Enzyme-linked Immunosorbent Assay For Detection Research Articles

Engineered Macrophages-Based uPA-Scavenger Load Gemcitabine to Prompt Robust Treating Cancer Metastasis.

The majority of cancer patients die of metastasis rather than primary tumors, and most patients may have already completed the cryptic metastatic process at the time of diagnosis, making them intractable for therapeutic intervention. The urokinase-type plasminogen activator (uPA) system is proved to drive cancer metastasis. However, current blocking agents such as uPA inhibitors or antibodies are far from satisfactory due to poor pharmacokinetics and especially have to face multiplex mechanisms of metastasis. Herein, an effective strategy is proposed to develop a uPA-scavenger macrophage (uPAR-MΦ), followed by loading chemotherapeutics with nanoparticles (GEM@PLGA) to confront cancer metastasis. Interestingly, significant elimination of uPA by uPAR-MΦ is demonstrated by transwell analysis on tumor cells in vitro and enzyme-linked immunosorbent assay detection in peripheral blood of mice with metastatic tumors, contributing to significant inhibition of migration of tumor cells and occurrence of metastatic tumor lesions in mice. Moreover, uPAR-MΦ loaded with GEM@PLGA shows a robust antimetastasis effect and significantly prolonged survival in 4T1-tumor-bearing mice models. This work provides a novel living drug platform for realizing a potent treatment strategy to patients suffering from cancer metastasis, which can be further expanded to handle other tumor metastasis markers mediating cancer metastasis.

Clinically Deployable Bioelectronic Sensing Platform for Ultrasensitive Detection of Transferrin in Serum Sample

Varying levels of transferrin (Tf) have been associated with different disease conditions and are known to play a crucial role in various malignancies. Regular monitoring of the variations in Tf levels can be useful for managing related diseases, especially for the prognosis of certain cancers. We fabricated an immunosensor based on graphene oxide (GO) nanosheets to indirectly detect Tf levels in cancer patients. The GO nanosheets were deposited onto an indium tin oxide (ITO)-coated glass substrate and annealed at 120 °C to obtain reduced GO (rGO) films, followed by the immobilization of an antibody, anti-Tf. The materials and sensor probe used were systematically characterized by UV-Visible spectroscopy (UV-Vis), X-ray diffraction (XRD), atomic force microscopy (AFM), and Fourier transform infrared spectroscopy (FTIR). Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) were also used for the stepwise sensor probe characterizations and Tf detection in serum samples, respectively. The anti-Tf/rGO/ITO immunosensor DPV output demonstrated an excellent Tf detection capability in the linear range of 0.1 mg mL-1 to 12 mg mL-1 compared to the enzyme-linked immunosorbent assay (ELISA) detection range, with a limit of detection (LOD) of 0.010 ± 0.007 mg mL-1. Furthermore, the results of the fabricated immunosensor were compared with those of the ELISA and autobioanalyzer techniques, showing an outstanding match with < 5% error and demonstrating the immunosensor's clinical potential.

A novel S2-derived peptide-based ELISA for broad detection of antibody against infectious bronchitis virus

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV). Vaccination is an effective approach for controlling IBV. Therefore, reliable immune monitoring for IB is critical for poultry. In this study, a novel peptide derived from S2 protein was used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of broadly cross-reactive antibodies against IBV. The peptide-based ELISA (pELISA) showed good specificity and sensitivity in detecting IBV antibodies against different serotypes. A semilogarithmic regression method for determining IBV antibody titers was also established. Antibody titers detected by pELISA and calculated with this equation were statistically similar to those evaluated by indirect fluorescence assay (IFA). Moreover, the comparison analysis showed a 96.07% compatibility between the pELISA and IDEXX ELISA. All these data demonstrate that the pELISA generated here can be as a rapid and reliable serological surveillance tool for monitoring IBV infection or vaccination.

Development and application of an indirect ELISA for the serological detection of bovine viral diarrhea virus infection based on the protein E2 antigen.

