MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY Volume 33, Number 5, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/mab.2014.0013
Development and Application of an ELISA Kit for the Detection of Milk Progesterone in Dairy Cows Ling Wu,1 Chuchu Xu,1 Cheng Xia,1,2 Yu Duan,1 Chuang Xu,1 Hongyou Zhang,1 and Jun Bao 2
Progesterone (P4) is a steroid gonadal hormone that is mainly produced from the corpus luteum and placenta and has various biological functions, especially reproductive regulation. It is important to establish a specific and sensitive P4 enzyme-linked immunosorbent assay (ELISA) for the study of ovary activity and reproductive disorders. Therefore, we prepared a monoclonal antibody (MAb) in a completed antigen (11a-OH-P4-HSOVA). Based on the MAb and our previously prepared completed antigen, a highly specific and sensitive ELISA was developed. In the present study, a competitive ELISA for the determination of P4 was described in dairy cow milk. It was found that P4 concentration in milk samples from five pregnant cows was significantly higher than that from five estrus cows. The diagnosis rate for pregnancy and non-pregnancy in 54 dairy cows were 93.3% and 95.8%, respectively, at 19 to 23 days after pregnancy by detecting milk P4 concentration. In summary, the developed ELISA is a potential tool for P4 research and offers an alternative, simple, rapid technique for detecting P4, especially in future large clinical investigations on pregnancy identification and reproductive disorders in dairy farms in China.
Introduction
P
rogesterone (P4) is a gonadal hormone that is mainly produced from the corpus luteum and placenta in humans, pigs, and cows. It has various versatile functions and is recognized as a multifunctional steroid hormone not only in the estrus and pregnancy but also on reproductive disorders, including persistent corpus luteum and corpus luteum cyst.(1,2) It has been reported that the methods of estrous cycle detection and pregnancy diagnosis in dairy cows include rectal palpation,(3) ultrasound inspection,(4) ELISA kits,(5) and immunosensors.(6) In dairy farms, pregnancy, ovarian activity, and breeding status are usually assessed by monitoring blood P4 concentrations. However, the detection of milk P4 concentrations in dairy cows has been widely used as a routine clinical examination in developed countries because it avoids stress and any undesirable effects caused by blood collection, rectal palpation and ultrasound inspection.(3,4) Studies have reported that P4 concentrations at 21 days post-mating were closely related to pregnancy status.(5,7) Additionally, the positive rate of non-pregnancy and pregnancy diagnoses by the assessment of P4 concentrations was 94% and 97%, respectively.(8) Cavestany and Foote reported that pregnancy rate was low in dairy cows with P4 concentrations 5 ng/mL.(10) 1 2
In China, there are no independent commercial kits for the measurement of milk P4 concentrations in dairy farms. In addition, the imported ELISA kits cannot be widely used in China because of their high cost. Here we successfully developed an enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody (MAb) to determine the P4 in the milk of pregnant cows. The result shows that the diagnosis rate of pregnancy and non-pregnancy was 93.3% and 95.8%, respectively. Therefore, the developed ELISA is a potential tool for P4 research and offers an alternative, simple, rapid technique for detecting P4 and reproductive disorders in dairy farms in China. Materials and Methods Reagents and animals
For the experiments, 96-well plates were purchased from Costar (Shanghai Bioleaf Biotech Co., Shanghai, China). An ELISA plate reader (ELX800) was supplied by Bio-Tek Instruments (Winooski, VT). Anti-progesterone monoclonal antibody (MAb) and progesterone complete antigen (11a-OHP4-HS-OVA, P4-OVA) were prepared in our laboratory.(11) Progesterone (P4) and anti-mouse-IgG-HRP were purchased from Sigma-Aldrich Co. (Shanghai, China). Bovine serum albumin (BSA) and polyoxyethylene-sorbiton monolaurate (Tween-20) were purchased from Beijing Biosynthesis Biotechnology (Beijing, China). Bovine ovalbumin (OVA) was
College of Animal Science and Veterinary Medicine, HeiLongjiang Bayi Agricultural University, Daqing, P.R. China. Synergetic Innovation Center of Food Safety and Nutrition, Northeast Agricultural University, Harbin, P.R. China.
