Enzyme-linked immunosorbent assay (ELISA) is an immunological assay widely used in basic science research, clinical application studies, and diagnostics. The ELISA technique relies on the interaction ...between the antigen (i.e., the target protein) versus the primary antibody against the antigen of interest. The presence of the antigen is confirmed through the enzyme-linked antibody catalysis of the added substrate, the products of which are either qualitatively detected by visual inspection or quantitatively using readouts from either a luminometer or a spectrophotometer. ELISA techniques are broadly classified into direct, indirect, sandwich, and competitive ELISA-all of which vary based on the antigens, antibodies, substrates, and experimental conditions. Direct ELISA relies on the binding of the enzyme-conjugated primary antibodies to the antigen-coated plates. Indirect ELISA introduces enzyme-linked secondary antibodies specific to the primary antibodies bound to the antigen-coated plates. Competitive ELISA involves a competition between the sample antigen and the plate-coated antigen for the primary antibody, followed by the binding of enzyme-linked secondary antibodies. Sandwich ELISA technique includes a sample antigen introduced to the antibody-precoated plate, followed by sequential binding of detection and enzyme-linked secondary antibodies to the recognition sites on the antigen. This review describes ELISA methodology, the types of ELISA, their advantages and disadvantages, and a listing of some multifaceted applications both in clinical and research settings, including screening for drug use, pregnancy testing, diagnosing disease, detecting biomarkers, blood typing, and detecting SARS-CoV-2 that causes coronavirus disease 2019 (COVID-19).
Koi herpesvirus disease (KHVD) caused by the koi herpesvirus (KHV) is difficult to diagnose in live fish, presenting a challenge to the koi industry. The enzyme‐linked immunosorbent assay (ELISA) ...method cannot be widely used to detect KHV because few commercial anti‐KHV antibody exists. Here, we developed an anti‐ORF132 polyclonal antibody and confirmed its reactivity via indirect immunofluorescence assay and Western blotting. A double‐antibody sandwich ELISA (DAS‐ELISA) was established to detect KHV, monoclonal antibody 1B71B4 against ORF92 was used as the capture antibody, and the detection antibody was the polyclonal antibody against the truncated ORF132. The lowest limit was 1.56 ng/ml KHV. Furthermore, the DAS‐ELISA reacted with KHV isolates, while no cross‐reactions occurred with carp oedema virus, spring viraemia of carp virus, frog virus 3 and grass carp reovirus. Two hundred koi serum samples from Guangdong, China, were used in the DAS‐ELISA test, and the positive rate of the koi sera was 13%. The clinical sensitivity and specificity of the DAS‐ELISA relative to the traditional PCR method were 66.7% and 97.6%, respectively. Our findings may be useful for diagnosing and preventing KHVD in koi and common carp.
Advanced glycation end-products (AGEs) can aggregate amid incessant inflammation, as may be available in patients with rheumatoid arthritis. d-Ribose reacts more promptly than glucose monosaccharide ...to the proteins and forms heterogeneous group of products known as AGEs. Obesity includes persons with provocative joint inflammation with increased lipid profile. Immunogenic evidences recommend a cross-sectional relationship between glycated LDL-Apo B100 and inflammation.
The point of this examination was to look at the connection between d-ribose glycated ApoB100 (ApoB100-AGE) with obesity and rheumatoid arthritis. The binding specificity of auto-antibodies against ApoB100-AGE antigen present in obesity and rheumatoid arthritis patient's serum were inspected by direct binding and was further established by competitive inhibition ELISA. In the present study, hydroxyl radical, superoxide radical, ketoamine moieties, hydroxyl-methyl furfural (HMF) and carbonyl substances were evaluated in the patients' serum via respective specific methods. The prevalence of auto-antibodies against ApoB100-AGE antigen was recorded to be 58% and 52.86% from obese and rheumatoid arthritis patient respectively in contrast to its native analogue (P < 0.001). Moreover, the autoantibodies present in obese and arthritis patients were found to be highly specific towards ApoB100-AGE as confirmed by inhibition ELISA.