Bovine viral diarrhea virus (BVDV) causes continuous economic losses to the livestock industry. Monitoring antibodies with enzyme-linked immunosorbent assay (ELISA) is a valuable tool to ensure the purification of BVDV in cattle. However, currently available ELISA kits based on the whole BVDV virion are both costly and time-consuming. The E2 protein has good immunogenicity, induces the secretion of neutralizing antibodies and is an essential immunogen for serological detection. We developed a novel recombinant E2 protein-based indirect ELISA (rE2-iELISA) and conducted a serological survey for BVDV antibodies in 2021-2022 in Beijing, China. The results showed that E2 protein was successfully expressed with high immunogenicity and the optimal rE2-iELISA displayed high sensitivity, reproducibility and specificity. Clinical testing of 566 serum specimens indicated that 318 BVDV positive samples and 194 BVDV negative samples were tested by rE2-iELISA and the IDEXX BVDV ELISA-Ab kit, with a positive coincidence rate of 93.3%, a negative coincidence rate of 86.3%, and an overall coincidence rate of 90.5%. This study established an rE2-iELISA method, which is a highly sensitive, specific and robust ELISA-test validated to detect anti-BVDV antibodies. These findings indicate that the newly developed rE2-iELISA method has the potential to be used as a rapid, reliable and cost-effective screening tool for BVDV infection and provides technical support for the evaluation of vaccine efficacy in cattle herds in the future.

Two-Dimensional Fe-N-C Single-Atomic-Site Catalysts with Boosted Peroxidase-Like Activity for a Sensitive Immunoassay.

Single-atomic-site catalysts (SASCs) with peroxidase (POD)-like activities have been widely used in various sensing platforms, like the enzyme-linked immunosorbent assay (ELISA). Herein, a two-dimensional Fe-N-C-based SASC (2D Fe-SASC) is successfully synthesized with excellent POD-like activity (specific activity = 90.11 U/mg) and is used to design the ELISA for herbicide detection. The 2D structure of Fe-SASC enables the exposure of numerous single atomic active sites on the surface as well as boosts the POD-like activity, thereby enhancing the sensing performance. 2D Fe-SASC is assembled into competitive ELISA kit, which achieves an excellent detection performance for 2,4-dichlorophenoxyacetic acid (2,4-D). Fe-SASC has great potential in replacing high-cost natural enzymes and working on various advanced sensing platforms with high sensitivity for the detection of various target biomarkers.

Development of a novel double-antibody sandwich quantitative ELISA for detecting SADS-CoV infection.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging swine enteric alphacoronavirus that can cause acute diarrhea, vomiting, dehydration, and death of newborn piglets. In this study, we developed a double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) for detection of SADS-CoV by using an anti-SADS-CoV N protein rabbit polyclonal antibody (PAb) and a specific monoclonal antibody (MAb) 6E8 against the SADS-CoV N protein. The PAb was used as the capture antibodies and HRP-labeled 6E8 as the detector antibody. The detection limit of the developed DAS-qELISA assay was 1ng/mL of purified antigen and 101.08TCID50/mL of SADS-CoV, respectively. Specificity assays showed that the developed DAS-qELISA has no cross-reactivity with other swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV). Three-day-old piglets were challenged with SADS-CoV and collected anal swab samples which were screened for the presence of SADS-CoV by using DAS-qELISA and reverse transcriptase PCR (RT-PCR). The coincidence rate of the DAS-qELISA and RT-PCR was 93.93%, and the kappa value was 0.85, indicating that DAS-qELISA is a reliable method for applying antigen detection of clinical samples. KEY POINTS: • The first double-antibody sandwich quantitative enzyme-linked immunosorbent assay for detection SADS-CoV infection. • The custom ELISA is useful for controlling the SADS-CoV spread.

Effect of VEGF on neurological impairment and prognosis of acute cerebral infarction patients: A retrospective case-control study.

