DISCUSSION
Since the first case of African swine fever occurred in August 2018, the Chinese pig industry has been devastated. In the absence of commercial vaccines and therapeutic drugs, early, effective, rapid, and convenient detection is essential for the prevention and control of ASF. Therefore, it is urgent to establish a rapid and efficient ASF antibody detection method.
At present, the commonly used methods of serological detection of antibodies include ELISA, indirect immunofluorescence test (IFA), western blotting (WB), and colloidal gold immunochromatographic detection technology. Compared with other methods, colloidal gold immunochromatographic detection technology is simple, portable, and has a rapid response; results can be observed with the naked eye within 5–10 min. Therefore, it is suitable for rapid high-throughput detection at the grassroots level or on-site (Lei et al., 2020; Sun et al., 2020; Yang et al., 2020a). Colloidal gold immunochromatographic detection technology has diagnostic significance for serological diagnosis. The ASFV structural proteins primarily include p30, p72, and p54 (Costard et al., 2009; Neilan et al., 2004). The p30 protein is generally expressed and secreted in the early stages of viral infection and has good antigenicity; thus, it can be used to detect infection early. The p72 protein is mainly expressed in the late stages of viral infection; it is highly conserved, and its secretion and expression show good antigenicity (Bao et al., 2019; Fernández-Pinero et al., 2013; Olasz et al., 2019). Considering the advantages of colloidal gold immunochromatographic test strips and the combined characteristics of p30 and p72 as diagnostic antigens, we established an ASFV antibody dual colloidal gold immunochromatographic detection method that can quickly and conveniently monitor the occurrence and development of ASF infection in real time.
The stability of a test strip plays a vital role in field applications. Accordingly, the quality of the gold solution and the purity and concentration of the raw materials (protein or antibody) all affected and restricted the stability of the test strip.
The quality of the gold solution is one of the decisive factors determining whether colloidal gold immunochromatographic detection technology can be successfully established. If the diameter variation range of the gold particles is too large, the stability of the test strip will be affected. At the same time, if the size of the gold particles is not uniform, the gold-labeled probes will easily precipitate, resulting in false positives. Therefore, preparing a colloidal gold solution with uniform particles and good dispersion is especially important for subsequent experiments. According to Wang et al. (2004), if the maximum absorption peak and its corresponding OD450nm value are between 0.8 and 1.2, it can be preliminarily considered that the gold solution meets the conditions. Therefore, to obtain a good-quality gold solution, we systematically explored the amount of reducing agent required and obtained a clear and bright wine-red gold solution. The maximum absorption peak λmax (520 nm) of the gold solution and its corresponding OD450nm value (0.925) were obtained using an ultraviolet scanner. These values indicated that the conditions were met. Many tests have proven that the purity and concentration of the raw materials directly affect the quality of the gold-labeled probe. The recombinant p30 protein prepared in this study had a His-tag and contained more imidazole after purification through a nickel column. At the same time, p30 protein is an inclusion body protein, and a high concentration of urea is used when it is dissolved. If the colloidal gold label contains imidazole and urea, a large amount of colloidal gold can accumulate, precipitate, or even cease activity. Therefore, the raw materials must be fully dialyzed to remove urea and imidazole before labeling the gold particles. The main component of the dialysate was 20 mM Tris-HCl. A small amount of salt can also be added to ensure a balance within the entire system.
To evaluate the practicability of the test strips prepared in this study, we tested 84 serum samples simultaneously using two commercial ELISA kits. The results indicated that the test strips and the two commercial ELISA kits had coincidence rates higher than 98%. Test strip specificity was evaluated by testing for cross-reactivity with pig-derived CSFV, PRRSV, FMDV-A, FMDV-O, and PCV-2 positive sera, which are clinically similar to ASF and easily confused for it. The results showed that the test strip had good specificity. The test strip sensitivity was assessed using different test dilutions of attenuated Δ181/UK strain immune serum, and the results revealed a high sensitivity of the test strips (p30 ELISA titer: 1:512, p72 ELISA titer: 1:256). Notably, the test strips were able to detect the growth and decline of antibodies from days 0 to 29 after challenge with an attenuated strain of ASFVΔ181/UK, enabling us to determine the period during which the antibodies corresponding to p30 and p72 appeared. The earliest appearance of p30 occurred on the 7th day, and the latest was on the 11th day. The earliest appearance time of p72 was generally the 9th day, and the latest was on the 15th day. Therefore, the present study demonstrated the dynamic monitoring of ASF by observing the growth and decline of the two antibodies.
Currently, the development of African swine fever primarily shows three trends. 1) Infection of a pig herd with a virulent ASFV strain will cause high mortality over a short period, with a fatality rate as high as 100%. Since disease duration is short and mortality is close to 100%, antibody testing is not applicable. 2) By monitoring the prevalence of ASF in seven Chinese provinces from June to December 2020, it was shown that there were natural mutations or deletions in the ASFV strains, resulting in a non-blood cell adsorption phenotype. Following virulence tests in pigs, it was observed that a toxic dose of 106 TCID50 could cause fatal acute or subacute disease in pigs. The remaining non-fatally diseased pigs will naturally recover after tolerance but become carriers for a long time, and the virus is easily transmitted since a 103TCID50 dose could cause non-lethal chronic or persistent infections. Compared with the former, the latter trend has a higher survival rate, but more pigs are infected, and transmission is greater. Therefore, it is easy to cause a second ASF pandemic, which will bring a new level of destruction to the world’s breeding industry. Since surviving animals can maintain high antibody levels for a long time, the detection of ASFV-specific antibodies is of great significance for determining the infection status of the host. Therefore, the test strips prepared in this study could be effectively applied to detect the presence of mutant strains. Dynamic monitoring of antibodies in pigs provides a powerful technical means for the prevention and control of ASF, ensuring that economic losses are reduced by minimizing the risk of transmission. 3) Through the continuous efforts of researchers, an effective ASF vaccine should be successfully developed in the near future, and antibodies that can inhibit or kill ASFV can be obtained in pig populations after comprehensive immunization. The test strips prepared in this study can be effectively used to evaluate the immune effects of vaccines. In addition, this study uses a test strip to detect two antibodies simultaneously, which not only achieves the effect of diversified detection but also lowers costs.