Enzyme-linked Immunosorbent Assay For Detection Research Articles

WITHDRAWN: Development of a double-antigen sandwich ELISA for African swine fever virus antibody detection based on K205R protein

African swine fever (ASF) is a highly fatal infectious disease that affects swine caused by the African swine fever virus (ASFV) and poses a significant threat to the swine industry, and its effective diagnosis is crucial due to the current lack of commercially available vaccine for disease prevention and control. In this regard, the K205R protein is a suitable diagnostic antigen for enzyme-linked immunosorbent assay (ELISA) owing to its continuous expression throughout all ASFV replication stages, high immunogenicity, and sequence conservation. The double-antigen sandwich ELISA (DAgS-ELISA) is capable of detecting all immunoglobulins present in the serum, and outperforms alternative methods by enabling earlier antibody detection. Therefore, in this study, the K205R protein was prokaryotically expressed, and the K205R protein digested His-tag was labeled with horseradish peroxidase (HRP) and named K205R-HRP. The DAgS-ELISA was conducted using a combination of digested His-tag proteins and K205R-HRP. This method exhibits exceptional properties, including high sensitivity and no cross-reactivity with other porcine antibodies, with both the intra- and inter-assay coefficients of variation (CV) below 10 %. Antibody kinetics experiments have demonstrated that this method is capable of detecting antibodies as early as 7 to 9 days post-infection, which is a significant improvement compared to the commercial kit that can only detect antibodies from 11 to 13 days post-infection. In 120 clinical serum tests, the Kappa value between the DAgS-ELISA and the commercial kit was 0.96 (almost perfect). Owing to its high sensitivity, impressive repeatability, and strong specificity, this method enables earlier antibody detection and is suitable for ASFV antibody testing.

CD30 Lateral Flow and Enzyme-Linked Immunosorbent Assays for Detection of BIA-ALCL: A Pilot Study

Introduction: Breast Implant-Associated Anaplastic Large Cell Lymphoma (BIA-ALCL) commonly presents as a peri-implant effusion (seroma). CD30 (TNFRSF8) is a consistent marker of tumor cells but also can be expressed by activated lymphocytes in benign seromas. Diagnosis of BIA-ALCL currently includes cytology and detection of CD30 by immunohistochemistry or flow cytometry, but these studies require specialized equipment and pathologists’ interpretation. We hypothesized that a CD30 lateral flow assay (LFA) could provide a less costly rapid test for soluble CD30 that eventually could be used by non-specialized personnel for point-of-care diagnosis of BIA-ALCL. Methods: We performed LFA for CD30 and enzyme-linked immunosorbent assay (ELISA) for 15 patients with pathologically confirmed BIA-ALCL and 10 patients with benign seromas. To determine the dynamic range of CD30 detection by LFA, we added recombinant CD30 protein to universal buffer at seven different concentrations ranging from 125 pg/mL to 10,000 pg/mL. We then performed LFA for CD30 on cryopreserved seromas of 10 patients with pathologically confirmed BIA-ALCL and 10 patients with benign seromas. Results: Recombinant CD30 protein added to universal buffer produced a distinct test line at concentrations higher than 1000 pg/mL and faint test lines at 250–500 pg/mL. LFA produced a positive test line for all BIA-ALCL seromas undiluted and for 8 of 10 malignant seromas at 1:10 dilution, whereas 3 of 10 benign seromas were positive undiluted but all were negative at 1:10 dilution. Undiluted CD30 LFA had a sensitivity of 100.00%, specificity of 70.00%, positive predictive value of 76.92%, and negative predictive value of 100.00% for BIA-ALCL. When specimens were diluted 1:10, sensitivity was reduced to 80.00% but specificity and positive predictive values increased to 100.00%, while negative predictive value was reduced to 88.33%. When measured by ELISA, CD30 was below 1200 pg/mL in each of six benign seromas, whereas seven BIA-ALCL seromas contained CD30 levels > 2300 pg/mL, in all but one case calculated from dilutions of 1:10 or 1:50. Conclusions: BIA-ALCL seromas can be distinguished from benign seromas by CD30 ELISA and LFA, but LFA requires less time (<20 min) and can be performed without special equipment by non-specialized personnel, suggesting future point-of-care testing for BIA-ALCL may be feasible.

