




































In ternationa l
Scholars
Journa ls

 

African Journal of Pig Farming ISSN 2375-0731 Vol. 2 (12), pp. 001-007, December, 2014. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

Full Length Research Paper 

 

A novel approach for very early pregnancy diagnosis in 
swine by anti-early pregnancy factor (EPF) antiserum 
blocking enzyme-linked immunosorbent assay (ELISA) 

 
Kai Quan1,2, Xingxu Zhao1*, Changxing Zhang2, Qiuliang Xu2, Hongfang Wei1, Junjie 

Hu1 and Yong Zhang1
 

 
1
College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, Gansu 730070, China. 

2
Zhengzhou College of Animal Husbandry Engineering, Zhengzhou, Henan 450011, China. 

 
Accepted 09 April, 2014 

 
Early pregnancy factor (EPF) is essential for the initiation and maintenance of pregnancy. Early pregnancy 
factor activity monitoring has been reported to be the effective method for very early pregnancy diagnosis. In 
this study, three BALB/c mice were immunized with the synthetic peptide segment corresponding to the amino 

acid sequence 36 to 55 of EPF (IG20) for anti-EPF antibodies. Mouse anti-EPF antiserum titers were evaluated by 

an indirect enzyme-linked immunosorbent assay (ELISA), and the titers were 6.4 × 10
3
. Serum samples were 

taken from 21 Yorkshire × Landrace crossbred sows (12 pregnant and 9 non-pregnant). The presence of EPF in 
these serum samples was detected by a blocking ELISA using the antigen-antibody (Ag-Abs) reaction between 

IG20-ovalbumin and mouse anti-EPF antiserum for very early pregnancy diagnosis, blank was used as negative 
controls. The optical density (OD) values were measured at 450 nm, and the OD ratios of negative control/serum 
sample (N/S) >2.1 were considered positive, and N/S <2.1 negative. When the test serum samples were in 1/4 
dilutions with PBS, twelve samples from pregnant swine were positive, nine non-pregnant serum samples were 
negative. Very early pregnancy can be determined by using the mouse anti-EPF antiserum blocking ELISA in 
swine. 
 
Key words: Very early pregnancy diagnosis, early pregnancy factor (EPF), Rosette inhibition test (RIT), blocking 
enzyme-linked immunosorbent assay (ELISA). 

 
INTRODUCTION 

 
Early and accurate diagnosis of pregnant sows and gilts 
has potential economic advantages by reducing non-
productive days and increasing reproductive efficiency in 
pig production. Currently, the porcine pregnancy 
diagnosis is mainly through the detection of return to 
estrus (Almond et al., 1986, 1987), as well as the scan of 
embryo with amplitude-depth (A-mode) (Pyorala, 1989), 
Doppler and Real-time (B-mode) ultrasonography (RTU) 
(Zambelli et al., 2006; Williams et al., 2008). However, 
these methods are only effective after 21 or 24 days of 
gestation (Krüger et al., 2002), and could not be used for  
 
 
 
*
Corresponding author. E-mail: quankai1115@126.com 

 
Abbreviation: EPF, Early pregnancy factor. 

 
 
 

 
very early pregnancy diagnosis (in pigs conceptuses 7 to 
15 days) in sows (Shaw et al., 1980). It is necessary to 
establish a fast and efficient method for very early 
pregnancy diagnosis in sows.  

Early pregnancy factor (EPF) is a pregnancy-
associated protein, which was firstly detected by rosette 
inhibition test (RIT) in mice serum as early as 4 h after 
mating (Morton et al., 1974). Early pregnancy factor has 
been shown to be essential for the initiation and 
maintenance of pregnancy, and it was assayed by the 
RIT through the decreasing ability in the formation of 
rosettes (Greco et al., 1992; Straube et al., 1989). The 
activity of EPF has been detected in maternal serum 
within 24 h of fertilization in all mammalian species tested 
and persisted for at least the first half of gestation (Morton 
et al., 1987), and EPF activity monitoring (RIT) has been 
reported to be the only method suitable for very 



 
 
 

 

early pregnancy diagnosis (Straube et al., 1988; Mesrogli 
et al., 1988). However, RIT was not a quantitative assay 
and has been proved cumbersome-and time-consuming 
(Koch et al., 1985).  