Due to the complex pathological mechanism of acute cerebral infarction, the role of vascular endothelial growth factor (VEGF) on the disease is not clear. Therefore, a retrospective case-control study was performed to explore the effect of VEGF on neurological impairment and prognosis of acute cerebral infarction patients. A total of 100 patients with acute cerebral infarction admitted to our hospital from April 2021 to April 2022 were selected. Blood samples from all patients would be routinely collected to detect the expression of serum VEGF. Pearson chi-square, Spearman correlation and univariate Logistic regression were used to analyze the clinical data to explore the relationship between VEGF expression and basic information, stroke degree, quality of life, and prognosis of patients. To determine whether VEGF can provide relevant basis for the early prevention and prognostic treatment of acute cerebral infarction. And multivariate logistic regression was used to calculate the odds ratio between each variable and VEGF expression. Pearson chi-square test and Spearman correlation coefficient showed that sex, degree of stroke, limb convulsions, loss of consciousness, hemiplegia, aphasia, mental functioning score, overall quality of life score, and short-term prognosis were significantly correlated with VEGF expression in 100 patients. Univariate logistic regression was used to describe the ORs and 95% confidence interval of subjects at the univariate level, and the degree of stroke (OR = 83.333, P < 0.001), tic of limbs (OR = 26.316, P < 0.001), loss of consciousness (OR = 23.256, P < 0.001), hemiplegia (OR = 62.500, P < 0.001), aphasia (OR = 76.923, P < 0.001), mental functioning score (OR = 7.937, P < 0.001), overall quality of life score (OR = 5.464, P < 0.001), short-term prognosis (OR = 37.037, P < 0.001) was significantly correlated with the high expression of VEGF. The level of serum VEGF was positively correlated with neurological impairment degree and prognosis in patients with acute cerebral infarction, the more severe the degree of stroke and the worse the prognosis.

Development of in-house ELISA for detection of antibodies against lumpy skin disease virus in cattle and assessment of its performance using a bayesian approach

Lumpy skin disease (LSD) is a contagious disease among cattle and buffalo worldwide. Currently, an enzyme-linked immunosorbent assay (ELISA) has been recognized as an efficient diagnostic tool that is less time-consuming and easier than the viral neutralization test to measure the antibody levels. In the present study, an in-house method of indirect ELISA was developed to detect the bovine antibodies against Lumpy skin disease virus (LSDV) and its performance was assessed using field samples. This in-house method has been compared with the commercial ELISA test kit for detection of bovine antibodies against LSDV. The sensitivity (Se) and the specificity (Sp) of the test were estimated using a Bayesian latent class model. Checkerboard titration was performed using the naturally LSDV-infected bovine sera and colostrum-deprived calf sera. The LSDV antigen concentrations (1 TCID50/mL), the sample serum (1:500), and goat anti-bovine immunoglobulin G (IgG) labeled with horseradish peroxidase (HRP) (1:10,000) were determined to be optimal for this assay. The calculated cut-off value was 0.067, and there were no differences in the results of tests that utilized positive and negative sera (p < 0.05). The characteristics of two diagnostic tests were evaluated using a conditional dependent and one-population Bayesian model. The Se value of an in-house indirect ELISA were almost similar to ELISA test kit. On the other hand, the Sp value of the in-house ELISA test was lower than that of the commercial ELISA test with the median values of 89% (95% PPI = 75.9–99.3%) and 91.4% (95% PPI = 85.3–95.5%), respectively. A posterior estimate for the prevalence was 66.9% (95% PPI = 60.8–83.3%) and higher than initially expected.

Prevalence of Barmah Forest Virus, Chikungunya Virus and Ross River Virus Antibodies among Papua New Guinea Military Personnel before 2019.

