Scientia Agricultura Sinica ›› 2026, Vol. 59 ›› Issue (12): 2750-2762.doi: 10.3864/j.issn.0578-1752.2026.12.016

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Development of Double-Antibody Sandwich ELISA for the Detection of Clostridium perfringens ε Toxin (ETX)

HAN FengYe1,2(), LIU Ying2, ZHU KaiYing2, YIN ChunSheng2, ZHANG QianYi2, WEN YongJun1, WANG FengXue1(), DU JiGe2()   

  1. 1 College of Veterinary Medicine of Inner Mongolia Agricultural University/Key Laboratory of Animal Diseases Clinical Diagnosis and Treatment Technology of Ministry of Agriculture and Rural Affairs, Hohhot 010000
    2 China Institute of Veterinary Drug Control, Beijing 100081
  • Received:2025-10-16 Accepted:2026-04-21 Online:2026-06-16 Published:2026-06-16
  • Contact: WANG FengXue, DU JiGe

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Abstract:

【Background】Clostridium perfringens Epsilon toxin (ETX), recognized as the world's third most potent known biological toxin, causes severe economic losses in the livestock industry due to ETX poisoning. Therefore, there is an urgent need to establish a diagnostic method for ETX that is highly specific, sensitive and easy to operate, and suitable for large-scale sample screening. 【Objective】This study aimed to produce mouse anti-ETX monoclonal antibodies (mAbs) and establish a double-antibody sandwich ELISA (DAS-ELISA) for ETX detection, thereby providing a material foundation for early ETX diagnosis, epidemic surveillance, and the formulation of prevention and control strategies.【Method】BALB/c mice were immunized with inactivated crude ETX (inactivated ETX, iETX) and non-toxic recombinant ETX (rETXm1) as immunogens. An indirect ELISA method and an indirect immunofluorescence assay, established using rETXm1 as the coating antigen, were employed for mAb screening. The obtained mAbs were tested for ETX neutralizing activity, and mouse monoclonal ascites were prepared. Purification of mouse ascites was performed using Protein A affinity chromatography, followed by identification via SDS-PAGE and Western-blot analysis. A DAS-ELISA for ETX detection was established using rabbit polyclonal anti-Clostridium perfringens ε toxin antibody (capture antibody) and neutralizing monoclonal antibody (detection antibody). Optimal reaction conditions were determined by optimizing capture and detection antibody concentrations via a checkerboard approach. The method's cutoff value, specificity, sensitivity, and reproducibility were validated prior to clinical application.【Result】A total of three hybridoma cell lines capable of stable passage and producing ETX-specific mAbs were selected and designated ETX-CH, ETX-ZH, and ETX-NH, respectively. Toxin neutralization assays revealed that ETX-ZH exhibited the highest neutralizing titer at the cellular level, while ETX-NH showed no neutralizing activity. In vivo toxin neutralization experiments in mice further demonstrated that ETX-ZH retained neutralizing activity against ETX in vivo. The purified ascites contained 2.3 mg·mL-1 of ETX-ZH protein. Using the checkerboard method, the optimal dilution for capture antibody was determined to be 1﹕100, and for detection antibody, 1﹕1 600. The optimized conditions for this method were: capture antibody coating at 4 ℃ for 12 h, blocking with 5% skim milk at 37 ℃ for 2 h, antigen incubation at 37 ℃ for 90 min, detection antibody incubation at 37 ℃ for 30 min, incubation with enzyme-labeled secondary IgG antibody at 37 ℃ for 1 h, and TMB color development at 37 ℃ for 15 min. The positive cutoff value was 0.161, and the negative cutoff value was 0.143. The assay demonstrated high specificity with no cross-reactivity to other Clostridium perfringens toxins or purified proteins. It exhibited good reproducibility, with an intra-assay coefficient of variation ≤4.69% and an inter-assay coefficient of variation ≤5.32%. The lower limit of detection for rETXm1 was 31.25 ng·mL-1, and for ETX it was 0.5 MLD. Testing of eighteen multi-valent dry powder vaccines containing ETX-like toxins yielded a 100% detection rate for ETX.【Conclusion】The DAS-ELISA method developed using the prepared ETX mAb exhibited high specificity and good reproducibility, making it suitable for ETX detection.