330
OPTIMIZED ELISA KIT FOR MILK PROGESTERONE
Table 1. Optimized Parameters of ELISA Based on Enzyme-labeled Antigen
331
Table 2. Sensitivity of Developed ELISA Kits OD 450 nm values
Parameters Complete antigen dilution ratio Coated solution
1:2000 PBS (pH 7.4) 4C overnight 1% casein 4C overnight 1:16000
Closed solution Enzyme-labeled antibody dilution ratio Cross reaction (min) TMB work solution (mg/mL) H2O2 work solution (mg/mL) Standard curve Sensitivity (ng/mL) Detecting range (ng/mL)
1.21–1.271
Optimum results
50 100 30 Y = - 2.4598x + 0.999 (R2 = 0.996) 0.12 0.12*40
obtained from Beijing Dingguo Biotechnology. All other solvents and reagents were of analytic grade. Whole milk samples from dairy cows collected at 19–23 days post-mating in an intensive dairy farm (Misan, Heilongjiang, China) were used in this study. However, these milk samples were treated into milk without hormones before analysis.(11) All animal procedures were implemented according to the University’s Institutional Animal Care and Use Committee. Composition and protocol of ELISA kits
The developed competitive ELISA kits consisted of a 96well plate, 1 mL of P4 (40 ng/mL), 8 mL of milk without hormones, 5 mL of enzyme-labeled working solution, 150 mL of substrate A (100 · TMB concentrated solution), 10 mL of substrate B (H2O2), 5 mL of termination buffer (2 MH2SO4), and 200 mL of washing buffer (PBS buffer containing 0.05% Tween-20). The ELISA protocol consists of eight steps: (1) the 96-well plate was washed two times (3 min/wash) with washing buffer; (2) standard P4 solutions were prepared in milk without hormones as different concentrations (40, 20, 10, 5,
Mean (X0) SD X0 - 3SD Regression equation Sensitivity (ng/mL)
1.241 0.0212 1.197 y = - 0.8616x + 0.3827; R2 = 0.9953 0.14
2.5, 1.25, 0.625, and 0 ng/mL); (3) enzyme was mixed with the standard P4 solutions or unknown samples, added to the wells (100 mL/well), and incubated at 37C for 50 min in a wet box; (4) liquid in excess was discarded and the wells were washed three times (3 min/wash) with washing buffer; (5) substrate A diluted in substrate B was added to the 96-well plate (100 mL/well), which was stored in the dark for 15 min; (6) termination buffer was added to each well (50 mL/well); (7) OD450nm was measured in a microplate reader; and (8) the curvilinear relationship between OD values and P4 concentrations was described by Logit (%B/B0) = Ln [(B/B0)/(1 B/B0)]. Optimization of ELISA kits
Several experimental parameters were examined in order to determine the assay specificity, sensitivity, repeatability, and accuracy.(11) Concentrations of P4 were determined by checkerboard titration. Other experimental parameters such as the blocking agent and reaction temperature and period were optimized sequentially using the above-described ELISA protocol. Criteria used to evaluate the assay performances were absorbance of the same P4 content. Application to milk samples
The milk samples from five estrus cows and five pregnant cows and 54 cows at 19–23 days after artificial insemination were diluted 5-fold in dilution buffer (1 · PBS), and applied to the detecting antibody coated plate (100 mL/well). The following steps were the same as in the above-described ELISA.
FIG. 1. ELISA inhibition curves for progesterone, estradiol, testosterone, and estriol. B0, OD value at 0 ng/mL progesterone; B, OD value at different progesterone concentrations.
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WU ET AL.
FIG. 2. Standard curve of the developed ELISA kit for milk samples. B0, OD value at 0 ng/mL progesterone; B, OD value at different progesterone concentrations; P, progesterone; R2, correlation index. Results and Discussion
standard curve identified that the combination between antibody and antigen was satisfactory (Fig. 2).