Barmah Forest virus (BFV), Chikungunya virus (CHIKV) and Ross River virus (RRV) belong to the Alphavirus genus of the family Togaviridae. All three virus infections have been reported in Papua New Guinea (PNG) previously, but the exact prevalence and distribution of these three alphaviruses in PNG has not been established. Sera collected from 204 PNG Military Personnel (PNGMP) study participants in April 2019 was tested for the presence of anti-BFV, anti-CHIKV and anti-RRV immunoglobulin G (IgG) antibodies using commercially available enzyme-linked immunosorbent assay (ELISA) IgG detection kits, as well as for specific neutralizing antibodies (NAb) against individual viruses. Overall, sero-positivity of the sera was anti-BFV IgG 12.3% (25/204), anti-BFV NAb 8.3% (17/204); anti-CHIKV IgG 47.1% (96/204), anti-CHIKV NAb 34.8% (71/204); and anti-RRV IgG 93.1% (190/204), anti-RRV NAb 56.4% (115/204), respectively. Of the 137/204 participants that were Nab-positive for at least one virus, we identified 4 BFV, 40 CHIKV and 73 RRV single infections, and 9 RRV+CHIKV and 11 BFV+RRV double infections. The lower proportion of NAb sero-positive compared to the ELISA IgG sero-positive assay samples suggests that the currently available commercial ELISA detection kits for these three alphaviruses may not be suitable for diagnostic/surveillance purposes in endemic areas such as PNG, due to serological cross-reactivity among these three alphaviruses. Laboratory testing using known positive control sera indicated no cross-neutralization between BFV and RRV; however, some RRV or BFV single infection human sera demonstrated low-level cross-neutralization against CHIKV (the ratio of RRV/CHIKV NAb titers or BFV/CHIKV ≥ 4). Our preliminary results indicate that the majority of PNGMP have previously been exposed to RRV, with mild exposure to CHIKV and low-level exposure to BFV, suggesting that multiple alphaviruses have been circulating among PNGMP. The transmission landscapes of these three alphaviruses across PNG should be prioritized for further investigation, including identification of specific vectors and hosts that mediate human spillover in order to mitigate future outbreaks. Ongoing education regarding precautionary and protective measures are needed to better protect individuals who travel to PNG.

Serological response to vaccination against coronavirus disease-19 in patients with inflammatory bowel disease.

Vaccination against coronavirus disease-19 (COVID-19) is effective in preventing the occurrence or reduction in the severity of the infection. Patients with inflammatory bowel disease (IBD) are on immunomodulators, which may alter serological response to vaccination against COVID-19. Accordingly, we studied (i) the serological response to vaccination against COVID-19 in IBD patients and (ii) a comparison of serological response in IBD patients with that in healthy controls. A prospective study was undertaken during a 6-month period (July 2021 to January 2022). Seroconversion was assessed among vaccinated, unvaccinated IBD patients and vaccinated healthy controls using anti-severe acute respiratory syndrome coronavirus 2 immunoglobulin G (anti-SARS-CoV-2 IgG) antibody detection enzyme-linked immunosorbent assay (ELISA) kit, and optical density (OD) was measured at 450nm. OD is directly proportional to the antibody concentration. One hundred andthirty-two blood samples were collected from 97 IBD patients (85 [87.6%] ulcerative colitis and 12 [12.4%] Crohn's disease). Forty-one of the seventy-one (57.7%) unvaccinated and 60/61 (98.4%) vaccinated IBD patients tested positive (OD > 0.3) for SARS-CoV-2 IgG antibodies. Fourteen of the sixteen (87.5%) healthy controls tested positive for SARS-CoV-2 IgG antibodies. Vaccinated IBD patients had higher ODs than unvaccinated IBD patients (1.31 [1.09-1.70] vs. 0.53 [0.19-1.32], p < 0.001) and 16 vaccinated healthy controls (1.31 [1.09-1.70] vs. 0.64 [0.43-0.78], p < 0.001). Three of the seventy-one (4.2%) unvaccinated IBD patients reported having recovered from COVID-19. Most IBD patients seroconvert after vaccination against SARS-CoV-2, similar to a healthy population. A large proportion of IBD patients had anti-SARS-CoV-2 antibodies even before vaccination, suggesting the occurrence of herd immunity.

Evaluation of In-House ELISA for Antirabies Antibodies Detection in Domestic Canine.

