1. Noelita Melo de Sousa, Aline Tchimbou Jean-François Beckers
Project D 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

2. 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.

3. 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 ; Check_Dairy_Cattle_ and_Why)

4. 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.

5. 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 !

6. 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)

7. 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)

8. 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)

9. 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)

10. 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

11. 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 !

12. 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)

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

14. 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.

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

16. 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.

17. 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)

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

19. 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)

20. 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

21. 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

22. 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

23. 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…

24. 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)

25. 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.

26. 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

27. 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

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

29. 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)

30. > 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

31. 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

32. 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

33. 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).

34. 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

35. 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

36. 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)

37. 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)

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

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

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

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

42. 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.

43. 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

44. 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

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

46. 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 % 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.

47. 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 % 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.

48. 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
Fertilization
parturition
Normal or endangered pregnancy …
Pregnancy
diagnosis
Clinical
Assay
Blood
Milk

49. Thank you for your attention!

50. 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