Generation and characterization of a monoclonal antibody against an African swine fever virus protein encoded by the A137R gene

The ongoing African swine fever (ASF) pandemic continues to have a major impact on global pork production and trade. Since ASF cannot be distinguished from other swine hemorrhagic fevers clinically, ASF-specific laboratory diagnosis is critical. Thus ASF virus (ASFV)-specific monoclonal antibodies (mAbs) are critical for the development of laboratory diagnostics. In this study, we report one ASFV-specific mAb, F88ASF-55, that was generated and characterized. This mAb recognizes the ASFV A137R-encoded protein (pA137R). Epitope mapping results revealed a highly conserved linear epitope recognized by this mAb, corresponding to amino acids 111–125 of pA137R. We explored the potential use of this mAb in diagnostic applications. Using F88ASF-55 as the detection antibody, six ASFV strains were detected in an enzyme-linked immunosorbent assay (ELISA) with low background. In immunohistochemistry (IHC) assays, this mAb specifically recognized ASFV antigens in the submandibular lymph nodes of animals experimentally infected with different ASFV strains. Although not all ASFV genotypes were tested in this study, based on the conserved ASFV epitope targeted by F88ASF-55, it has the potential to detect multiple ASFV genotypes. In conclusion, this newly generated ASFV pA137R-specific mAb has potential value in ASF diagnostic tool development. It can be used in ELISA, IHC, and possibly-immunochromatographic strip assays for ASFV detection. It also suggests that pA137R may be a good target for diagnostic assays to detect ASFV infection.

Ferroptosis Inhibitor Regulates the Disease Progression of Systematic Lupus Erythematosus Mice Model Through Th1/Th2 Ratio.

Systematic lupus erythematosus (SLE) is an autoimmunemediated disease. So far, there is no relevant report on ferroptosis in SLE research, and the role of T helper 1 (Th1) and T helper 2 (Th2) cells in SLE is still unclear. This study employed SLE mice models with and without ferroptosis inhibitors (Liproxstatin‑1) and normal control mice. Treated mice were analyzed with hematoxylin and eosin (H&E) staining, immunohistochemical detection of glutathione peroxidase 4 (GPX4), malondialdehyde (MDA) detection, ELISA(enzyme-linked immunosorbent assay) detection of Th1 and Th2 cytokines and flow cytometry detection of Th1 and Th2 ratio. The results showed that compared with the normal group, the SLE group exhibited significantly higher expression of anti-double-stranded deoxyribonucleic acid (anti-dsDNA), MDA and Th1 cytokines, significantly lower expression of GPX4 and Th2 cytokines and increased Th1/Th2 ratio. Similarly, compared with the SLE group, the SLE + liproxstatin-1 group showed significantly low expression of anti-dsDNA, MDA and Th1 cytokines, significantly high expression of GPX4 and Th2 cytokines and reduced Th1/Th2 ratio. These results demonstrate that ferroptosis may be involved in promoting SLE development. Therefore, inhibiting ferroptosis may be a potential treatment for SLE. Similarly, the Th1/Th2 ratio may have a role in promoting SLE development.

Development and diagnostic evaluation of indirect enzyme linked immunosorbent assay for Epidermophyton floccosum infection in humans

BackgroundEpidermophyton floccosum is an anthrophophilic dermatophyte widely distributed in the tropics and subtropics. It can invade keratinized tissues of humans and cause superficial mycoses called dermatophytosis (ringworm). ObjectiveThe main objective of this study was to develop an in-house indirect enzyme-linked immunosorbent assay (ELISA) and to evaluate its performance for the immunological diagnosis of E. floccosum infection in humans. MethodsAn in-house indirect ELISA was developed using partially purified E. floccosum antigens, pre immunized rabbit serum as negative control, immunized rabbit polyclonal antibodies as positive control, enzyme labeled goat anti rabbit antibodies and goat anti human antibodies. A total of 50 serum samples from E. floccosum infected patients as confirmed by direct microscopy and culture and 30 samples from humans without history of dermatophyte infection that served as controls were used to evaluate the performance of an in-house indirect ELISA developed in this study. Analytical and diagnostic performance characteristics were determined to evaluate its diagnostic value. ResultsThe diagnostic sensitivity, specificity, positive and negative predictive values of E. floccosum indirect ELISA were 90.00 %, 83.33 %, 90.83 %, 83.83 % respectively. The performance of indirect ELISA assay was compared with gold standard diagnostic tests such as KOH hydrolysis test and fungal culture. The correlation coefficients of E. floccosum indirect ELISA with KOH hydrolysis and fungal culture method were 0.612** and 0.826** (P < 0.01) respectively indicating significant correlation between these tests. ConclusionThis revealed the great potentiality of E. floccosum indirect ELISA in early, specific and precise detection of E. floccosum infection in humans.

