Synthesized in fetal cotyledons Glycoproteins MM between 35 and 70 kDa Belong to the family of aspartic proteases Also synthesized in intercaruncular area (Touzard et al. 2013)

Migration of binucleated cells… Binucleate cell juste after fusion with a maternal uterine epithelial cell (*) Granulated binucleate cell has migrated up to the microvillar junction (arrow) Trinucleate (or maternal giant) cell in the maternal uterine epithelium Micrography of the cow placentome. The interuption of microvillar jonction between trophectoderm and uterine epithelium is shown by arrow ( ). Wooding & Beckers (1987)

The expression of different PAGs is programmed in time and space The PAGs Till now, 22 cDNAs were identified in bovine placenta The expression of different PAGs is programmed in time and space Green et al. (2000)

Concentrations of PAG in plasma/serum in cow Profile of PAGs concentrations during pregnancy Days of pregnancy Days before and after calving Low concentrations Concentrations of PAG in plasma/serum in cow Zoli et al. (1992)

Different RIA systems are avalaible Homologuous boPAG67kDa as standard and tracer Heterologous RIA 2 : Antiserum raised against caPAG55+59 RIA 1 : Antiserum raised against boPAG67kDa RIA 3 : Antiserum raised against caPAG55+62 By using heterologous systems, higher concentrations of PAGs are detected between Day 22 and Day 60

Assay of PAG in blood samples… PAGs molecules are stable Long half life in vivo Also stable in blood samples during transportation Blood samples can be sent by post !

PAG in the milk samples The assay by the classical RIA allows the quantification of concentrations in milk of ewes and goats. Pregnancy diagnosis is valuable from Day 32 after mating. Goat Ewe Gonzalez et al. (2001) El Amiri et al, (submitted) Unfortunately, in cow, milk concentrations of PAG are much more lower (20 to 30 times) than in serum. Moreover, in bovine species, blood concentrations are relatively low till the 6th month of pregnancy. Gajewski et al. (2008); Friedrich et Holtz (2008)

Day of pregnancy Plasmatic concentrations are low till the 6th month after fertilization

An ASSAY in milk samples The challenge: Milk contains high concentrations of lipids, casein micelles, cells, membrane fragments etc… A competitive assay in milk can be compared to a « jogging in a marsh land ». Non specific effects after the speed of reaction between molecules and stability of reagents.

Objective of the study Increasing the sensitivity of the technique ! Increasing the robustness of the technique ! …development of an Immuno-Radio-Metric-Assay (IRMA)

The IRMA technique « sandwich » assay Characteristics: The IRMA sandwich is not a competitive technique. It is based in consecutive saturation steps. An antibody is coated to the tube and used for the immobilisation of the antigen. An other antibody bount to biotin is added in order to fix to the antigen and quantify the antigen present in the sample.

The IRMA technique « sandwich » assay Characteristics: IRMA technique can be very sensitive and robust: Detected concentrations can be 50 to 100 times lower than those detected in competition systems (Canfield et al, 1987)

Ab capture (coated) Anti-PAG immunoglobulins (capture Ab) are coated on the internal well of the tube

Ab capture (coated) Ag to be dosed Anti-PAG immunoglobulins (capture Ab) are coated on the internal well of the tube Sample (volume till 4 mL) is incubated in the coated tube (stirring or rotation are possible)

A first wash (multistep 3, 4 or 5 rinsing) eliminates the sample Ab capture (coated) Ag to be dosed Ab revelation (marqued) 125 Iodine Anti-PAG immunoglobulins (capture Ab) are coated on the internal well of the tube Sample (volume till 4 mL) is incubated in the coated tube (stirring or rotation are possible) A first wash (multistep 3, 4 or 5 rinsing) eliminates the sample

A first wash (multistep 3, 4 or 5 rinsing) eliminates the sample Ab capture (coated) Ag to be dosed Ab revelation (marqued) 125 Iodine Anti-PAG immunoglobulins (capture Ab) are coated on the internal well of the tube Sample (volume till 4 mL) is incubated in the coated tube (stirring or rotation are possible) A first wash (multistep 3, 4 or 5 rinsing) eliminates the sample The tube is than incubated with an other antibody radiolabelled with 125 Iodine