The amino acid sequence of EPF is fully identical to the 
extra-cellular form of chaperonin10 (cpn10) (Alice et al., 
1996; Morton, 1998), human, bovine and swine share the 
same sequence of EPF (Englebretsen et al., 2002; Fotini 
et al., 2003). In the present study, we have undertaken 
production of anti-EPF antibodies to synthetic peptide 
segment corresponding to the amino acid 36 to 55 of EPF 

(IG20). Serum samples were collected from 12 pregnant 

and 9 non-pregnant sows, the presence of EPF in these 
sera samples was detected by a blocking enzyme-linked 
immunosorbent assay (ELISA) using the antigen-antibody 

(Ag-Abs) reaction between IG20 and mouse anti-EPF 

antiserum, then, the optical density (OD) values were 
measured at 450 nm, and pregnancy was determined by 
the OD ratios of control/serum sample (N/S) (Loffeld et 
al., 1989; Shu et al., 2011; Xie et al., 2007). 
 

 
MATERIALS AND METHODS 
 
Preparation of serum samples 

 
The blood samples (each 2 ml) were taken from the ear vein of 21 
Yorkshire × Landrace crossbred sows (12 pregnancies and 9 non-
pregnancies) after second parity. Twelve blood samples of pregnant 
sows were collected on the sixth day after artificial insemination 
(AI), and pregnancy was confirmed by B-ultrasound after the 40 
days of AI. Nine blood samples of non-pregnant sows were 
collected on the sixth day after estrus (no insemination). These 
samples were allowed to clot for about 1 h at room temperature, 
and the sera were separated by centrifugation at 1 000 ×g for 30 
min, and deactivated at 56°C for 30 min, then stored at -20°C until 
testing. 

 

Rosette inhibition test 

 
The human lymphocytes were collected from one of the lymphocyte 
donors. Lymphocytes were separated through a gradient 
centrifugation by the Ficoll-Hypaque (Sigma Chemical Co., St. 
Louis, MO, USA) method and processed according to the 
manufacturer’s instructions. The isolated lymphocytes were 

adjusted to 4 × 10
6
 cells per ml with Hanks balanced salt solution 

(Hanks', pH7.4, Ca
2+

 and Mg
2+

 free). Sheep red blood cells (sRBC) 
were obtained from fresh defribrinated sheep blood according to the 
description of Ghaffari et al. (2008), and sRBC were adjusted to a 

final concentration (1 × 10
8
 cells per ml) with Hanks'.  

Rosette inhibition test was performed as established by Ghaffari 
et al. (2008) and Athanasas-Platsis et al. (2000) with minor 
modification. Serum samples of each pig (100 µl) were mixed with 
human lymphocytes suspensions (500 µl) and incubated at 3°C for 
60 min. The reaction solutions were divided into twelve equivalents 

(50 µl each sample), then 100 µl serially diluted (1 : 2
1
 × 10

3
, 1 : 2

2
 

× 10
3
, to 1 : 2

12
 × 10

3
) rabbit antihuman-lymphocyte globulin (ALG) 

were added, and incubated at 37°C for 60 min, washed 3 times with 
Hanks', respectively. Fifty micolitres sRBC were added and 
centrifuged immediately at 800 ×g for 5 min. Then one 100 µl 0.8% 
glutaraldehyde was added to fix the lymphocytes and erythrocytes. 
Over 200 lymphocytes and rosettes were counted and recorded. 

 
 

  
 
 

 
Each sample was repeated three times.  

The number of rosettes formed was determined and expressed 
as percentage of the number of rosettes formed without ALG. 
Results are recorded as rosette inhibition titers: the highest dilution 

of ALG [log2 (reciprocal dilution × 10
-3

)] to give rosette formation of 
<75% (samples without ALG = 100% rosette formation). 