Key words: ETX, monoclonal antibody, polyclonal antibody, neutralising activity, double antibody sandwich ELISA

Fig. 1

Indirect immunofluorescence of monoclonal antibodies A: ETX-CH; B: ETX-ZH; C: ETX-NHThe red arrow indicates the presence of distinct green fluorescent particle signals within the cell cytoplasm"

Fig. 2

Monoclonal antibody subclass identification"

Table 1

MDCK cell neutralization assay"

单抗名称
Monoclonal antibody name		 与不同稀释倍数的毒素结合中和MDCK细胞
Binding and neutralization of MDCK cells with toxins at different dilution ratios
100 200 400 600 800 1600
ETX-CH + + + - - -
ETX-ZH + - - - - -
ETX-NH + + + + + +

Fig. 3

Cell neutralization assay lysate"

Fig. 4

Neutralization assay of hybridoma cells in animals"

Fig. 5

SDS-PAGE of purified monoclonal antibody M: Protein molecular weight standard; 1: ETX-ZH; 2: ETX-NH"

Fig. 6

Western-blot identification of mAbs M: Protein molecular weight standard; 1, 3: Ending enzyme-treated blank control; 2: ETX-ZH; 4: ETX-NH"

Table 2

Results of antibody dilution tests at different dilutions"

多克隆抗体稀释度
Polyclonal antibody dilution		 单克隆抗体稀释度 Monoclonal antibody dilution
1:100 1:200 1:400 1:800 1:1600 1:3200
1:50 1.464 1.131 1.109 1.077 1.007 0.998
0.445 0.178 0.180 0.130 0.111 0.137
P/N 3.290 6.350 6.160 8.280 9.070 7.280
1:100 1.382 1.079 1.010 1.057 1.012 0.954
0.376 0.192 0.153 0.125 0.104 0.125
P/N 3.680 5.620 6.600 8.470 9.730 7.630
1:200 1.324 1.051 1.083 0.934 0.915 0.918
0.358 0.241 0.182 0.125 0.118 0.141
P/N 3.700 4.360 5.950 7.470 7.750 6.510
1:400 1.363 1.031 0.934 0.858 0.798 0.837
0.391 0.257 0.192 0.143 0.134 0.161
P/N 3.490 4.010 4.860 6.000 5.960 5.200
1:800 1.179 0.857 0.689 0.688 0.701 0.722
0.264 0.196 0.176 0.106 0.122 0.133
P/N 4.470 4.370 3.910 6.490 5.750 5.430
1:1600 1.097 0.756 0.639 0.634 0.613 0.675
0.246 0.152 0.128 0.109 0.118 0.145
P/N 4.460 4.970 4.990 5.820 5.200 4.660
1:3200 1.105 0.785 0.680 0.608 0.620 0.667
0.214 0.151 0.100 0.090 0.114 0.162
P/N 5.160 5.200 6.800 6.760 5.440 4.120

Fig. 7

Optimal reaction condition"

Table 3

The specificity test of the DAS-ELISA"

待检样品Sample to be tested OD450nm值OD450nmvalue 结果Result
D型产气荚膜梭菌天然毒素Clostridium perfringens crude toxin Type D 1.13 +
诺维梭菌C64-1株天然毒素Clostridium novyi C64-1 strain crude toxin 0.11 -
破伤风梭菌C66-1株天然毒素Clostridium tetani C66-1 strain crude toxin 0.11 -
肉毒梭菌C型C62-4株天然毒素Clostridium botulinum C62-4 strain crude toxin 0.11 -
腐败梭菌C55-1株天然毒素Clostridium sordellii C55-1 strain crude toxin 0.13 -
破伤风梭菌蛋白Clostridium tetani protein 0.14 -
腐败梭菌蛋白Clostridium sordellii protein 0.12 -
小反刍兽疫蛋白Peste des petits ruminants (PPR) protein 0.13 -
羊口疮蛋白Orf protein 0.11 -
山羊痘蛋白Goatpox protein 0.11 -
狂犬病毒蛋白 Rabies protein 0.10 -
PBS 0.09 -

Fig. 8

Sensitivity determination of the DAS-ELISA method The red arrow indicates the minimum detectable levels of recombinant protein (A) and native toxin (B) when P/N ≥2.1"

Table 4

Repeatability assay for the DAS-ELISA(n=3)"

样品
Sample		 批内变异系数Intra-batch CV 批间变异系数Inter-batch CV
平均数
$\overline{x}$
±SD		 变异系数 CV(%) 平均数
$\overline{x}$
±SD		 变异系数CV (%)
1 1.406+0.0491 3.49 1.435+0.061 4.25
2 1.492+0.070 4.69 1.505+0.08 5.32
3 1.519+0.047 3.09 1.530+0.059 3.86
4 0.874+0.028 3.20 0.859+0.023 2.68
5 0.617+0.009 1.46 0.601+0.014 2.33

Table 5

Testing of vaccine samples"

待检样品
Sample to be tested		 OD450nm值
OD450nmvalue		 结果
Result		 待检样品
Sample to be tested		 OD450nm值
OD450nmvalue		 结果
Result
1 1.01 + 10 0.83 +
2 1.12 + 11 0.89 +
3 0.99 + 12 0.90 +
4 0.98 + 13 1.00 +
5 1.05 + 14 0.96 +
6 1.20 + 15 0.96 +
7 1.19 + 16 1.05 +
8 1.07 + 17 1.05 +
9 0.84 + 18 1.08 +
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