African swine fever (ASF) is a highly detrimental viral disease caused by African swine fever virus (ASFV). The occurrence and prevalence of this disease have become a serious threat to the global swine industry and national economies. At present, the detection volume of African swine fever is huge, more sensitive and accurate detection techniques are needed for the market. pp62 protein, as a protein in the late stage of infection, has strong antigenicity and a high corresponding antibody titer in infected pigs. In this study, the CP530R gene was cloned into expression vector pET-28a to construct a prokaryotic expression plasmid, which was induced by IPTG to express soluble pp62 protein.
Western blot analysis showed that it had great reactivity. Using the purified recombinant protein as an antigen, an indirect ELISA method for detecting ASFV antibody was established. The method was specific only to ASFV-positive serum, 1:1600 diluted positive serum could still be detected, and the coefficients of variation (CV) of the intra assay and inter assay were both <10%. It turns out that the assays had excellent specificity, sensitivity, and repeatability. This provides an accurate, rapid, and economical method for the detection of ASFV antibody in clinical pig serum samples.
Preparation and epitope mapping of monoclonal antibodies against African swine fever virus P30 protein
- African swine fever virus (ASFV) causes acute, febrile, and highly contagious diseases in swine. Early diagnosis is critically important for African swine fever (ASF) prevention and control in the absence of an effective vaccine. P30 is one of the most immunogenic proteins that are produced during the early stage of an ASFV infection. This makes P30 a good serological target for ASF detection and surveillance. In this study, two P30-reactive monoclonal antibodies (mAbs), 2H2 and 5E8, were generated from mice immunized with recombinant P30 protein (rP30). Epitope mapping was performed with overlapping polypeptides, alanine mutants, and synthetic peptides.
- The mapping results revealed that 2H2 recognized a region located in the N-terminal, 16-48 aa. In contrast, 5E8 recognized a linear epitope in the C-terminal, 122-128 aa. Further analysis indicated that the epitope recognized by 2H2 was highly conserved in genotypes I and II, while the 5E8 epitope was conserved in most genotypes and the Ser to Pro change at position 128 in genotypes IV, V, and VI did not affect recognition.
- Overall, the results of this study provide valuable information on the antigenic regions of ASFV P30 and lay the foundation for the serological diagnosis of ASF and vaccine research. KEY POINTS: • Two specific and reactive mAbs were prepared and their epitopes were identified. • 2H2 recognized a novel epitope highly conserved in genotypes I and II. • 5E8 recognized a seven-amino acid linear epitope highly conserved in most genotypes.
Lateral Flow Assay for the Detection of African Swine Fever Virus Antibodies Using Gold Nanoparticle-Labeled Acid-Treated p72
African swine fever is a widespread and highly contagious disease in the porcine population, which is caused by African swine fever virus (ASFV). The PCR and ELISA detection methods are the main conventional diagnostic methods for ASFV antigen/antibody detection in the field. However, these methods have limitations of expensive equipment, trained technicians, and time-consuming results. Thus, a rapid, inexpensive, accurate and on-site detection method is urgently needed. Here we describe a double-antigen-sandwich lateral-flow assay based on gold nanoparticle-conjugated ASFV major capsid protein p72, which can detect ASFV antibody in serum samples with high sensitivity and specificity in 10 min and the results can be determined by naked eyes.
A lateral flow assay was established by using yeast-expressed and acid-treated ASFV p72 conjugated with gold nanoparticles, which are synthesized by seeding method. A high coincidence (97.8%) of the assay was determined using clinical serum compared to a commercial ELISA kit. In addition, our lateral flow strip can detect as far as 1:10,000 diluted clinically positive serum for demonstration of high sensitivity. In summary, the assay developed here was shown to be rapid, inexpensive, accurate and highly selective. It represents a reliable method for on-site ASFV antibody detection and may help to control the ASFV pandemic.
Rapid Analysis of Bacterial Contamination in Platelets without Pre-Enrichment Using Pig Serum-Derived Antibodies
As the shelf life of platelets collected from donated blood is very short, approximately 5 days, the determination of bacterial contamination in platelets has become necessary. In this study, rapid analysis of Gram-positive and Gram-negative bacterial contamination in platelet samples was presented without pre-enrichment using pig serum-derived antibodies against the outer membrane proteins (OMP) of Gram-negative bacteria and antibodies against lipoteichoic acid (LTA) on the surface of Gram-positive bacteria.