 

Preparation of immunogens 
 

A peptide segment (IMLPEKSQGKVLQATVVAVG, IG20) 
corresponding to the amino acid sequence 36 to 55 of EPF 
(accession no. NP776771) was synthesized on 0.3 mmol/g Fmoc-  
wang resin (GL Biochem Ltd., Shanghai, China) using a 

Symphony
®

 Multiplex Peptide Synthesizer (Protein International 
Technologies, Inc., St. Louis, MO, USA) (Ahlborg, 1995). A cysteine 
residue was added at the N-terminal of the peptide segment to 
facilitate conjugation. The peptide was purified by reversed-phase 
high performance liquid chromatography (RP-HPLC) coupled to  
pneumatically assisted electro spray ionization mass spectrometry 

(ESI-MS) (Quattro. Micro
TM

 Api, Waters Corp., Milford, MA, USA) 
with the solid-phase purification over 95% (Sanz-Nebot et al., 
2001).  

The synthetic peptide was conjugated to a carrier protein of IgG-
free bovine serum albumin (BSA, Sigma Chemical Co., St. Louis, 
MO, USA) using a hetero-bifunctional cross-linker succinimidyl 4-
(Nomaleinimido-methyl) cyciohexane-l-car-boxylate (Sulfo-SMCC, 
Pierce Biotechnology Inc., Rockford, IL, USA) according to the 
method described by Alexander et al. (1984). Briefly, 4 mg of BSA 
was dissolved in 0.5 ml phosphate buffered solution (PBS, 0.1 M 
sodium phosphate, 0.15 M sodium chloride, 0.1 M EDTA, pH 7.2), 
then mixed with 1 mg sulfo-SMCC dissolved in 50 µl DMSO. The 
mixture was kept at room temperature for 30 min, and then the 
buffered solution dialysis was conducted at 4°C overnight. The 
dialysis fluid was changed three times to remove excess coupling 
agent. The SMCC-activated BSA (SMCC-BSA) solution was  
adjusted to a concentration of 5 mg/ml with PBS and stored at - 

20°C. Two milligrams of IG20 were dissolved in 50 µl 
dimethylformamide (DMF) and added 150 µl conjugation buffer (5  
mM EDTA buffered solution 0.01 M PB, pH 7.2). Thereafter, 10 
mg/ml of IG20 was mixed with SMCC-BSA and kept at room  
temperature for 30 min, to have the peptide segment conjugated to 

carrier protein to form the immunogen IG20-BSA. The IG20-BSA 
was identified by sodium dodecyl sulfate polyacrylamide gel  
electrophoresis (SDS-PAGE). Ovalbumin (OVA, Sigma Chemical 

Co., St. Louis, MO, USA) was conjugated to IG20 (IG20-OVA, 
coating antigen) as the similar procedure described earlier. 

 

Preparation of mice antiserum to EPF 

 
Three BALB/c mice (female, 8 weeks of age) were immunized with 

IG20-BSA conjugates. The first dose consisted of 50 µg of IG20-
BSA conjugates for injection subcutaneously as an emulsion of 
PBS and Freund’s complete adjuvant (FCA, Sigma Chemical Co., 
St. Louis, MO, USA). The boosters were given at two weeks 

intervals with the same dosage of IG20-BSA conjugates emulsified 
in Freund’s incomplete adjuvant (FIA, Sigma Chemical Co., St. 
Louis, MO, USA) (Ebensen et al., 2007).  

Blood samples (20 µl) were collected from tail veins 14 days after 
third immunization. Anti-EPF antibody titer was determined by the 
indirect ELISA using the following procedure (Kit et al., 1990; Sun et 
al., 2007). 96 well ELISA plates (Costar) were coated with 1 µg/ml 

IG20-OVA diluted in carbonate buffer, pH 9.6, in a 100 µl per well 
and incubated overnight at 4°C. Thereafter, the plates were washed 
four times with PBST and repeat between each of the following 
steps, and blocked with 5% chicken serum at 37°C for 1 h, 50 µl 
1/100 serial diluted anti-sera were added to the blocked plates and 



       
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Figure 1. The Rosette inhibition titers (RIT) range of pregnant and non-pregnant porcine serum samples. 

