Sensitive Sandwich Enzyme-linked Immunosorbent Assay Research Articles

Development of novel monoclonal antibodies for blocking NF-κB activation induced by CD2v protein in African swine fever virus

BackgroundCD2v, a critical outer envelope glycoprotein of the African swine fever virus (ASFV), plays a central role in the hemadsorption phenomenon during ASFV infection and is recognized as an essential immunoprotective protein. Monoclonal antibodies (mAbs) targeting CD2v have demonstrated promise in both diagnosing and combating African swine fever (ASF). The objective of this study was to develop specific monoclonal antibodies against CD2v.MethodsIn this investigation, Recombinant CD2v was expressed in eukaryotic cells, and murine mAbs were generated through meticulous screening and hybridoma cloning. Various techniques, including indirect enzyme-linked immunosorbent assay (ELISA), western blotting, immunofluorescence assay (IFA), and bio-layer interferometry (BLI), were employed to characterize the mAbs. Epitope mapping was conducted using truncation mutants and epitope peptide mapping.ResultsAn optimal antibody pair for a highly sensitive sandwich ELISA was identified, and the antigenic structures recognized by the mAbs were elucidated. Two linear epitopes highly conserved in ASFV genotype II strains, particularly in Chinese endemic strains, were identified, along with a unique glycosylated epitope. Three mAbs, 2B25, 3G25, and 8G1, effectively blocked CD2v-induced NF-κB activation.ConclusionsThis study provides valuable insights into the antigenic structure of ASFV CD2v. The mAbs obtained in this study hold great potential for use in the development of ASF diagnostic strategies, and the identified epitopes may contribute to vaccine development against ASFV.

Development of a specific and sensitive sandwich enzyme-linked immunosorbent assay for the quantification of dasatinib

This study sought to develop a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for pharmacokinetic studies of dasatinib, a tyrosine kinase inhibitor. Anti-dasatinib antibodies were obtained from mice or rabbits by using two partial structures of dasatinib as haptens: 2-amino-N-(2-chloro-6-methylphenyl)-thiazole-5-carboxamide and 2-{4-(2-hydroxyethyl)-1-piperazinyl}-isonicotinic acid. The best combination of two antibodies for sandwich ELISA of dasatinib was determined using four anti-dasatinib antibodies derived from mice and rabbits. Using two dasatinib-specific rabbit antibodies, we successfully developed an ultra-specific and highly sensitive sandwich ELISA that is hardly affected by the main metabolite of dasatinib. The sandwich ELISA showed a linear detection range from 320 pg/mL to 1000 ng/mL. Serum dasatinib concentrations lower than 320 pg/mL were reproducibly measurable using the sandwich ELISA. The ELISA was specific to dasatinib and there were no cross-reactivities with the major metabolites 4′-hydroxy dasatinib and dasatinib carboxylic acid. The developed sandwich ELISA will be a valuable tool for pharmacokinetic studies of dasatinib. Furthermore, this study revealed that rabbit antibodies can sandwich drug molecules of a smaller size than mouse antibodies in sandwich ELISA.

A sensitive sandwich enzyme-linked immunosorbent assay (sELISA) for the detection of walnut residues in processed food

A sensitive sandwich enzyme-linked immunosorbent assay (sELISA) for the detection of walnut residues in processed food

A Pilot Study of Biliary Atresia Newborn Screening Using Dried Blood Spot Matrix Metalloproteinase-7.

