Horseradish Peroxidase-Conjugated-Nanobody-Based cELISA For The Rapid and Sensitive Clinical Detection of African Swine Fever Virus Antibodies

Background: African swine fever (ASF), which is caused by the ASF virus (ASFV), is a highly contagious hemorrhagic disease that affects pigs and has the potential to cause mortality in almost 100% of domestic pigs and wild boars. Due to the lack of an effective vaccine, the control of ASF must depend on early, efficient, cost-effective detection and strict control and elimination strategies. Traditional molecular and serological testing methods are generally associated with high testing costs, complex operations and high technical requirements. As a promising alternative diagnostic tool to traditional antibodies, nanobodies (Nb) have the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity. The application of Nbs in the detection of ASFV antibodies in the serum has not yet been reported, to the best of our knowledge. Results: Using a phage display technology, one specific Nb against the ASFV p54 protein that exhibited high specificity and affinity to the protein, Nb8, was successfully screened. A HEK293T cell line stably expressing Nb8-horseradish peroxidase (HRP) fusion protein was established using the lentiviral expression system. Following the optimization of the reaction conditions, the Nb8-HRP fusion protein was successfully used to establish a competitive enzyme-linked immunosorbent assay (cELISA) to detect ASFV-specific antibodies in pig serum, for the first time. The cut-off value for the cELISA was 15.78%. A total of 209 serum samples were tested using the developed cELISA and a commercial ELISA kit. The specificity of the cELISA was 98.97%, and the limit of detection was 1:320 in inactivated ASFV antibody-positive reference serum samples, with the coincidence rate between the two methods being 98.56%. Conclusions: A specific, sensitive and repeatable cELISA was successfully developed based on the unique Nb8 as a probe, providing a promising method for the detection of anti-ASFV antibodies in clinical pig serum.

Upconverting phosphor technology-based lateral flow assay for the rapid and sensitive detection of anti-Trichinella spiralis IgG antibodies in pig serum

Background Trichinella spiralis is a zoonotic food-borne parasite. A disease caused by infection with T. spiralis is called trichinellosis in humans. It is important to investigate the epidemic situation and the surveillance of herds and then prevent infection in humans. Therefore, this study is to develop a rapid and sensitive diagnostic method for on-site test in domestic and wild animals. Methods Upconverting phosphor nanoparticles (UCNPs), an excellent optical label, were conjugated with the excretory-secretory (ES) antigens from T. spiralis muscle larvae (ML) or goat anti-rabbit IgG, and a lateral flow (LF) assay based on these probes (UCNPs-ES/goat anti-rabbit IgG) was developed for the rapid and sensitive detection of anti-T. spiralis IgG antibodies in pig serum. The assay is named the UPT-LF-ES assay. In addition, the probes were characterized, and the assay was optimized. A cut-off threshold of the assay was also identified by using 169 known negative pig samples. Performance of the assay to T. spiralis with different infective numbers, cross-reactivity with other parasitic infections, the single-blinded experiment, and coincidence were evaluated with the assay. Results The UPT-LF-ES assay was successfully constructed and optimized based on the probes of UCNPs-ES/goat anti-rabbit IgG. In the pigs infected with 100, 1000, and 10,000 ML, positive results were first presented at 35 days post-infection (dpi), 30 dpi, and 25 dpi, respectively. The assay had no cross-reaction with other parasitic infections. A single-blinded experiment indicated that the sensitivity and specificity of the UPT-LF-ES assay were 100% and 100%, respectively, the area under the receiver operating characteristic (ROC) curve was 1.000. In addition, the value detected by the UPT-LF-ES assay was significantly different between positive and negative samples. Moreover, compared with the “gold standard” magnetic stirrer method, the coincidence rate of the UPT-LF-ES assay was 87.27%, and the kappa (K) coefficient was 0.7454, showing a substantial agreement. Conclusions The UPT-LF-ES assay is a useful point-of-care test (POCT) with T. spiralis in the detection of pig, which contributes to preventing human trichinellosis. Graphical Abstract

Development and Optimization of an Enzyme Immunoassay to Detect Serum Antibodies against the Hepatitis E Virus in Pigs, Using Plant-Derived ORF2 Recombinant Protein