Development of an indirect ELISA for detection of the adaptive immune response of black carp (Mylopharyngodon piceus)

Black carp (Mylopharyngodon piceus) is an important fishery resource and the main breeding target in China. Due to the lack of an assay of immunoglobulin M (IgM) antibodies in black carp, there is no effective method to evaluate adaptive immune response, which limits immunological studies and vaccine development. The present study used mAbs (monoclonal antibodies) against serum IgM of grass carp as capture antibodies. The results of Western blot analysis indicated that these antibodies had strong affinity and specificity to IgM heavy chain in black carp serum and were used to detect the antibody titer, optimize the conditions, perform a sensitivity test, and develop an indirect ELISA (enzyme-linked immunosorbent assay) to detect specific IgM antibodies in the serum. This detection method has good specificity and is effective only for grass carp (Ctenopharyngodon idella) and black carp and not for crucian carp (Carassius aumtus), silver carp (Hypophthalmichthys molitrix), bighead carp (Hypophthalmichthys nobilis), mandarin fish (Siniperca chuatsi), black bream (Megalobrama skolkovii), or yellow catfish (Pseudobagrus fulvidraco). The lowest antigen detection level was 0.05 μg/ml. The error of experimental repetition in the same sample was 1.61–4.61%. The levels of specific IgM in black carp serum were steadily increased after immunization, peaked on day 28, and then slowly decreased. Indirect ELISA can be applied to detect the changes in specific antibodies in black carp serum. Moreover, indirect ELISA provides a convenient and reliable serological detection method for immunological research and evaluation of immune effects of a vaccine in black carp.

Synergistic effects of gold nanoparticles enhanced the sensitivity of enzyme-linked immunosorbent assay for latex allergens

Hevea brasiliensis rubber elongation factor (Hev b1), a non-glycosylated protein, is a notable allergen in natural latex products. This study aims to assess enzyme-linked immunosorbent assay (ELISA) combined with gold nanoparticles (AuNPs) as a Hev b1 detection substrate. AuNP synthesis precursor and reducing agent concentrations were optimized using factorial design and multiple linear regression (MLR). The optimized conditions yielded spherical AuNPs with an average particle size of 73 ± 23 nm. This substrate enhanced detection sensitivity by around 50% compared to the limit of detection of the bare substrate, in the linear range of 0.25–1.75 μg/mL. Additionally, the heightened absorbance intensity observed during Hev b1 ELISA signifies both augmented surface immobilization of the analyte and optical induction arising from the aggregation of suitably small AuNPs (<80 nm) into dimer clusters, supported by discrete dipole approximation (DDA). Overall, the findings highlighted the synergistic effects of AuNPs in enhancing the performance of ELISA for Hev b1 detection.

One-pot synthesis of AuPt@FexOy nanoparticles with excellent peroxidase-like activity for development of ultrasensitive colorimetric lateral flow immunoassay of cardiac troponin I

Detection of cardiac troponin I (cTnI) plays a critical role in diagnosing acute myocardial infarction (AMI). In this report, a new kind of spherical AuPt@FexOy core@shell nanoparticles (termed as AuPt@FexOy NPs) were one-pot synthesized by a redox interaction-engaged strategy (RIES) without the addition of any surfactants or reducing agents. The as-synthesized AuPt@FexOy NPs not only retain the plasmonic activity of gold nanoparticles (AuNPs), but also possess excellent catalytic activities of platinum nanoparticles (PtNPs) and FexOy nanoclusters. The features of AuPt@FexOy NPs enable greatly enhance the colorimetric detection sensitivity of lateral flow immunoassay (LFIA) through integrating AuPt@FexOy NPs labeling procedure and catalyzing oxidation of chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) signal amplification strategy. The as-developed colorimetric LFIA (termed as AuPt@FexOy-LFIA) exhibits the limit of detection (LOD) as 26.0 pg mL−1 cTnI under the TMB signal amplification mode. In particular, the detection results of cTnI in 40 clinical seral samples by AuPt@FexOy-LFIA are correlated well with those of cTnI in the same samples by commercial enzyme-linked immunosorbent assay (ELISA) detection kit (R2 = 0.97, slope = 1), demonstrating the highly reliable analytical performance and good application prospect of AuPt@FexOy-LFIA.