The anti-OMP antibodies against Gram-negative bacteria were isolated using sequential incubation with (1) the modified Gram-negative bacteria ClearColi, which lacks lipopolysaccharide (LPS) on the outer membrane, and (2) the Gram-positive bacteriaBacillus subtilis to filter away nonspecifically bound proteins from ClearColi. The anti-lipoteichoic acid (LTA) antibodies against Gram-positive bacteria were isolated using sequential incubation with (1) the Gram-positive bacteriaB. subtilis and (2) the Gram-negative bacteria Escherichia coli BL21 to filter away nonspecifically bound proteins fromB. subtilis.
The feasibility of using the antibodies isolated from pig serum against Gram-negative and Gram-positive bacteria was demonstrated using flow cytometry. Finally, detection of the contamination of platelets with Gram-negative and Gram-positive bacteria using the impedance immunosensor based on these isolated antibodies was successfully demonstrated.
Guinea Pig Anti Pig Insulin Polyclonal Antibody |
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Pig RBC antibody |
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PIG-Y Antibody |
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PIG-Y Antibody |
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PIG-Y Antibody |
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PIG-Y Antibody |
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PIG-Y Antibody |
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PIG-Y Antibody |
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| 4955-01mg | ProSci | 0.1 mg | 523.7 EUR |
Pig CD5 Antibody |
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| GWB-Q01221 | GenWay Biotech | 0.25 mg | Ask for price |
Pig IgM Antibody |
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Pig IgA Antibody |
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| GWB-B0BCC6 | GenWay Biotech | 1 mg | Ask for price |
Pig IgM Antibody |
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| GWB-C0C73E | GenWay Biotech | 1 mg | Ask for price |
PIG-Y Antibody |
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| MBS9414487-01mL | MyBiosource | 0.1mL | 495 EUR |
PIG-Y Antibody |
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| MBS9414487-5x01mL | MyBiosource | 5x0.1mL | 2075 EUR |
PIG-Y Antibody |
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| MBS9411870-01mL | MyBiosource | 0.1mL | 495 EUR |
PIG-Y Antibody |
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| MBS9411870-5x01mL | MyBiosource | 5x0.1mL | 2075 EUR |
PIG-Y Antibody |
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| MBS151115-01mg | MyBiosource | 0.1mg | 445 EUR |
PIG-Y Antibody |
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| MBS151115-5x01mg | MyBiosource | 5x0.1mg | 1965 EUR |
PIG-Y Antibody |
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| MBS150136-01mg | MyBiosource | 0.1mg | 445 EUR |
PIG-Y Antibody |
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| MBS150136-5x01mg | MyBiosource | 5x0.1mg | 1965 EUR |
Production and application of mouse monoclonal antibodies targeting linear epitopes in pB602L of African swine fever virus
African swine fever (ASF) is an acute hemorrhagic disease of domestic pigs. The causative agent of ASF, ASF virus (ASFV), is a double-stranded DNA virus, the sole member in the family Asfarviridae. The non-structural protein pB602L of ASFV is a molecular chaperone of the major capsid protein p72 and plays a key role in icosahedral capsid assembly. This protein is antigenic and is a target for developing diagnostic tools for ASF. To generate monoclonal antibodies (mAbs) against pB602L, a prokaryotically expressed recombinant pB602L protein was produced, purified, and used as an antigen to immunize mice.
A total of eight mouse mAbs were obtained, and their binding epitopes were screened by Western blot using an overlapping set of polypeptides from pB602L. Three linear epitopes were identified and designated epitope 1 (366ANRERYNY373), epitope 2 (415GPDAPGLSI423), and epitope 3 (498EMLNVPDD505). Based on the epitope recognized, the eight mAbs were placed into three groups: group 1 (B2A1, B2F1, and B2D10), group 2 (B2H10, B2B2, B2D8, and B2A3), and group 3 (B2E12). The mAbs B2A1, B2H10, and B2E12, each representing one of the groups, were used to detect pB602L in ASFV-infected porcine alveolar macrophages (PAMs) and pig tissues, using an indirect fluorescence assay (IFA) and immunohistochemical staining, respectively. The results showed that pB602L was detectable with all three mAbs in immunohistochemical staining, but only B2H10 was suitable for detecting the proteins in ASFV-infected PAMs by IFA. In summary, we developed eight anti-pB602L mouse mAbs recognizing three linear epitopes in the protein, which can be used as reagents for basic and applied research on ASFV.