•The keto-amine moieties, HMF and carbonyl substance were higher in obese and Arthritic patient sera.•High degree of specificity in 80% obese and 73% rheumatoid sera against Apo-B100-AGE-IgG was obtained.•In obese and Rheumatoid sera, inhibition with glycated ApB100 was 87% and 64% respectively.
CD226 is an important receptor constitutively expressed on most immune cells, performing vital functions in immune responses. However, the levels of soluble CD226 (sCD226) and its roles in primary ...Sjögren syndrome (pSS) remain unclear. In this study, we developed two novel mouse anti-human CD226 monoclonal antibodies (mAbs) and established a novel sandwich enzyme-linked immunosorbent assay (ELISA) system, which proved to be highly effective in detecting human sCD226. We then analyzed the expression of sCD226 in the plasma of pSS patients. Our results showed that the levels of sCD226 were significantly lower in patients with pSS compared to healthy controls. The significant decline was also observed in active group and the patients with high levels of IgG or positive anti-SSB. Additionally, reduced sCD226 was found to be negatively correlated with the disease activity of pSS and several clinical manifestations, including arthralgia, fatigue, decayed tooth and interstitial lung disease (ILD). Furthermore, receiver operator characteristics (ROC) curve analysis showed that sCD226 displayed outstanding capacity in discriminating pSS and predicting the disease activity. Altogether, plasma sCD226 emerges as a promising candidate for diagnostic markers in the context of pSS.
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•We successfully developed an innovative sandwich ELISA system for detection of human sCD226.•Reduced plasma sCD226 levels are associated with with disease activity in pSS.•Soluble CD226 emerges as a promising candidate for therapeutic targeting in the context of pSS.
This study focuses on the development and initial assessment of an indirect IgG enzyme-linked immunosorbent assay (ELISA) specifically designed to detect of anti-SARS-CoV-2 antibodies. The unique ...aspect of this ELISA method lies in its utilization of a recombinant nucleocapsid (N) antigen, produced through baculovirus expression in insect cells. Our analysis involved 292 RT-qPCR confirmed positive serum samples and 54 pre-pandemic healthy controls. The process encompassed cloning, expression, and purification of the SARS-CoV-2 N gene in insect cells, with the resulted purified protein employed in our ELISA tests. Statistical analysis yielded an Area Under the Curve of 0.979, and the optimized cut-off exhibited 92 % sensitivity and 94 % specificity. These results highlight the ELISA's potential for robust and reliable serological detection of SARS-CoV-2 antibodies. Further assessments, including a larger panel size, reproducibility tests, and application in diverse populations, could enhance its utility as a valuable biotechnological solution for diseases surveillance.
An Alexandrium affine strain (AAJQ-1) from San José Island, Gulf of California was characterized for growth and toxicology. Fivefold of f/2 + Se cultures were incubated for 34 days in a temperature ...gradient (21–29 °C). Aliquots were collected every third day for cell counting, toxin determination, and nutrient analyses. In this study ELISA method was used to evaluate the PSP toxin production due to the lower detection limit than the HPLC method. The highest cell density (6724 cells mL−1) and growth rate (0.22 day−1) were obtained at 27 °C and they were related to temperature in all treatments. Cell density showed negative correlation with nitrate at temperatures ≥23 °C, and with orthophosphate 27 °C, furthermore, these correlations promote the toxin production (0.05–0.45 fmol STX cell−1); beyond that nitrite at high temperature seems to promote toxin production, which has not been sufficiently documented.
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•Low growth rates for an A. affine strain isolated from the Mexican Pacific Ocean.•Highest population growth was promoted at warm temperatures (≥25 °C).•Very low ELISA PSP toxin detection from 0.05 to 0.45 fmol PSP cell−1.•Maximum PSP toxin production occurs in the stationary phase at 21 °C.•Negative correlation between nitrate and temperatures above 23 °C.