Hepatitis E is an emerging global disease, mainly transmitted via the fecal–oral route in developing countries, and in a zoonotic manner in the developed world. Pigs and wild boar constitute the primary Hepatitis E virus (HEV) zoonotic reservoir. Consumption of undercooked animal meat or direct contact with infected animals is the most common source of HEV infection in European countries. The purpose of this study is to develop an enzyme immunoassay (EIA) for the detection of anti-hepatitis E virus IgG in pig serum, using plant-produced recombinant HEV-3 ORF2 as an antigenic coating protein, and also to evaluate the sensitivity and specificity of this assay. A recombinant HEV-3 ORF2 110-610_6his capsid protein, transiently expressed by pEff vector in Nicotiana benthamiana plants was used to develop an in-house HEV EIA. The plant-derived HEV-3 ORF2 110-610_6his protein proved to be antigenically similar to the HEV ORF2 capsid protein and it can self-assemble into heterogeneous particulate structures. The optimal conditions for the in-house EIA (iEIA) were determined as follows: HEV-3 ORF2 110-610_6his antigen concentration (4 µg/mL), serum dilution (1:50), 3% BSA as a blocking agent, and secondary antibody dilution (1:20 000). The iEIA developed for this study showed a sensitivity of 97.1% (95% Cl: 89.9–99.65) and a specificity of 98.6% (95% Cl: 92.5–99.96) with a Youden index of 0.9571. A comparison between our iEIA and a commercial assay (PrioCHECK™ Porcine HEV Ab ELISA Kit, ThermoFisher Scientific, MA, USA) showed 97.8% agreement with a kappa index of 0.9399. The plant-based HEV-3 ORF2 iEIA assay was able to detect anti-HEV IgG in pig serum with a very good agreement compared to the commercially available kit.

Horseradish peroxidase-conjugated-nanobody-based blocking ELISA for the rapid and sensitive clinical detection of African swine fever virus antibodies

Background African swine fever (ASF), which is caused by the ASF virus (ASFV), is a highly contagious hemorrhagic disease that affects pigs and has the potential to cause mortality in almost 100% of domestic pigs and wild boars. Due to the lack of an effective vaccine, the control of ASF must depend on early, efficient, cost-effective detection and strict control and elimination strategies. Traditional molecular and serological testing methods are generally associated with high testing costs, complex operations and high technical requirements. As a promising alternative diagnostic tool to traditional antibodies, nanobodies (Nb) have the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity. The application of Nbs in the detection of ASFV antibodies in the serum has not yet been reported, to the best of our knowledge. Results Using a phage display technology, one specific Nb against the ASFV p54 protein that exhibited high specificity and affinity to the protein, Nb83, was successfully screened. Nb83 was labeled with horseradish peroxidase (HRP) to create an Nb83-HRP fusion protein in 293T cells. Following the optimization of the reaction conditions, the Nb83-HRP fusion protein was successfully used to establish a blocking enzyme-linked immunosorbent assay (ELISA) to detect ASFV-specific antibodies in pig serum, for the first time. The cutoff value for the blocking ELISA was 39.16%. A total of 210 serum samples were tested using the developed blocking ELISA and a commercial ELISA kit. The specificity of the blocking ELISA was 100%, and the limit of detection was 1:5,120 in inactivated ASFV antibody-positive reference serum samples, with the coincidence rate between the two methods being 98.57%. Conclusions A specific, sensitive and repeatable blocking ELISA was successfully developed based on the unique Nb as a probe, providing a promising method for the detection of anti-ASFV antibodies in clinical pig serum.

Combining mass spectrometric platforms for lipidome investigation - Application to the characterisation of disruptions in the lipid profile of pig serum upon β-agonist treatment

In the last decade, many mass spectrometric fingerprinting methods dedicated to lipidomics have been proposed: either non-targeted approaches, coupled with annotation methods, or targeted strategies, aiming at specifically monitoring a limited number of substances.In a general public health perspective and through a strategy combining non-targeted and targeted lipidomics MS-based approaches, this study aims at identifying disrupted patterns in serum lipidome upon growth promoter treatment in pig and evaluating the relative contributions of the three platforms involved.Pig serum samples collected during an animal experiment involving control and treated animals, whose food had been supplemented with ractopamine, were extracted and characterised using three MS strategies: Non-targeted RP LC-HRMS; the targeted Lipidyzer™ platform (differential ion mobility associated with shotgun lipidomics) and a homemade LC-HRMS triglyceride platform.The three different platforms showed complementarity insight into lipid characterisation, which, applied to a selected set of samples, enabled highlighting specific lipid profile patterns involving various lipid classes, mainly in relation with cholesterol esters, sphingomyelins, lactosylceramide, phosphatidylcholines and triglycerides.Thanks to the combination of both non-targeted and targeted MS approaches, the exploration of various compartments of the pig serum lipidome could be performed, including commonly characterised lipids (Lipidyzer™), triglyceride isomers (Triglyceride platform) -whose accurate analysis was considered an analytical challenge, and unique lipid features (non-targeted LC-HRMS). Thanks to their respective characteristics, the complementarity of the three tools could be demonstrated for public health purposes, with enhanced lipidome coverage, level of characterisation and applicability.