Optimization of ELISA kits for detecting P4
It is well known that optimization of the ELISA kit includes specificity, sensitivity, repeatability, and accuracy.(12,13) In this study, a direct competition ELISA method, named enzyme-labeled antigen method, was developed by a self-made progesterone complete antigen (P4-OVA), enzyme-labeled antigen (P4-HRP), and enzyme-labeled antibody (MAb-HRP) to detect cow milk progesterone, and its operative conditions were optimized (Table 1). The appropriate dilution of complete antigen and enzymelabeled antibody (MAb-HRP) are key factors determining the sensitivity and working range of the completive ELISA.(14) In order to determine the optimum reaction condition of the completive ELISA kit as a detecting antibody (MAbHRP), complete antigen were mixed together at different concentrations.(15) In Table 1, the rational number of the OD450 nm absorbance was obtained when the complete antigen and MAb-HRP were diluted with 1:2000 and 1:16000, respectively. The optimization method for other factors is also shown in Table 1. The linear dynamic range was between 0.12 and 40 ng/mL. The detection limitation of P4 was 0.12 ng/mL. Stability tests for the developed ELISA kit were performed at 37C or 4C, respectively. The results revealed that the ELISA kit could be stored at 4C for >6 months. Additionally, the ELISA kit had no cross-reactivity with estradiol, estriol, or testosterone (Fig. 1). The sensitivity of the kit was 0.14 ng/mL (Table 2), the intra-assay variation was 4.11%, the inter-assay variation was 6.26%, the recovery rate was 93.80–107.60%, and the co-efficient of variation was 5.65%. Further study by serially diluted P4 in milk to make a
Application of ELISA kit to milk samples
For detecting milk progesterone during pregnancy and estrous cycle, progesterone concentrations in five milk samples from pregnant or estrus cows were determined using the developed optimum ELISA kit in Table 1. In Table 3, the results revealed that milk progesterone concentrations were
Development and Application of an ELISA Kit for the Detection of Milk Progesterone in Dairy Cows Ling Wu,1 Chuchu Xu,1 Cheng Xia,1,2 Yu Duan,1 Chuang Xu,1 Hongyou Zhang,1 and Jun Bao 2
Progesterone (P4) is a steroid gonadal hormone that is mainly produced from the corpus luteum and placenta and has various biological functions, especially reproductive regulation. It is important to establish a specific and sensitive P4 enzyme-linked immunosorbent assay (ELISA) for the study of ovary activity and reproductive disorders. Therefore, we prepared a monoclonal antibody (MAb) in a completed antigen (11a-OH-P4-HSOVA). Based on the MAb and our previously prepared completed antigen, a highly specific and sensitive ELISA was developed. In the present study, a competitive ELISA for the determination of P4 was described in dairy cow milk. It was found that P4 concentration in milk samples from five pregnant cows was significantly higher than that from five estrus cows. The diagnosis rate for pregnancy and non-pregnancy in 54 dairy cows were 93.3% and 95.8%, respectively, at 19 to 23 days after pregnancy by detecting milk P4 concentration. In summary, the developed ELISA is a potential tool for P4 research and offers an alternative, simple, rapid technique for detecting P4, especially in future large clinical investigations on pregnancy identification and reproductive disorders in dairy farms in China.