A first wash (multistep 3, 4 or 5 rinsing) eliminates the sample Ab capture (coated) Ag to be dosed Ab revelation (marqued) 125 Iodine Anti-PAG immunoglobulins (capture Ab) are coated on the internal well of the tube Sample (volume till 4 mL) is incubated in the coated tube (stirring or rotation are possible) A first wash (multistep 3, 4 or 5 rinsing) eliminates the sample The tube is than incubated with an other antibody radiolabelled with 125 Iodine After incubation, a last wash is performed

A first wash (multistep 3, 4 or 5 rinsing) eliminates the sample Labelled fraction signal Antibody concentration Ab capture (coated) Ag to be dosed Ab revelation (marqued) 125 Iodine Anti-PAG immunoglobulins (capture Ab) are coated on the internal well of the tube Sample (volume till 4 mL) is incubated in the coated tube (stirring or rotation are possible) A first wash (multistep 3, 4 or 5 rinsing) eliminates the sample The tube is than incubated with an other antibody radiolabelled with 125 Iodine After incubation, a last wash is performed And the radioactivity is counted…

The IRMA technique « sandwich » assay Difference regarding classical RIA: IRMA technique uses antibodies in excess IRMA technique uses different antibodies for capture and revelation Aims of IRMA technique is to capture a maximum of the antigens present in the sample Direct IRMA was used in order to select the best « pairs » of antibodies (capture and revelation Ab)

The IRMA technique « sandwich » assay Advantages: In IRMA system, using coated tubes for the « capturing » immunoglobulins, the sample can be eliminated (washed) after the first incubation. The assay continues in a clean medium minimizing non specific interferences… and interactions with the tracer.

Table summarizing the anti-PAG antisera available for coating and radiolabelling Espèce Antigène Pregnancy AS Bovine boPAG-1 (67kDa) 4-6 months R726 " R727 R735 R736 R737 R738 R739 R740 R741 R742 R743 R744 R745 Espèce Antigène Pregnancy AS Caprine caPAG55+62kDa D48 - D69 R706 " R707 caPAG55+59kDa R708 R709 caPAG R902 Espèce Antigène Pregnancy AS Ovine ovPAG 55kDa > D100 R778 ovPAG 57+59kDa " R779 ovPAG 57kDa R780 ovPAG 58+61kDa D60 - D100 R805 ovPAG 58+66kDa R806 R807 ovPAG 58+60kDa R808 R809 Espèce Antigène Age gestat. AS Bovine boPAG-2 R435 " R436 R437 R438 Espèce Antigène Pregnancy AS Bubaline wbPAG69kDa 8 months R858 wbPAG61+73kDa " R859 wbPAG58-76kDa R860

Table summarizing the anti-PAG antisera available for coating and radiolabelling Espèce Antigène Pregnancy AS Bovine boPAG-1 (67kDa) 4-6 months R726 " R727 R735 R736 R737 R738 R739 R740 R741 R742 R743 R744 R745 Espèce Antigène Pregnancy AS Caprine caPAG55+62kDa D48 - D69 R706 " R707 caPAG55+59kDa R708 R709 caPAG R902 Espèce Antigène Pregnancy AS Ovine ovPAG 55kDa > D100 R778 ovPAG 57+59kDa " R779 ovPAG 57kDa R780 ovPAG 58+61kDa D60 - D100 R805 ovPAG 58+66kDa R806 R807 ovPAG 58+60kDa R808 R809 Espèce Antigène Age gestat. AS Bovine boPAG-2 R435 " R436 R437 R438 Espèce Antigène Pregnancy AS Bubaline wbPAG69kDa 8 months R858 wbPAG61+73kDa " R859 wbPAG58-76kDa R860

Purification of specific antibodies A very important step! In order to immunopurify specific antibodies we need large amounts oh highly purified PAG.