The Rosette inhibition titers (mean ± SD). Pregnant (◇): 8.0 ± 0.5**, non-pregnant (■): 6.0 ± 0.4. ** was P 

< 0.01 (t-test) between pregnant and non-pregnant serum samples. 
 

 
incubated at 3 °C for 15 min, then 50 µl 1000-fold diluted 
horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG 
(Abnova Corp., Heidelberg, Germany) was added and again 
incubated at 37°C for 30 min. The substrate-chromogen solution 

(TMB-H2O2) was added, and the OD (450nm) value was measured 
using a micro-plate reader. 50 µl 1 µg/ml OVA was used as 
negative controls and test was repeated three times. 

 

Blocking ELISA detection of EPF presence in serum samples 

 
The procedures of blocking ELISA was conducted as previously 
described (Kit et al., 1990; Sun et al., 2007). ELISA plates were 
sealed with 5% chicken serum. Hereafter, 50 µl of each porcine 
sera sample (9 non-pregnant and 12 pregnant sows, blank group 
served as negative control) were serially diluted (1/2 to 1/64) with 
PBS, then mixed with 50 µl 800-fold diluted mice anti-EPF sera in 
each well, and incubated at room temperature for 30 min. The 
mixed solutions were transferred to another ninety-six well ELISA 

plates which were coated with 100 µl 0.5 µg/ml IG20-OVA and 
sealed with 5% chicken serum, incubated at 37°C for 15 min. 
Horseradish peroxidase-conjugated rabbit anti-mouse IgG was 

added, incubated at 37°C for 30 min, TMB-H2O2 was added and 
the OD value at 450 nm was measured by micro-plate reader. The 
test was repeated three times. 
 
 
Statistical analysis 

 
Rosette-inhibition titers of 9 non-pregnant and 12 pregnant serum 
samples were analyzed using the SPSS Statistics 17.0 ANOVA and 
t-test. The OD values of mouse anti-EPF antiserum blocking ELISA 
in serials diluted samples (nine non-pregnancies, twelve 
pregnancies and negative controls) were analyzed using the SPSS 
Statistics 17.0 ANOVA. 

 
 

 

RESULTS 

 

The rosette-inhibition test titers 

 

Early pregnancy factor activities of 9 non-pregnant and 
12 pregnant porcine serum samples were tested by RIT 
using human lymphocytes. There were significant 
differences between pregnant and non-pregnant sera 
(P<0.01) (Figure 1). 
 

 

Evaluation of mice antiserum responses to EPF 
 

The conjugations (IG20-BSA and IG20-OVA) were 
identified by SDS-PAGE. The calculated molecular 

combined ratios of IG20/BSA and IG20/OVA were 15:25 
and 5:15 (Figure 2), respectively. Three BALB/c mice 

were immunized with IG20-BSA, and the antibody 
responses to EPF were measured by indirect ELISA. The 

serum anti-EPF antibody titers were 1:6.4 × 10
3
 (Figure 

3). 
 

 

Evaluation of blocking ELISA 

 

When the test serum samples were diluted 1/4 with PBS, 
the OD ratios of negative control/serum sample (N/S) 
were over 2.1 (positive) in 12 pregnant serum samples, 
and was less than 2.1 (negative) in nine non-pregnant 
serum samples (Loffeld et al., 1989; Shu et al., 2011; Xie 



   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

Figure 2. The SDS-PAGE analysis of conjugated products. 
The molecular weight of IG20-BSA ranged from 97.4 to 110 
kDa, and IG20-OVA was from 66 to 97 kDa.  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 3. The titers of anti-EPF serum antibody. When the OD values of anti-EPF serum antibody 
were 2.1 times negative, they were positive; and hence negative. 

 

 

et al., 2007). However, when the sera were 1/2 diluted, 
six of the nine non-pregnant serum samples were 
presented as a false-positive. The serum samples were 
1/8 dilution or more, the non-pregnant and partly 
pregnant serum samples showed negative (Figure 4 and 
Table 1). 

 
 

 

DISCUSSION 

 

Accurate and early detection of pregnant and non-
pregnant sows has become a key to good breeding 
management because it is an essential factor for 
monitoring and controlling fertility in pigs. In commercial 



    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 4. The OD ratios of N/S (negative control/porcine serum sample). N/S > 2.1 were 
considered positive (+), and N/S value < 2.1 negative (-). 

