Detection and Quantification of SARS-CoV-2 Receptor Binding Domain Neutralization by a Sensitive Competitive ELISA Assay

This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure requires a small amount of S-protein/RBD and angiotensin converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include (1) serum heat treatment to prevent non-specific interactions, (2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, (3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and (4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 h for >30 serum samples; this includes the 7.5 h of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the ‘gold standard’ assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious and time consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R2 = 0.975, n = 25), demonstrating high sensitivity.

Expression of plasma IFN signaling-related miRNAs during acute SARS-CoV-2 infection and its association with RBD-IgG antibody response

Background Coronavirus disease 2019 (COVID-19) is a huge challenge worldwide. Although previous studies have suggested that type I interferon (IFN-I) could inhibit the virus replication, the expression characteristics of IFN-I signaling-related miRNAs (ISR-miRNAs) during acute severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and its relationship with receptor-binding domain (RBD) IgG antibody response at the recovery phase remain unclear. Methods Expression profiles of 12 plasma ISR-miRNAs in COVID-19 patients and healthy controls were analyzed using RT-qPCR. The level of RBD-IgG antibody was determined using the competitive ELISA. Spearman correlation was done to measure the associations of plasma ISR-miRNAs with clinical characteristics during acute SARS-CoV-2 infection and RBD-IgG antibody response at the recovery phase. Results Compared with the healthy controls, COVID-19 patients exhibited higher levels of miR-29b-3p (Z = 3.15, P = 0.002) and miR-1246 (Z = 4.98, P < 0.001). However, the expression of miR-186-5p and miR-15a-5p were significantly decreased. As the results shown, miR-30b-5p was negatively correlated with CD4 + T cell counts (r = − 0.41, P = 0.027) and marginally positively correlated with fasting plasma glucose in COVID-19 patients (r = 0.37, P = 0.052). The competitive ELISA analysis showed the plasma level of miR-497-5p at the acute phase was positively correlated with RBD-IgG antibody response (r = 0.48, P = 0.038). Conclusions Our present results suggested that the expression level of ISR-miRNAs was not only associated with acute SARS-CoV-2 infection but also with RBD-IgG antibody response at the recovery phase of COVID-19. Future studies should be performed to explore the biological significance of ISR-miRNAs in SARS-CoV-2 infection.

Bluetongue Virus Infections in Cattle Herds of Manabí Province of Ecuador

Bluetongue (BT) is a viral disease transmitted by Culicoides (Diptera: Ceratopogonidae) to domestic and wild ruminants. Infections in cattle are mainly subclinical, but severe necrotic and hemorrhagic illness and death may occur depending on the strain of the virus and other factors; cattle act as a reservoir for the virus. Although the Ecuadorian coast has climatic conditions that favor the presence of the vector, there are few serologic or virologic BTV studies available. Manabí is a coastal province in which livestock farming is mostly implemented in the northern part. We conducted two studies to assess, for the first time, the presence of active BTV infections in Manabí province. We collected 430 serum samples from 38 randomly selected farms between March and July 2019 to perform BTV competitive ELISA. In addition, six seropositive farms were selected to place eight sentinel BTV-naive calves. All these calves were blood sampled and the presence of BTV RNA and antibodies was tested for by RT-PCR and competitive ELISA, respectively, once a week for 6–8 weeks until seroconversion was evidenced. A high individual seroprevalence (99%) was obtained, and all investigated farms had BTV seropositive animals. All sentinel calves became BTV viremic and seroconverted. The first viremia appeared after 2–5 weeks from arrival at the farm; they seroconverted 1–3 weeks later. We demonstrate for the first time that there is a high level of BTV circulation north of Manabí, with active infections on these farms. Integrated control strategies such as hygienic measures on farms to reduce midge populations would be advisable for the owners as mitigation measures.

Development of a nanobody-based competitive ELISA for efficiently and specifically detecting antibodies against genotype 2 porcine reproductive and respiratory syndrome viruses

Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes considerable economic loss to the global pig industry. Efficient detection assay is very important for the prevention of the virus infection. Nanobodies are the advantages of small molecular weight, simple genetic engineering, and low production cost for promising diagnostic application. In this study, to develop a nanobody-based competitive ELISA (cELISA) for specifically detecting antibodies against PRRSV, three nanobodies against PRRSV-N protein were screened by Camel immunization, library construction, and phage display. Subsequently, a recombinant HEK293S cell line stably secreting nanobody-horseradish peroxidase (HRP) fusion protein against PRRSV-N protein was successfully constructed using the lentivirus transduction assay. Using the cell lines, the fusion protein was easily produced. Then, a novel cELISA was developed using the nanobody-HRP fusion protein for detecting antibodies against PRRSV in pig sera, exhibiting a cut-off value of 23.19% and good sensitivity, specificity, and reproducibility. Importantly, the cELISA specifically detect anti-genotype 2 PRRSV antibodies. The cELISA showed more sensitive than the commercial IDEXX ELISA kit by detecting the sequential sera from the challenged pigs. The compliance rate of cELISA with the commercial IDEXX ELISA kit was 96.4%. In addition, the commercial IDEXX ELISA kit can be combined with the developed cELISA for the differential detection of antibodies against genotype 1 and 2 PRRSV in pig sera. Collectively, the developed nanobody-based cELISA showed advantages of simply operation and low production cost and can be as an assay for epidemiological investigation of genotype 2 PRRSV infection in pigs and evaluation after vaccination.