Purification of specific antibodies The lessons of immunoaffinity: Antibodies for « capture » have to be finely selected… When crude antiserum was coated, the frequence of false positive results increased Antibodies for radiolabelling are also to be finely selected (this aspect was known from litterature)

> 40 mg have been purified Purification of bovine PAG in high amounts Placenta aged of 4 to 6 months (2.6 kg) Extraction in PBS Acidic precipitation Ammonium sulfate precipitation 20-40% 40-80% DEAE cellulose DE-52 40 mM 80 mM 160mM Sephadex G-75 Mono S DEAE G75 Coomassie blue staining of the Mono S fraction having the highest specific activity > 40 mg have been purified

Purification of specific antibodies Steps: Activation of 4B-Sepharose using the cyanogen bromid Coupling of 40 mg of highly purified PAG to 20 mL of activated gel (washing and stabilization of the gel) Purification of the immunoglobulins anti-PAG

Weight of eluted proteins (mg) Table showing the recovery after immunopurification of specific antibodies AS Origin of the antigen Treated volume Evaluated peak) Weight of eluted proteins (mg) Recovery (mg/mL of treated AS) ip-Ig727 Bovine 12 mL 39 à 60 11,4 0,95 ip-Ig742 36 à 57 7,7 0,64 ip-Ig708 Caprine 21 à 28 8,5 0,71 ip-Ig780 Ovine 27 à 34 12,4 1,03 ip-Ig860 Buffalo 37 à 58 16,4 1,36

Purification of specific anti-PAG immunoglobulins Equilibration buffer: PBS 50 mM, pH 7.4 Washing buffer: The same PBS added with NaCl 18 g/L Elution solution: Glycine-HCl (pH 2.8) Buffer added in the collection tubes: NaHCO3 0.1 M (pH 8.0) Profile of O.D. (280 nM) obtained after affinity chromatography for PAG (8 mL gel PAG-Sepharose 4B). Loading buffer was PBS 50 mL + 15 mM NaCl (pH 7.4). Elution was realized with Glycine-HCl (pH 2.8).

Three different techniques of radiolabelling were used: Results of IRMA based on the radiolabelled IgG and coated immunopurified IgG Three different techniques of radiolabelling were used: Iodogen Lactoperoxydase Chloramine T

A direct IRMA based on radiolabelled antibodies Results appeared of low quality Non specific binding was high Specific binding was never higher than 30% Explanation: Iodine can be fixed on the FAB part of the immunoglobulin, this event was known from litterature

Results using the direct IRMA CDR Binding rate ~ 30% : Lower than expected Possible explanation: Higher concentration of tyrosine in the « complementary determining region » (≈ 20%) => when iodine fixes in this part the antigen recognition site is altered Nikula et al., 1995 Fixation of iodine on the FAB region is the weak point of the direct radiolabelling of IgG Ac capture (coated) Ag to be dosed Ac revelation (radiolabelled)

Revisitation of the protocol Biotinylation of the revelation antibody Final revelation using the radiolabelled streptavidin Structure of the tetrameric complexe formed by streptavidin (Streptomyces avidinii) and biotin (B7vitamin)

Revisitation of the protocol: IRMA using biotinylated Ab and radiolabelled streptavidin Ab capture Ag to be dosed Ab biotinylated

Revisitation of the protocol: IRMA using biotinylated Ab and radiolabelled streptavidin Ab capture Ag to be dosed Ab biotinylated

Revisitation of the protocol: IRMA using biotinylated Ab and radiolabelled streptavidin Ab capture Ag to be dosed Ab biotinylated Radiolabelled streptavidin

IRMA using biotiniled Ab and radiolabelled streptavidin Ab captured Ag to be dosed Ab biotinylated Radiolabelled streptavidin Antigen concentration Binding ratio

First results after using biotinylated Ab for revelation and radiolabelled streptavidin Binding of the tracer is greater than 70% Binding rate of radiolabelled streptavidin. Ip-Ig860 was coated, ip-Ig727 was biotinylated (1:8.000). Standard was boPAG67kDa ranging from 0.1 to 50 ng/mL.