 

 
Table 1. The OD values of mouse anti-EPF antiserum blocking ELISA using the serum samples of serial dilution (the mean 
values of 3 times repeated for each sample).  

 

Parameter Number 
 Diluted time of sows’ serum sample  

Negative  

1/2 1/4 1/8 1/16 1/32 1/64 
 

   
 

 1 0.258* 0.380* 0.814 1.102 1.135 1.132 1.300 
 

 2 0.146* 0.397* 0.468 0.561 0.562 0.874 0.772 
 

 3 0.145* 0.183* 0.482 0.696 0.722 0.698 0.919 
 

 4 0.148* 0.298* 0.692 0.52 0.644 0.897 0.735 
 

 5 0.098* 0.440* 0.610* 0.704 0.835 1.021 1.321 
 

Pregnancy 
6 0.114* 0.297* 0.544 0.663 1.061 0.574 0.565 

 

7 0.125* 0.183* 0.388* 0.697 0.782 0.666 0.932 
 

 
 

 8 0.188* 0.268* 0.792 0.631 0.844 0.837 0.766 
 

 9 0.135* 0.480* 0.714 0.704 0.931 1.031 1.032 
 

 10 0.146* 0.357* 0.631 0.769 0.665 0.672 0.874 
 

 11 0.135* 0.283* 0.566 0.893 0.721 0.789 0.728 
 

 12 0.178* 0.252* 0.591 0.722 0.746 0.863 0.639 
 

 1 0.321* 0.438 0.603 0.794 0.761 0.862 0.835 
 

 2 0.342* 0.533 0.692 0.834 0.996 0.822 0.921 
 

 3 0.465 0.612 0.772 0.865 0.82 0.944 0.965 
 

 4 0.372* 0.552 0.685 0.903 0.804 0.835 0.932 
 

Non-pregnancy 5 0.467* 0.631 0.782 0.734 0.863 1.061 1.166 
 

 6 0.494 0.611 0.612 0.734 0.892 0.782 1.032 
 

 7 0.322* 0.461 0.722 0.776 0.7312 0.844 0.874 
 

 8 0.479 0.490 0.694 0.811 0.904 0.931 0.728 
 

 9 0.351* 0.589 0.731 0.921 0.869 0.665 0.939 
 

 
* The OD values < 2.1 times of negative, means positive. 

 

 

swine farms, the most common strategy for identification 
of non-pregnant females is detection of estrus via daily 
boar exposure from 17 to 23 days after breeding, 

 
 

 

followed by the use of ultrasound between 28 and 45 
days of gestation (Almond et al., 1986; Zambelli et al., 
2006). Although, this regimen is often used with a high 



 
 
 

 

degree of accuracy, its usefulness is compromised by the 
inability to determine pregnancy status within 15 days 
after breeding (Shaw et al., 1980). Of course, the 
measurement of human chorionic gonadotrophin (hCG) 
was widely used for very early pregnancy diagnoses in 
human (Thomas et al., 1986), and the plasma hormone 
(including prostaglandin-F2, hCG, progesterone and 
estrone sulfate) concentrations have been reported in 
very early pregnancy diagnoses in swine (Almond et 
al.,1986; Krüger et al.,2002), these plasma hormones 
concentrations had obvious changes from a non-pregnant 
to pregnant swine, but there was no statistical difference 
due to the high variation of the hormone levels between 
individuals (Almond et al., 1987). Hence, these hormones 
were not having the cut-off value as a neutral standard 
concentration range for identified non-pregnant or 
pregnant sows. The use of hormones as a diagnostic 
method may not be applicable in commercial units 
because of the accuracy in reducing the test.  