Introduction
P
rogesterone (P4) is a gonadal hormone that is mainly produced from the corpus luteum and placenta in humans, pigs, and cows. It has various versatile functions and is recognized as a multifunctional steroid hormone not only in the estrus and pregnancy but also on reproductive disorders, including persistent corpus luteum and corpus luteum cyst.(1,2) It has been reported that the methods of estrous cycle detection and pregnancy diagnosis in dairy cows include rectal palpation,(3) ultrasound inspection,(4) ELISA kits,(5) and immunosensors.(6) In dairy farms, pregnancy, ovarian activity, and breeding status are usually assessed by monitoring blood P4 concentrations. However, the detection of milk P4 concentrations in dairy cows has been widely used as a routine clinical examination in developed countries because it avoids stress and any undesirable effects caused by blood collection, rectal palpation and ultrasound inspection.(3,4) Studies have reported that P4 concentrations at 21 days post-mating were closely related to pregnancy status.(5,7) Additionally, the positive rate of non-pregnancy and pregnancy diagnoses by the assessment of P4 concentrations was 94% and 97%, respectively.(8) Cavestany and Foote reported that pregnancy rate was low in dairy cows with P4 concentrations 5 ng/mL.(10) 1 2
In China, there are no independent commercial kits for the measurement of milk P4 concentrations in dairy farms. In addition, the imported ELISA kits cannot be widely used in China because of their high cost. Here we successfully developed an enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody (MAb) to determine the P4 in the milk of pregnant cows. The result shows that the diagnosis rate of pregnancy and non-pregnancy was 93.3% and 95.8%, respectively. Therefore, the developed ELISA is a potential tool for P4 research and offers an alternative, simple, rapid technique for detecting P4 and reproductive disorders in dairy farms in China. Materials and Methods Reagents and animals
For the experiments, 96-well plates were purchased from Costar (Shanghai Bioleaf Biotech Co., Shanghai, China). An ELISA plate reader (ELX800) was supplied by Bio-Tek Instruments (Winooski, VT). Anti-progesterone monoclonal antibody (MAb) and progesterone complete antigen (11a-OHP4-HS-OVA, P4-OVA) were prepared in our laboratory.(11) Progesterone (P4) and anti-mouse-IgG-HRP were purchased from Sigma-Aldrich Co. (Shanghai, China). Bovine serum albumin (BSA) and polyoxyethylene-sorbiton monolaurate (Tween-20) were purchased from Beijing Biosynthesis Biotechnology (Beijing, China). Bovine ovalbumin (OVA) was
College of Animal Science and Veterinary Medicine, HeiLongjiang Bayi Agricultural University, Daqing, P.R. China. Synergetic Innovation Center of Food Safety and Nutrition, Northeast Agricultural University, Harbin, P.R. China.
330
OPTIMIZED ELISA KIT FOR MILK PROGESTERONE
Table 1. Optimized Parameters of ELISA Based on Enzyme-labeled Antigen
331
Table 2. Sensitivity of Developed ELISA Kits OD 450 nm values
Parameters Complete antigen dilution ratio Coated solution
1:2000 PBS (pH 7.4) 4C overnight 1% casein 4C overnight 1:16000
Closed solution Enzyme-labeled antibody dilution ratio Cross reaction (min) TMB work solution (mg/mL) H2O2 work solution (mg/mL) Standard curve Sensitivity (ng/mL) Detecting range (ng/mL)
1.21–1.271
Optimum results
50 100 30 Y = - 2.4598x + 0.999 (R2 = 0.996) 0.12 0.12*40
obtained from Beijing Dingguo Biotechnology. All other solvents and reagents were of analytic grade. Whole milk samples from dairy cows collected at 19–23 days post-mating in an intensive dairy farm (Misan, Heilongjiang, China) were used in this study. However, these milk samples were treated into milk without hormones before analysis.(11) All animal procedures were implemented according to the University’s Institutional Animal Care and Use Committee. Composition and protocol of ELISA kits
The developed competitive ELISA kits consisted of a 96well plate, 1 mL of P4 (40 ng/mL), 8 mL of milk without hormones, 5 mL of enzyme-labeled working solution, 150 mL of substrate A (100 · TMB concentrated solution), 10 mL of substrate B (H2O2), 5 mL of termination buffer (2 MH2SO4), and 200 mL of washing buffer (PBS buffer containing 0.05% Tween-20). The ELISA protocol consists of eight steps: (1) the 96-well plate was washed two times (3 min/wash) with washing buffer; (2) standard P4 solutions were prepared in milk without hormones as different concentrations (40, 20, 10, 5,