A high-sensitivity ELISA for detection of human FGF18 in culture supernatants from tumor cell lines

Fibroblast growth factor 18 (FGF18) is elevated in several human cancers, such as gastrointestinal and ovarian cancers, and stimulates the proliferation of tumor cells. This suggests that FGF18 may be a promising candidate biomarker in cancer patients. However, the lack of a high-sensitivity enzyme-linked immunosorbent assay (ELISA) does not permit testing of this possibility. In this study, we generated monoclonal antibodies against human FGF18 and developed a high-sensitivity ELISA to measure human FGF18 at concentrations as low as 10 pg/mL. Of the eight tumor cell lines investigated, we detected human FGF18 in culture supernatants from four tumor cell lines, including HeLa, OVCAR-3, BxPC-3, and SW620 cells, albeit the production levels were relatively low in the latter two cell lines. Moreover, the in-house ELISA could detect murine FGF18 in sera from mice overexpressing murine Fgf18 in hepatocytes, although the sensitivity in detecting murine FGF18 was relatively low. This FGF18 ELISA could be a valuable tool to validate FGF18 as a potential biomarker for cancer patients and to test the contribution of FGF18 for various disease models invivo and in vitro.

Hydrogels loaded with atenolol drug metal–organic framework showing biological activity

Abstract Using the starting reactants of Zn(OAc)2 and 2-mercaptonicotinic acid (H2L), we successfully synthesized a fresh Zn(ii) compound, that is, [Zn3(L)3] n (1), under hydrothermal conditions and then successfully loaded with atenolol. Hualuronic acid/carboxymethyl chitosan hydrogels loaded with atenolol drug metal–organic framework were prepared based on a chemical synthesis method. The microscopic morphology of the hydrogels was investigated, and scanning electron microscopic results showed that the hydrogels had a highly porous morphology with good penetration between the pores. For the new hydrogels, the enzyme-linked immunosorbent assay detection was conducted, and the production of sVCAM-1 and TNF-a in vascular endothelial cells was determined. Besides, the levels of inflammatory response in the vascular endothelial cells were also determined with the real-time reverse transcriptase polymerase chain reaction assay.

Histopathology and antibody responses describe the seasonal pattern of dicrocoeliosis in small ruminants in the Himalayan ranges of Pakistan

In some parts of the world, Dicrocoelium spp. lancet flukes cause significant production loss in pastoral livestock, and accurate diagnosis of infection is important. The aims of the present study were to describe the histopathology and to investigate the transmission patterns of Dicrocoelium amongst ten sheep and goat farms in Khyber Pakhtunkhwa and Gilgit Baltistan, Pakistan. The liver histology and indirect enzyme-linked immunosorbent assay (ELISA) analyses followed standard procedures. The liver histopathology showed intensive tissue destruction and biliary hyperplasia associated with presence of adult flukes, severe inflammatory cell infiltration, congestion of blood vessels, damaged hepatocytes, and sinusoids in the infected areas. The time of onset of infection was investigated by ELISA detection of antibodies in sheep (n = 164) and goats (n = 152). Colostral transfer of Dicrocoelium antibodies from seropositive mothers was detected in sheep and goats up to 16 weeks of age. In both sheep and goats, the estimated time of infection differed between farms and years. Infection was seen in both sheep flocks and goat herds, with high variation between flocks and herds, and the highest infection rate in lambs. Dicrocoelium infection was most prevalent in sheep and goats in September (n = 84) and August (n = 63) respectively. This study concluded Dicrocoelium causes severe inflammation and necrosis of liver tissues in sheep and goats. Colostral transfer of antibodies can be detected up to about ten weeks of age. Higher infection rates are observed during August and September in sheep than in goats, putatively due to effects of different grazing and browsing behaviors on the ingestion of ants. The results will aid in the development of effective disease control strategies to ensure optimal growth and productivity of sheep and goats.

Differentiation of Salmonella vaccinated and infected animals by serological detection of antibody to T3SS effector SsaK in an indirect ELISA

The differentiation of animals that are vaccinated and those that are naturally infected with Salmonella is difficult by conventional serological tests. We have shown here an indirect Enzyme-linked immunosorbent assay for detection of Salmonella infection based on the presence of a Type III secretory effector SsaK in the sera.

Sandwich Enzyme-linked Immunosorbent Assay (ELISA) to Quantify Monoclonal Antibody (B[a]P-13) for Herbal Medicine Products