The earliest specific indication for fertilization and the 
continuing presence of a viable conceptus is a serum 
constituent, which was originally detected in mice (Morton 
et al., 1974). This substance is known as the EPF and 
has also been described in pigs (Greco et al., 1992, Koch 
et al., 1985). It has early appearance (within hours) after 
mating or insemination (Shaw et al., 1980), and rapid 
disappearance following induced death or removal of the 
embryos (Morton et al., 1987).These factors suggest that 
EPF may be the most useful tool for very early pregnancy 
diagnosis. The objectives of this study were to evaluate 
the effectiveness of the EPF test for detecting the 
pregnant sows, and to compare the reliability of RIT and 
anti-EPF antiserum blocking ELISA for very early 
pregnancy diagnosis.  

The rosette inhibition titers of pregnant sows were 
significant higher than in non-pregnant gilts. This was 
consistent with the report of Koch (1985). The RIT test 
was based on the ability of ALG to inhibit the formation of 
spontaneous rosettes between human lymphocytes and 
sRBC. This means that the lymphocytes are spon-
taneously form rosettes, a flower-like arrangement in 
which a lymphocyte has several red blood cells attached 
to it. Lymphocytes from pregnant animals form fewer 
rosettes than those from non-pregnant animals. 
Therefore, with the aid of EPF diagnostics, a pregnancy 
can be detected at a very early stage. However, rosette 
inhibition test is not a quantitative assay, and the assay is 
not suitable for high-throughput diagnostic and commer-
cial applications, and RIT proved cumbersome, time-con-
suming and was easy to disturb with a particular disad-
vantage which is the indirect character of the test and its 
possible interference by other substances (Koch et al., 
1985). To replace the RIT with a more user-friendly 
assay, we developed an anti-EPF antiserum blocking 
ELISA for very early pregnancy diagnosis.  

The production of anti-EPF antibody is indispensable 
for very early pregnancy diagnosis by anti-EPF antiserum 

 
 

  
 
 

 

blocking ELISA. To obtain anti-EPF antiserum of high titer 

and specificity, the antigenic peptide (36 to 55, IG20) based 

on prediction of DNAMAN has been synthesized. Three 

BALB/c mice were immunized by IG20-BSA, and the mouse 

anti-IG20 antiserum titers were 6.4 × 10
3
. Using anti-IG20 

antiserum, a blocking ELISA was constructed for very early 
pregnancy diagnosis in sows. When the porcine sera 
samples were 1/4 dilution, the pregnancy can be accurately 
determined (Figure 4). The concen-tration of EPF efficiently 

blocked the Ag-Abs reaction between IG20-OVA and mouse 

anti-EPF antiserum by a blocking ELISA in pregnant serum 
samples. At the same time, the non-pregnancy ones were 
not obviously influenced by the OD values of blocking 
ELISA. The ones with pregnancy were positive and non-
pregnant serum samples were negative, the pregnancy can 
be deter-mined by the anti-EPF antiserum blocking ELISA. 

However, some homologous substances of IG20 (HS-
IG20) were in the porcine serum samples (Betsou et al., 
2003), when the serum samples were 1/2 dilution, with 
the concentration increases of HS-IG20, the cross-reac-
tion was enhanced between mouse anti-EPF antiserum 
and HS-IG20 (Leder et al., 1994), and it was blocking the 
reaction between antibody and antigen (the coating 
material, IG20-OVA). So, some non-pregnant serum 
sam-ples were presented as false-positive. On the 
contrary, when the porcine sera samples were 1/8 to 1/64 
dilution, the concentration of EPF was limited in serum 
samples, and it could not efficiently block the Ab-Abs 
reaction between IG20-OVA and mouse anti-EPF 
antiserum by blocking ELISA, the presence of EPF in 
these serum samples were not detected.  

In conclusion, the synthetic segment including the 
residue 36 to 55 of EPF can be used as an antigen to 
produce the anti-EPF polyclonal antibody. Using this 
antibody, we developed an anti-EPF antiserum blocking 
ELISA for very early pregnancy diagnosis. When the test 
serum samples were in 1/4 dilutions, the very early preg-
nancy can be detected in sows. 

 

ACKNOWLEDGEMENTS 

 

This study was supported by the Basic and Edge 
Technology Fund of Henan, China (grant number: 
082300433201). We would like to thank all the staff of 
Henan Key Laboratory of Animal Immunology for their 
kind support. 

 
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