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. 2024 Mar 11;21(1):64.
doi: 10.1186/s12985-024-02302-4.

Development of a time-resolved fluorescence immunoassay kit for detecting canine coronavirus and parvovirus through double labeling

Laiqing Li  1 , Cuicui Chen  2 , Huankun Liang  2 , Wenqi Dong  3 , V N Leontiev  1 , Igor Vitalievich Voytov  4
Affiliations

Development of a time-resolved fluorescence immunoassay kit for detecting canine coronavirus and parvovirus through double labeling

Laiqing Li et al. Virol J. .

Abstract

Objective: Canine enteric coronavirus (CCV) and canine parvovirus type 2 (CPV-2) are the main pathogens responsible for acute gastroenteritis in dogs, and both single and mixed infections are common. This study aimed to establish a double-labeling time-resolved fluorescence immunoassay (TRFIA) to test and distinguish CCV and CPV-2 diseases.

Methods: A sandwich double-labeling TRFIA method was established and optimized using europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. CCV/CPV-2 antigens were first captured by the immobilized antibodies. Then, combined with Eu3+/Sm3+-labeled paired antibodies, the Eu3+/Sm3+ fluorescence values were detected after dissociation to calculate the CCV/CPV-2 ratios. The performance, clinical performance and methodology used for laboratory (sensitivity, specificity, accuracy and stability) testing were evaluated.

Results: A double-label TRFIA for CCV and CPV-2 detection was optimized and established. The sensitivity of this TRFIA kit was 0.51 ng/mL for CCV and 0.80 ng/mL for CPV-2, with high specificity for CCV and CPV-2. All the accuracy data were less than 10%, and the recovery ranged from 101.21 to 110.28%. The kits can be temporarily stored for 20 days at 4 °C and can be stored for 12 months at temperatures less than - 20 °C. Based on a methodology comparison of 137 clinically suspected patients, there was no statistically significant difference between the TRFIA kit and the PCR method. Additionally, for CCV detection, the clinical sensitivity was 95.74%, and the clinical specificity was 93.33%. For CPV-2 detection, the clinical sensitivity was 92.86%, and the clinical specificity was 96.97%.

Conclusion: In this study, a double-label TRFIA kit was prepared for CCV and CPV-2 detection with high laboratory sensitivity, specificity, accuracy, stability, clinical sensitivity and specificity. This kit provides a new option for screening/distinguishing between CCV and CPV-2 and may help improve strategies to prevent and control animal infectious diseases in the future.

Keywords: Canine; Coronavirus; Double labeling; Kit; Parvovirus; Time-resolved fluorescence immunoassay.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Schematic diagram of the TRFIA detection principle and steps
Fig. 2
Fig. 2
Optimization of the immunoreaction time
Fig. 3
Fig. 3
Standard curves for CCV and CPV-2 from this TRFIA
Fig. 4
Fig. 4
Stability of the kits (4 and 37 °C)
See this image and copyright information in PMC

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