Mean (X0) SD X0 - 3SD Regression equation Sensitivity (ng/mL)
1.241 0.0212 1.197 y = - 0.8616x + 0.3827; R2 = 0.9953 0.14
2.5, 1.25, 0.625, and 0 ng/mL); (3) enzyme was mixed with the standard P4 solutions or unknown samples, added to the wells (100 mL/well), and incubated at 37C for 50 min in a wet box; (4) liquid in excess was discarded and the wells were washed three times (3 min/wash) with washing buffer; (5) substrate A diluted in substrate B was added to the 96-well plate (100 mL/well), which was stored in the dark for 15 min; (6) termination buffer was added to each well (50 mL/well); (7) OD450nm was measured in a microplate reader; and (8) the curvilinear relationship between OD values and P4 concentrations was described by Logit (%B/B0) = Ln [(B/B0)/(1 B/B0)]. Optimization of ELISA kits
Several experimental parameters were examined in order to determine the assay specificity, sensitivity, repeatability, and accuracy.(11) Concentrations of P4 were determined by checkerboard titration. Other experimental parameters such as the blocking agent and reaction temperature and period were optimized sequentially using the above-described ELISA protocol. Criteria used to evaluate the assay performances were absorbance of the same P4 content. Application to milk samples
The milk samples from five estrus cows and five pregnant cows and 54 cows at 19–23 days after artificial insemination were diluted 5-fold in dilution buffer (1 · PBS), and applied to the detecting antibody coated plate (100 mL/well). The following steps were the same as in the above-described ELISA.
FIG. 1. ELISA inhibition curves for progesterone, estradiol, testosterone, and estriol. B0, OD value at 0 ng/mL progesterone; B, OD value at different progesterone concentrations.
332
WU ET AL.
FIG. 2. Standard curve of the developed ELISA kit for milk samples. B0, OD value at 0 ng/mL progesterone; B, OD value at different progesterone concentrations; P, progesterone; R2, correlation index. Results and Discussion
standard curve identified that the combination between antibody and antigen was satisfactory (Fig. 2).
Optimization of ELISA kits for detecting P4
It is well known that optimization of the ELISA kit includes specificity, sensitivity, repeatability, and accuracy.(12,13) In this study, a direct competition ELISA method, named enzyme-labeled antigen method, was developed by a self-made progesterone complete antigen (P4-OVA), enzyme-labeled antigen (P4-HRP), and enzyme-labeled antibody (MAb-HRP) to detect cow milk progesterone, and its operative conditions were optimized (Table 1). The appropriate dilution of complete antigen and enzymelabeled antibody (MAb-HRP) are key factors determining the sensitivity and working range of the completive ELISA.(14) In order to determine the optimum reaction condition of the completive ELISA kit as a detecting antibody (MAbHRP), complete antigen were mixed together at different concentrations.(15) In Table 1, the rational number of the OD450 nm absorbance was obtained when the complete antigen and MAb-HRP were diluted with 1:2000 and 1:16000, respectively. The optimization method for other factors is also shown in Table 1. The linear dynamic range was between 0.12 and 40 ng/mL. The detection limitation of P4 was 0.12 ng/mL. Stability tests for the developed ELISA kit were performed at 37C or 4C, respectively. The results revealed that the ELISA kit could be stored at 4C for >6 months. Additionally, the ELISA kit had no cross-reactivity with estradiol, estriol, or testosterone (Fig. 1). The sensitivity of the kit was 0.14 ng/mL (Table 2), the intra-assay variation was 4.11%, the inter-assay variation was 6.26%, the recovery rate was 93.80–107.60%, and the co-efficient of variation was 5.65%. Further study by serially diluted P4 in milk to make a
Application of ELISA kit to milk samples
For detecting milk progesterone during pregnancy and estrous cycle, progesterone concentrations in five milk samples from pregnant or estrus cows were determined using the developed optimum ELISA kit in Table 1. In Table 3, the results revealed that milk progesterone concentrations were