Indonesia is known to be endemic for rabies in several areas, especially in Sumatra, Kalimantan, Sulawesi, and Flores Islands. Currently, vaccinating dogs has been shown to be the most cost-effective strategy for preventing rabies in humans. Postvaccination monitoring should be carried out to evaluate the success of vaccination by measuring antibody titers in serum of vaccinated dogs. Serological methods for monitoring rabies-specific antibody titers can be carried out using enzyme-linked immunosorbent assay (ELISA) methods as recommended by the World Organization for Animal Health (WOAH). Therefore, the development of the in-house ELISA (BukTi-Vet) that we have carried out in order to support postvaccination monitoring in dogs needs to be evaluated for its diagnostic performance compared to commercial ELISA kits. The diagnostic performance of each ELISA kit was evaluated using 111 known positive and 47 negative serums. Each known positive and negative serum will be tested using the three rabies ELISA kits used in this study. BukTi-Vet is an in-house ELISA for the detection of rabies-specific IgG antibodies that have been developed with sensitivity, specificity, and accuracy of 98.19%, 97.87%, and 98.1%, respectively. Based on the value of its positive and negative clinical utility index, BukTi-Vet is excellent for use in immunoassays directed for confirmatory (0.97) as well as screening (0.94) tests. BukTi-Vet shows a very good agreement with both Platelia II and RFFIT, so it is convincing to be further refined into a diagnostic kit. Tests of field sera from dogs vaccinated with various vaccines should be performed to provide more complete information on diagnostic performance. BukTi-Vet showed a very good agreement with RFFIT, while Pusvetma and Platelia II only showed good agreement. The average value of BukTi-Vet compatibility with RFFIT can reach 94%.

Epidemiology of Rotavirus Infection in Dairy Calves in Central Ethiopia

Rotavirus is a major pathogen responsible for diarrheal disease in calves and neonates resulting in loss of productivity and economic losses in dairy farms in developed and developing countries. In Ethiopia, the diarrheal disease caused by Rotavirus is not so far studied adequately. Across-sectional study was conducted from October 2019 to May 2020 to estimate the prevalence, assess associated factors, and isolate Rotavirus in calves below two months of age in the central part of Ethiopia, namely Holeta, Sululta, Bishoftu, and Sebeta. A total of 404 fecal samples, comprising 213 diarrheic and 191 non-diarrheic cases, were collected from calves of less than two months of age by simple random sampling technique. Rotavirus infection was detected using antigen detection Enzyme-linked immunosorbent assay technique. All samples positive for ELISA were propagated in Madin Darby bovine kidney cells. Data was analyzed using STATA version 13. Fisher’s exact test and logistic regression analysis were used to determine the association between prevalence of rotavirus and potential risk factors. An overall rotavirus prevalence of 1.24% (95%CI 0.40 – 2.86%) was found. The prevalence of rotavirus in diarrheic calves (1.41%) was not significantly different from the prevalence of rotavirus in non-diarrheic calves (1.04%) (p&gt;0.05). Similarly, the origin, breed, sex, age, management, and type of farm had no significant association with the prevalence of rotavirus infection of calves (p&gt;0.05). After 3 subsequent passages, progressive cytopathic effect i.e. Cell swelling and obscure cell boundaries, detachment and floatation of cells, cytoplasmic vacuolationas, increased cell granularity and cytoplasmic stranding were observed in all samples. The presence of rotavirus in dairy calves might entail morbidity and mortality in calves. Moreover, rotavirus infection might also lead to zoonotic transmission. Therefore, further detailed molecular epidemiological studies are warranted. Good hygienic and husbandry practices are essential to limit the spread of infection.

Anti-tissue Transglutaminase Enzyme-Linked Immunosorbent Assay for Screening of Celiac Disease in Moroccan Pediatric Patients

Celiac disease (CD) is an immune-mediated systemic disorder elicited by gluten which induces an enteropathy with atrophy of intestinal villi. No prevalence information was available for CD in Moroccan children because the diagnosis is based on multiple invasive duodenal biopsies. To simplify CD diagnosis, the objective of this work was to develop an Enzyme-linked Immunosorbent Assay (ELISA) for IgA and IgG tissue transglutaminase (tTG) antibodies detection suitable for Moroccan children. The concentration of the coated antigen and serum dilutions were optimized for the development of IgA anti- tTG and IgG anti-tTG ELISA detection in serum. The cut-off was calculated and the developed ELISAs were used for detecting IgA and IgG-tTG antibodies in 97 serum samples from 97 children aged 1 to 13 years, with variable clinical characteristics. After adjusting optimal working conditions of ELISA, the cut-offs were 0.04 and 0.156 AU for detecting anti-tTG and IgA anti-tTG IgG respectively. The use of IgG anti-tTG as CD marker (100 % of sensitivity) was better than the use of IgA anti-tTG in ELISA tests (90.3% sensitivity). In addition, the ELISA test shows a clear correlation between the IgG tTG antibodies titer and the degree of duodenal damage, estimated by the Marsh stages classification in patients with confirmed CD. Our results suggest that the assessment of the adapted IgG anti-tTG ELISA test can be used for CD in initial screening in Moroccan children after validation with a larger number of samples with CD for confirmation.