Rapid and accurate detection of bacterial pathogens is critical in controlling disease outbreaks affecting farmed fish. The present study aimed to develop a novel serological diagnostic approach ...using nano‑silver based Enzyme-linked immunosorbent assay (ELISA) for speedy detection of Aeromonas veronii infections in Nile tilapia. A. veronii isolates used in ELISA assays were recovered from moribund Nile tilapia during a disease outbreak in a private fish farm in Egypt. A. veronii isolates were identified based on alignment analysis of the gyrB and 16S rRNA gene sequences. A. veronii antisera used in ELISA assays were prepared in tilapia, and the bacterial antigens were formalin-killed. The cut-off values were 0.46 and 0.48 in traditional and nano-based ELISA. There were no cross-reactions with bacterial isolates (Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria, Pseudomonas fluorescens, and Vibrio vulnificus). The lowest antigen concentration that produced positive results after checkerboard titration in indirect-ELISA (i-ELISA) and dot ELISA was 15 μg and 250 ng of prepared antigen, respectively. Nano-ELISA and nano-based dot-ELISA antigen concentration was 10 μg and 100 ng, respectively. Sera concentration was 1:100 in indirect-ELISA and dot-ELISA, while it was 1:50 in nano-based ELISA and nano dot-ELISA. The i-ELISA successfully detected anti-Aeromonas IgG antibodies with 83.33% sensitivity and 66.67% specificity, while in the dot-ELISA, the sensitivity and specificity were 83.33% and 100%, respectively. Nano dot-ELISA had 100% sensitivity, specificity, and accuracy. Nano dot-ELISA assays have higher specificity, sensitivity, and accuracy than traditional ELISAs in detecting A. veronii. Further studies are needed to develop a rapid test kit for on-site field diagnosis.
•In nano -ELISA the concentration of antigen was 10 μg while in nano-based Dot- ELISA was 100 ng.•The concentration of sera was 1:100 in i -ELISA and Dot ELISA while in nano-based ELISA and nano Dot ELISA 1:50 dilution of the sera.•The results of i-ELISA could detect anti-Aeromonas IgG antibodies with 83.33% sensitivity and 66.67% specificity; while in Dot-ELISA the sensitivity and specificity were 83.33% and 100% respectively. While the accuracy was 83.33% in i-ELISA and Dot- ELISA.
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•Low density lipoprotein modified by crotonaldehyde shows loss of α-helix and gain in β content.•Oxidation of low density lipoprotein generates non fibrillar and amorphous ...aggregates.•Oxidized low density lipoprotein is highly immunogenic and generates specific immune response in immunized rabbits.•Immunological studies confirm the presence of circulating antibodies in cancer patients.
Crotonaldehyde (CA), a prominent component of cigarette smoke (CS) is a pervasive environmental pollutant that is a highly toxic, unsaturated aldehyde. Exposure to CA-rich pollutants has been linked to the emergence of many malignancies in humans. To better understand the role of CA in biomolecule modification, this study investigated the detailed structural alterations in low-density lipoprotein (LDL) modified by CA, as well as the immunogenicity of the modified protein in experimental animals and the search for autoantibodies in various cancers patients.In vitro, results indicated alterations in secondary and tertiary structures; examined using UV–visible, fluorescence, far-UV circular dichroism, and Fourier transform infrared spectroscopy techniques. Changes in the oxidation status of LDL were studied by carbonyl content assay and NBT assay. ThT binding assay, scanning, and transmission electron microscopy were used to study aggregate formation. The findings revealed significant structural damage in LDL modified by CA. The modification resulted in the unmasking of hydrophobic clusters, the loss of the protein α-helix, and the formation of β-pleated sheet structure. The amyloid aggregate formation was confirmed through ThT microscopy and electron spectroscopy. Rabbits immunized with crotonaldehyde; lead to structural changes in the LDL; that acted as extra antigenic determinants, eliciting strong antibody response. Immunoglobulin response is highly specific for modified LDL as demonstrated by the ELISA. The presence of antibodies against CA-modified LDL was confirmed by the immunoglobulin content of blood sera from human subjects with lung cancer, and competitive ELISA demonstrated the specificity of these antibodies. This study offers insights into the CA-mediated LDL modification and immunogenicity in lung cancer that will have diagnostic importance.