The IRMA technique: the strategy and the improvement of reagents Quality and purity of reagents increase the sensitivity and the robustness of the technique Generally it was considered that the antiserum used for coating can be « crude »… Our data shown that all parameters have to be improved

IRMA using biotinylated Ab and radiolabelled streptavidin Coated Ab and biotinylated Ab were purified by immunoaffinity on pure PAG-Sepharose column Radiolabelled streptavidin was added to reveal the reaction This system was used on milk sample to determine PAG concentrations during pregnancy and postpartum

IRMA using biotinylated Ab and radiolabelled streptavidin Selection of pairs: Ig coated Ig biotiniled ip-Ig727 ip-Ig742 ip-Ig708 ip-Ig780 ip-Ig860

Preliminary results: Profiles of PAG in milk (measured by IRMA technique) Profiles of PAG (ng/mL) measured by IRMA in bovine milk samples. One milliliter of milk sample was incubated (ON, RT) in StarNunc tubes previously coated with 300 µL of anti-caPAG immunopurified AS#708 (0.32 µg/mL of ip-Ig708). A first wash (5 rinsing with 0.9% NaCl + 0.05% Tween 20) was done to eliminate the sample. Thereafter, the tubes were incubated (ON, RT) with 0.3 mL of biotinylated immunopurified AS #727 (dilution 1:8,000). A second wash was performed (5 times) and finally, radiolabelled streptavidin was added (0.3 mL, 150,000 cpm). Tubes were incubated 2 h (RT) before final washing and measurement of radioactivity.

Preliminary results: Profiles of PAG in milk (measured by IRMA technique) Profiles of PAG (ng/mL) measured by IRMA in bovine milk samples. One milliliter of milk sample was incubated (ON, RT) in StarNunc tubes previously coated with 300 µL of anti-caPAG immunopurified AS#708 (0.32 µg/mL of ip-Ig708). A first wash (5 rinsing with 0.9% NaCl + 0.05% Tween 20) was done to eliminate the sample. Thereafter, the tubes were incubated (ON, RT) with 0.3 mL of biotinylated immunopurified AS #727 (dilution 1:8,000). A second wash was performed (5 times) and finally, radiolabelled streptavidin was added (0.3 mL, 150,000 cpm). Tubes were incubated 2 h (RT) before final washing and measurement of radioactivity.

Perspectives 1 2 3 4 5 6 7 8 9 Normal or endangered pregnancy … Dry off Clinical Assay Blood Milk Clinical Assay Blood Clinical Assay Blood Milk AI ET => PAG assays integrated in program… AVORTEMENT 1 2 3 4 5 6 7 8 9 Fertilization parturition Normal or endangered pregnancy … Pregnancy diagnosis Clinical Assay Blood Milk

Thank you for your attention!

Acknowledgements... Participants: Contribution: Dr. N.M. Sousa (Post-Doc) Purification PAG / IRMA Mlle A. Njanjo (Lab technician) Purification PAG / IRMA Mlle M Bériot (FVM trainee) Purification PAG Mlle. L. Earley (FVM student) Care of rabbits Mlle N. Ponslet (FVM student) Care of rabbits M. C. Delhalle (ouvrier) Collect slaughterhouse Farmers from plateau de Herve… Equipe: Mme R. Fares (Secretary) Dr. C. Gatez (DVM) Mme A. Bella (DVM) Mme Ch. Poullet Mr. B. Glaude (Ing.) Mr. M. Machado Collaborateurs: Mr. Y. Colemonts (CER Marloie) Prof. O. Szenci (University of Ullo-Dora major, Hungary) Dr. O. Barbato (University of Perugia, Italy) Prof. F. Lopez-Gatius (University of Lleida, Spain) Dr. B. El Amiri (INRA-Settat, Marocco) Prof. Z. Gajewski (University of Warsaw, Poland) Dr. K. Klisch (University of Nottingham, UK) Prof. F. Gonzalez (University Canaries Islands, Spain) DGA – Ministry of Wallonne Region (Agricultural Direction) Laboratory of Animal Endocrinology and Reproduction