Development and evaluation of recombinant E2 protein based IgM capture enzyme-linked immunosorbent assay (ELISA) and double antigen sandwich ELISA for detection of antibodies to Chikungunya virus

Chikungunya virus (CHIKV) reemerged and caused millions of human infections since 2004. The disease could be established, when the virus has been introduced to areas where the appropriate vectors are endemic. The differential diagnosis of CHIKV infection varies based on place of residence, travel history, and exposures. Serological tests are commonly used to diagnose CHIKV infection, but their availability and assessments of the performance of the diagnostics have been limited. To develop and evaluate antibodies detection methods for chikungunya diagnosis and serological investigation. Recombinant E2 protein based IgM capture enzyme-linked immunosorbent assay (Mac-ELISA) and double antigen sandwich ELISA (Das-ELISA) for detection of antibodies to Chikungunya virus were developed and evaluated. The repeatability was evaluated by testing of three reference sera at single dilutions in triplicated for 5 times. The sensitivity, specificity, accuracy, and agreement of the MAC-ELISA and Das-ELISA were obtained by comparing the detection results of 225 serum samples (45 positive; 180 negative) with a real-time RT-PCR assay and an IFA commercial tests manufactured by Euroimmun. The established ELISA assays were standardized by determining the optimal concentrations of the key reagents. The coefficient values of repeat testing were within 10% and 20% for intraassay and interassay precision, respectively. A sensitivity of 60.0% and 52.5%, a specificity of 96.2% and 96.8%, and an accuracy of 89.8% and 88.9% were obtained for the Mac-ELISA and Das-ELISA, respectively, when compared to a CHIKV qRT-PCR method. And a sensitivity of 100%, a specificity of 97.5% and 99.5%, and an accuracy of 97.8% and 99.6% were yielded respectively when using the IIFT as a reference method, which showed a highly consistence to the commercial IIFT assay with a Kappa value greater than 0.90. The Mac-ELISA and Das-ELISA based on recombinant E2 protein of CHIKV were developed and standardized, which could detect IgM or total antibodies against CHIKV in 2-3 hours with acceptable sensitivities and specificities. These assays can be used for laboratory diagnosis and serological investigation of CHIKV infections to evaluate the risk of CHIKV transmission.

Establishment of indirect ELISA method for Salmonella antibody detection from ducks based on PagN protein

BackgroundSalmonella as an important food-borne zoonotic bacterial pathogen, infection in ducks is a recessive infection, however, it can also cause high mortality and threat to food safety. Preventing and controlling the infection and transmission of Salmonella in ducks critically require rapid and sensitive detection method. Full-length Salmonella-specific protein PagN was induced and expressed in E.coil BL21 and was purified as an antigen to establish an indirect enzyme-linked immunosorbent assays (iELSA) detection kit.ResultsThe recombinant PagN protein has a molecular weight of 43 kDa containing a His-tag, was recognized by an anti-Salmonella positive serum by Western blot assay. The optimal concentration of PagN as a coating antigen in the iELISA was 1 μg/mL, and the optimal dilution of enzyme-labeled secondary antibody was 1:4000 (0.025 μg/mL). The cutoff OD450 value was established at 0.268. The iELISA kit showed high selectivity since no cross-reaction with E. coli, Staphylococcus aureus and Streptococcus was observed. iELISA method and Dot-blot test were performed on 100 clinical sera samples collected from duck farms, and the actual coincidence rate was 89% (89/100). 613 duck serum samples from 3 different farms were tested using established method and commercial ELISA kit. The concordance between the two methods was 94.1%.ConclusionAnti-PagN based iELISA can serve as a useful tool for diagnosis of Salmonella infection.