Abstract: Sandwich enzyme-linked immunosorbent assay (ELISA) to quantify monoclonal antibody (B[a]P-13) Background: Only a few studies have focused on the analysis using specific antibodies in the sandwich ELISA method to each B[a]P in herbal medicine products. In contrast to the sandwich ELISA method, many competitive ELISA methods using specific antibodies such as benzo[a]pyrene monoclonal antibody (B[a]P-13) and a goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, horseradish peroxidase (HRP) were developed. Introduction: The objective of this study was to develop and validate the method for the response of the benzo[a]pyrene monoclonal antibody (B[a]P-13) and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody (HRP) to prepare the immunogen and its application to detect the benzo[a]pyrene in various herbal medicine products. Methods: This research method includes preparation of B[a]P-protein conjugates, sampling and extraction procedure for herbal medicines, sandwich ELISA procedure, evaluation of cross-reactivity for determination, matrix effect of the organic solvents, correlation of benzo[a]pyrene detection ELISA compared to HPLC-FLD in herbal medicine products. Results: The sandwich ELISA method for B[a]P was validated in linearity (R2 &gt; 0.99), the limit of detection (LOD) (0.080.19 μg/kg) and limit of quantification (LOQ) (0.240.57 μg/kg), accuracy (95.58117.06 %), and precision (3.8010.26 %). The cross-reactivity (CR) was found for B[a]P (100%), CHR (39%), B[b]F (27%), and B[a]A (41%). As a solvent, acetonitrile (MeCN) was used to express the normalized sandwich ELISA calibration curves with benzo[a]pyrene monoclonal antibody (B[a]P-13). The antigen-antibody binding in sandwich ELISA was decreased about 10 times with increasing the salt content (0.0060.18 mol/L phosphate to 20400 mmol/L). The pH range from 6 to 9 was not considered to affect the performance of the sandwich ELISA. Correlation of B[a]P detection in herbal medicines with ELISA compared to HPLC-FLD expressed good correlation (R2 = 0.991) and the slope of the graph for the ELISA (B[a]P-equivalents μg/kg) value divided by the HPLC-FLD (B[a]P μg/kg) value was 0.7292. Conclusion: Therefore, sandwich ELISA method using benzo[a]pyrene monoclonal antibody (B[a]P-13) could be an alternative screening method for detection of B[a]P in herbal medicine products.

Evaluation of Five Non-Culture-Based Methods for the Diagnosis of Meningeal Sporotrichosis

Sporotrichosis is the main subcutaneous mycosis worldwide. Several complications, including meningeal forms, can be observed in immunocompromised individuals. The sporotrichosis diagnosis is time-consuming due to the culture's limitations. The low fungal burden in cerebrospinal fluid (CSF) samples is another important drawback in the diagnosis of meningeal sporotrichosis. Molecular and immunological tests can improve the detection of Sporothrix spp. in clinical specimens. Therefore, the following five non-culture-based methods were evaluated for the detection of Sporothrix spp. in 30 CSF samples: (i) species-specific polymerase chain reaction (PCR); (ii) nested PCR; (iii) quantitative PCR; (iv) enzyme-linked immunosorbent assay (ELISA) for IgG detection; and (v) ELISA for IgM detection. The species-specific PCR was unsuccessful in the diagnosis of the meningeal sporotrichosis. The other four methods presented substantial levels of sensitivity (78.6% to 92.9%) and specificity (75% to 100%) for the indirect detection of Sporothrix spp. Both DNA-based methods presented similar accuracy (84.6%). Both ELISA methods were concomitantly positive only for patients with sporotrichosis and clinical signs of meningitis. We suggest that these methods should be implemented in clinical practice to detect Sporothrix spp. in CSF early, which may optimize treatment, augment the chances of a cure, and improve the prognosis of affected individuals.

Estrogen-Like Effect of Bazi Bushen Capsule in Ovariectomized Rats

This study aims to show the estrogen-like effect of Bazi Bushen capsule (BZBS), a Chinese herbal compound, in ovariectomized mice. Female Sprague-Dawley (SD) rats were randomly divided into six groups: a sham-operated group, a model group (OVX), a progynova group, and BZBS groups (1, 2, and 4 d/kg/d). An ovariectomy was performed on all rats except those in the sham-operated group. Micro-computed tomography (micro-CT) scanning, hematoxylin and eosin (H&E) staining, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) detection were performed after 4 months of BZBS treatment. As a result, compared with the OVX group, rats treated with BZBS showed an increased number and area of trabecular bone and bone marrow cells, and a decreased number of adipose cells. The bone volume, trabecular number, and trabecular thickness of the right tibia in the medication groups increased and the trabecular space decreased. The 17β-estradiol and serum calcium levels in the medication groups were elevated, but the levels of serum phosphorus, sclerostin, β-CTX, and TRACP-5b were decreased. In the medication groups, the RANKL and sclerostin levels were decreased, while the osteoprotegerin (OPG) level was increased. In conclusion, this protocol systematically evaluated the therapeutic effects and potential molecular mechanisms of Chinese herbal compounds in ovariectomized rats with a variety of techniques.

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.