Development and Application of an ELISA Kit for the Detection of Milk Progesterone in Dairy Cows

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 (11α-OH-P4-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 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 96-well plate, 1 mL of P4 (40 ng/mL), 8 mL of milk without hormones, 5 mL of enzyme-labeled working solution, 150 μL 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, 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 μL/well), and incubated at 37°C 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 μL/well), which was stored in the dark for 15 min; (6) termination buffer was added to each well (50 μL/well); (7) OD_450nm 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 μL/well). The following steps were the same as in the above-described ELISA.

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 enzyme-labeled 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 (MAb-HRP), 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 37°C or 4°C, respectively. The results revealed that the ELISA kit could be stored at 4°C 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 standard curve identified that the combination between antibody and antigen was satisfactory (Fig. 2).

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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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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.

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 <0.5 ng/mL in the estrous cows. During pregnancy, milk samples contained an average progesterone concentration of 4.48 ng/mL.

For measuring milk progesterone from pregnant and non-pregnant cows, 54 fresh milk samples were collected from cows at 19–23 days post-mating to assess the progesterone concentrations using the developed ELISA kits in Table 4. In this experiment, 1.25 ng/mL and 0.625 ng/mL were used to distinguish pregnancy from non-pregnancy, respectively. Thirty pregnant and 24 non-pregnant cows were monitored by rectal palpation at 60 days post-mating. The accuracy of the non-pregnancy and pregnancy diagnoses was 95.8% (23/24) and 93.3% (28/30), respectively. Results showed that the ELISA for milk P4 produced good accuracy for pregnancy or non-pregnancy.