Diagnostic Accuracy of Methods for Detection of Antibodies against Type I Interferons in Patients with Endocrine Disorders.

Autoantibodies against type 1 interferons (IFN-I) are a highly specific marker for type 1 autoimmune polyglandular syndrome (APS-1). Moreover, determination of antibodies to omega-interferon (IFN-ω) and alpha2-interferon (IFN-α2) allows a short-term diagnosis in patients with isolated and atypical forms of APS-1. In this study, a comparison of three different methods, namely multiplex microarray-based, cell-based and enzyme-linked immunosorbent assays for detection of antibodies against omega-interferon and alpha2-interferon, was carried out. A total of 206 serum samples from adult patients with APS-1, APS-2, isolated autoimmune endocrine pathologies or non-autoimmune endocrine disorders, and healthy individuals were analyzed. In the APS-1 patient cohort (n = 18), there was good agreement between the results of anti-IFN-I antibody tests performed by three methods, with 100% specificity and sensitivity for microarray-based assay. Although only the cell-based assay can determine the neutralizing activity of autoantibodies, the microarray-based assay can serve as a highly specific and sensitive screening test to identify anti-IFN-I antibody positive patients.

Profiling Plasma Cytokines by A CRISPR-ELISA Assay for Early Detection of Lung Cancer

Cytokines play crucial roles in tumorigenesis and are potential biomarkers for cancer diagnosis. An Enzyme-linked Immunosorbent Assay (ELISA) is commonly used to measure cytokines but has a low sensitivity and can only detect a single target at a time. CRISPR-Associated Proteins (Cas) can ultra-sensitively and specifically detect nucleic acids and is revolutionizing molecular diagnostics. Here, we design a microplate-based CRISPR-ELISA assay to simultaneously profile multiple cytokines, in which antibodies are coupled with ssDNA to form antibody-ssDNA complexes that bridges CRISPR/Cas12a and ELISA reactions. The ssDNA triggers the Cas12a collateral cleavage activity and releases the fluorescent reporters to generate amplified fluorescent signals in the ELISA detection of cytokines. The CRISPR-ELISA assay can simultaneously measure multiple cytokines with a significantly higher sensitivity compared with conventional ELISA. Using the CRISPR-ELISA assay to profile plasma cytokines in 127 lung cancer patients and 125 cancer-free smokers, we develop a panel of plasma cytokine biomarkers (IL-6, IL-8, and IL-10) for early detection of the disease, with 80.6% sensitivity and 82.0% specificity. The CRISPR-ELISA assay may provide a new approach to the discovery of cytokine biomarkers for early lung cancer detection.

Application of a truncated ORF2 protein-based ELISA for diagnosis of hepatitis E in an endemic area.

Enterically transmitted waterborne hepatitis E (HE) caused due to hepatitis E virus (HEV) prevails as a significant public health problem endemic to India. Due to short-term viremia/fecal excretion and poor in vitro transmissibility of HEV, HE diagnosis depends on detection of specific IgM antibodies in serum. Present study evaluated performances of two in-house and six commercial IgM detection enzyme-linked immunosorbent assays (ELISAs) using sera collected from volunteers/acute hepatitis patients (n = 716). The in-house ELISAs were based on complete and truncated open reading frame 2 (ORF2) proteins containing neutralizing epitope/s region of genotype 1 HEV (ORF2p, 1-660 amino acid (a.a.) and T1NEp, 458-607 a.a., respectively). The commercial ELISAs included Wantai (China), MP Diagnostics (MPD) (Singapore), DIA.PRO Diagnostics (Italy), MBS (Italy), abia (Germany), and ImmunoVision (USA). T1NE ELISA showed 97.0% positive percent agreement (PPA), 99.4% negative percent agreement (NPA), and 98.6% concordance (κ = 0.97, P = 0.0000) with ORF2 ELISA. ORF2, T1NE, Wantai, and MPD ELISAs agreed on results for 88% of sera tested. Two percent sera showed reactivity in each combination of three and two of aforementioned four ELISAs. Remaining 8% sera were single ELISA reactive. PPA and NPA value ranges were 76.3-99.0% and 84.8-99.5%, respectively. Pairwise concordances between all the eight ELISAs ranged from 88.0 to 100% (κ: 0.74-1.00). Both the in-house ELISAs agreed better with Wantai over MPD ELISA. In conclusion, both ORF2 and T1NE ELISAs were equally efficient in diagnosing HEV infections. T1NEp proved to be an excellent tool in HE sero-diagnosis and is worth exploring in development of simple rapid tests. KEY POINTS: • In-house ELISA based on bacterially expressed neutralizing epitope/s region protein • In-house ELISA based on complete ORF2 protein expressed in insect cells • Comparison of two in-house and six commercial anti-HEV IgM antibody detection ELISAs.