Competitive ELISA for a serologic test to detect dengue serotype-specific anti-NS1 IgGs using high-affinity UB-DNA aptamers

Serologic tests to detect specific IgGs to antigens related to viral infections are urgently needed for diagnostics and therapeutics. We present a diagnostic method for serotype-specific IgG identification of dengue infection by a competitive enzyme-linked immunosorbent assay (ELISA), using high-affinity unnatural-base-containing DNA (UB-DNA) aptamers that recognize the four categorized serotypes. Using UB-DNA aptamers specific to each serotype of dengue NS1 proteins (DEN-NS1), we developed our aptamer–antibody sandwich ELISA for dengue diagnostics. Furthermore, IgGs highly specific to DEN-NS1 inhibited the serotype-specific NS1 detection, inspiring us to develop the competitive ELISA format for dengue serotype-specific IgG detection. Blood samples from Singaporean patients with primary or secondary dengue infections confirmed the highly specific IgG detection of this format, and the IgG production initially reflected the serotype of the past infection, rather than the recent infection. Using this dengue competitive ELISA format, cross-reactivity tests of 21 plasma samples from Singaporean Zika virus-infected patients revealed two distinct patterns: 8 lacked cross-reactivity, and 13 were positive with unique dengue serotype specificities, indicating previous dengue infection. This antigen-detection ELISA and antibody-detection competitive ELISA combination using the UB-DNA aptamers identifies both past and current viral infections and will facilitate specific medical care and vaccine development for infectious diseases.

Investigation of Neospora caninum Seroprevalence and Associa-tion with Reproductive Problems in Cows in Burdur Province of Turkey

Background: An apicomplexan protozoon Neospora caninum, causative agent of neosporosis, is recognized as one of the most common and important cause of sporadic and endemic bovine abortion and reduced reproductivity in dairy and beef cattle worldwide. The present study was conducted to investigate the relationship between N. caninum seroprevalence and infertility problems in 400 cows in Burdur city, Turkey. Methods: Blood samples were collected from vena jugularis into sterile serum tubes from 49 aborted, 58 infertil, 48 pregnant and 245 healthy cows for the findings of reproductive anamnesis during a period of March 2010 to March 2011. Sera samples were analyzed by competitive ELISA kit. Results: The seroprevalences were 7.7%, 6.4% and 4.2% in 2-4, ≤2 and ≥4 age groups respectively and no statistically significance observed between age groups. Seropositivity rates were 5.7%, 5.1%, 4.5%, 3.6% in Holstein, Montofon, cross-breeds and Simental breeds respectively. Seroprevalence differences was not statistically significant among cattle breeds. Antibodies to N. caninum were found in rates of 16.3%, 6.9%, 6.3%, 2.4% in aborted, infertile, pregnant and healthy cows respectively and there was a significant difference (P<0.01) between aborted and healthy animals. Seroprevalences were Yeşilova 10%, Gölhisar and Ağlasun 8%, Bucak, Çavdır and Kemer 4%, Karamanlı and Burdur Centrum 2%, according to districts. Conclusion: The seroprevalence of N. caninum was revealed in Burdur region. It was emphasized that N. caninum infection should not be ignored in reproductive problems, especially in abortion cases.

Serological survey and isolation of Mycoplasma mycoides subspecies mycoides from cattle slaughtered at Katsina state central abattoir, Nigeria

serological survey for the detection of antibodies to Mycoplasma mycoides subspecies mycoides (Mmm) from cattle slaughtered at Katsina Central Abattoir was conducted from March to October 2012 using competitive ELISA (CIRAD) version: P05410/04 antibody detection kit. A total of 300 cattle sera were screened for Mmm antibodies out of which 22 (7.3%) obtained from 15 (68.2%) cows and 7 (31.8%) bulls were positive. Age distribution indicated that 3 (13.6%) were less than 2 years, 11 (50%) were between 2 and 4 years of age, while 8 (36.4%) were above 4 years. The study confirmed the susceptibility of some cattle breeds from all ages and sexes to Mmm. However, middle-aged cows were shown to be more associated with the infections though not statistically significant (P˃0.01). Similarly, a total of 130 pneumonic lung tissues were used for isolation and biochemical characterization of Mmm out of which 2 (1.5%) samples one each from adult White Fulani and Red Bororo cows were found to be positive. The study established baseline information on the status of Mmm infection in Katsina State. Continuous disease surveillance and annual mass vaccination programme using effective vaccines were recommended.