Low specificity and sensitivity often restrict the fluoroquinolones (FQs) detection when traditional IgG antibodies are used as recognition elements in immunoassay. Herein, shark-derived ...single-domain antibodies (ssdAbs) were used as new recognition elements to enhance the sensitivity and specificity for norfloxacin detection in immunoassay. Firstly, NOR-specific clones were isolated by phage display technology. Finally, five clones were successfully isolated from a NOR-immunized library. Secondly, a soluble expression system was constructed to produce ssdAbs. The 1N9 ssdAb exhibits the best affinity to the NOR complete antigen, with a limit of detection of 0.392 ng/mL and the cross-reaction rates of less than 8.240 %, indicating its high sensitivity and specificity as a new recognition element in NOR detection. More importantly, the ELISA result proved that in NOR-negative fish matrix, no serious interference was observed, the 1N9 ssdAb could still recognize complete antigen NOR-OVA effectively. While in icELISA, 1N9 ssdAb could recognize NOR in different NOR-spiked concentrations between 10 and 1000 ng/mL, with the recovery rates between 93.66 % and 116.49 % with high reproducibility. Our study provides a new and effective recognition element with great potential for NOR detection.
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•29 NOR-specific clones were isolated and identified from a NOR-immunized library.•1N9 ssdAb showed a high sensitivity to NOR, with the IC10 value of 0.392 ng/mL.•1N9 ssdAb showed rare recognitions to other fluoroquinolones.•1N9 ssdAb performed excellently in NOR-negative/spike fish matrix.
Type 1 diabetes (T1DM) is a common chronic disease in childhood. Increasing insulin resistance in puberty gives rise to higher doses of insulin usage in treatment. Of this reason new approaches in ...treatment are needed. Noopept researches suggest it to have anti-diabetic properties. We tried to determine the effects of noopept on pubertal diabetes.
The research was made with 60 prepubertal, 28 day-old, male, Sprague Dawley rats. The rats were divided into randomised 6 groups (n = 10/group). i) Control, ii) Diabetes Control, iii) Noopept Control, iv) Diabetes + Noopept, v) Diabetes + Insulin, vi) Diabetes + Insulin + Noopept. T1DM model was induced by streptozotocin on postnatal 28th day. 0.5 mg/kg noopept and 1 IU insulin were administered intraperitoneally for 14 days. Blood glucose and body weight measurements, puberty follow-up and MWM tests were performed. Hippocampus, hypothalamus and testis were evaluated histologically. Hypothalamic GnRH and kisspeptin were studied immunohistochemically. Serum LH, FSH and insulin, hippocampal homogenate NGF and BDNF levels were determined by ELISA.
Delayed puberty was normalized by noopept (p < 0.05). Blood glucose levels were lower in noopept-administered diabetic groups (p < 0.05). Noopept decreased HOMA-IR in insulin administered diabetic group (p < 0.05). Number of degenerated cells in hippocampus and testis were higher in diabetes control group when compared with other groups (p < 0.05). GnRH immunoreactivity in Diabetes + Noopept group was increased when compared to insulin + noopept group (p = 0.018). There was no difference in kisspeptin, serum LH, FSH, hippocampal NGF-BDNF levels and spatial learning assessment among groups (p > 0.05).
Noopept may have positive effect in treatment of pubertal diabetes.