Generation of a Large Repertoire of Monoclonal Antibodies against Dengue Virus NS1 Antigen and the Development of a Novel NS1 Detection Enzyme-Linked Immunosorbent Assay.

Commercial dengue virus (DENV) nonstructural-1 (NS1) Ag detection immunoassays often perform poorly, particularly in secondary DENV infection. To develop a highly sensitive NS1 ELISA, we generated a large repertoire of anti-DENV NS1 mouse mAbs (n = 95) that falls into 36 mAb classes based on binding specificities. The identified mAb pair, capable of efficiently detecting NS1 from four DENV serotypes in an immunoassay, was selected based on multiparametric analysis. The selected mAbs have subnanomolar affinities for NS1 with recognition sites outside the immunodominant wing domain. The assay was converted to an ELISA kit, which showed higher analytical sensitivity (3-fold to 83-fold) for NS1 from four DENV serotypes than commercial Platelia NS1 ELISA (Bio-Rad Laboratories). Compared to RT-PCR, the developed NS1 ELISA showed 78.57% (66 of 84) sensitivity, whereas Platelia NS1 ELISA showed a sensitivity of 60.71% (51 of 84). In a subgroup of RT-PCR-positive secondary dengue samples, our ELISA showed a sensitivity of 70.18% (40 of 57), whereas Platelia ELISA detected only 47.37% (27 of 57) samples. Furthermore, unlike Platelia ELISA, our test equally detected NS1 from four serotypes; Platelia ELISA performed poorly for the DENV-2 serotype, in which only 8 of 21 (38.10%) samples were detected compared with 17 of 21 (80.95%) in our ELISA. Moreover, our ELISA showed 100% specificity in 342 challenging dengue-negative samples. The large and diverse mAb repertoire generated against DENV NS1 and the appropriate selection of mAbs allowed us to establish an ELISA that can efficiently detect NS1 Ag even in secondary dengue and without serotype level bias.

Preparation of Monoclonal Antibodies against the Viral p54 Protein and a Blocking ELISA for Detection of the Antibody against African Swine Fever Virus.

African swine fever virus (ASFV) causes a highly contagious viral disease in domestic and wild pigs, leading to serious economic losses. As there are no vaccines or drugs available, early accurate diagnosis and eradiation of infected animals are the most important measures for ASFV prevention and control. Therefore, improvement of available diagnostic assays and development of novel effective techniques are required. This study is devoted to generating a new detection platform of blocking monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) against ASFV p54 protein. Seven monoclonal antibodies against recombinant p54 protein were produced and four epitopes were identified. Three blocking ELISAs were developed with 6A5 and 6F9 mAbs labeled with HRP, respectively, of which the 6A5/6F9-based blocking ELISA displayed the best detection performance, with an AUC of 0.986, sensitivity of 98.36% and specificity of 92.36% in ROC analysis. Moreover, it has an excellent agreement at 96.59% (198/205) when compared to the commercial blocking ELISA (kappa value = 0.920). The method also has high repeatability, with CV <10%, and no cross reaction with the serum antibodies against PRV, PRRSV, CSFV, PCV2 or SVA. This indicates that the 6A5/6F9-based blocking ELISA has high accuracy with good sensitivity and specificity, suitable for viral detection, field surveillance and epidemiological studies.