Timely diagnosis is a critical challenge and is associated with improved survival of biliary atresia (BA) patients. We aimed to measure matrix metalloproteinase-7 (MMP-7) levels in BA patients within 3 days of birth using the dried blood spot (DBS) method and evaluate its potential as a screening tool. The study enrolled 132 patients, including 25 patients diagnosed with BA and 107 non-BA patients with other congenital or perinatal conditions from the National Taiwan University Children Hospital. The stored DBS samples collected from 48 to 72 hours of life were retrieved from newborn screening centers. MMP-7 on the DBS was quantified using a sensitive sandwich enzyme-linked immunosorbent assay (ELISA). The MMP-7 levels of BA patients on the DBS were significantly higher than those of non-BA patients (19.2 ± 10.4 vs 5.6 ± 2.7 ng/mL, P value < 0.0001). MMP-7 levels in non-BA patients, including 5 patients with hepatobiliary structural anomaly, 9 patients with intrahepatic cholestasis, and 93 patients with other perinatal diseases, were 11.6 ± 4.2 ng/mL, 6.9 ± 3.0 ng/mL, and 5.2 ± 2.1 ng/mL, respectively. The DBS MMP-7 level showed good accuracy for identifying BA, with an area under the curve of 93.7% [95% confidence interval (CI): 87.7%-99.7%]. The MMP-7 cutoff at 8.0 ng/mL showed a sensitivity of 92.0% (95% CI: 75.0%-98.6%) and specificity of 92.5% (95% CI: 85.9%-96.1%) for detecting BA from other congenital or perinatal diseases. MMP-7 DBS analysis can be used to distinguish BA from other conditions as early as 3 days of age.

Development of a newly immunoassay specific for mouse presepsin (sCD14-ST)

Presepsin (sCD14-ST) is used as a marker for sepsis diagnosis. The production mechanism of presepsin is unique in that it is produced through phagocytosis of microorganisms. However, some studies have demonstrated that non-infected patients had increased presepsin levels and that presepsin is related to the risk or severity of diseases. This study was designed to describe a sensitive sandwich enzyme-linked immunosorbent assay for mouse presepsin developed to investigate the association of presepsin with diseases. Polyclonal antibodies were generated from peptide-immunized rabbit antiserum. Mouse presepsin standard was prepared using the recombinant method as an Fc-fusion protein. The linear detection range of the method was 4.7–300 pg/mL with a detection limit of 1.4 pg/mL. The assay detected mouse presepsin where mouse soluble CD14 (sCD14) was digested by cathepsin D proteinase and the cross-reactivity of sCD14 was not observed. The normal levels of mouse presepsin and sCD14 were compared; 65.9 ± 21.4 pg/mL and 43.2 ± 7.2 ng/mL were determined, respectively. Moreover, the levels of presepsin and sCD14 were compared with a lipopolysaccharide (LPS)-injected sepsis mouse model. The newly developed analytical method had high specificity to presepsin and is an efficient tool for studying the association between presepsin and diseases.

Development of a sensitive sandwich ELISA with broad species specificity for improved fish allergen detection

A sensitive Enzyme-linked Immunosorbent Assay (ELISA) with improved broad species specificity was developed for the detection of southern hemisphere fish residues in processed foods. The polyclonal antibodies were raised against parvalbumins from 13 fish species representing 7 fish orders selected for their molecular diversity and immunoreactivity profile. The optimized ELISA-2 (based on the rabbit capture antibody (RB#4) - sheep detection antibody (S2#4) pair) displayed an improved detection limit of 0.6 μg/L (3.7 μg of /kg). Our immunoreactivity-directed species selection approach in the strategized antibody production significantly improved the detection of no or weakly immunoreactive fish species previously not detected immunochemically. Of 37 commercially important fish species tested, the ELISA-2 could detect 28 fish species (76%). The optimized sample extraction with a buffer additive achieved good protein recoveries of 87.2 – 117.3% (within the AOAC recommended range). The ELISA-2 was able to detect fish residues in five highly processed food products.

Combined Antibody Tagged HRP Gold Nanoparticle Probe for Effective PCV2 Screening in Pig Farms.

IntroductionPorcine circovirus type 2 (PCV2) causes immune repression and intercurrent infections in pigs, resulting in a huge economic loss to the pig breeding industry. Additionally, the spread of PCV2 in pig farms can pollute the living environment of the residents in the farm’s vicinity, which increases the rate of infections. Therefore, rapid and sensitive detection methods are needed for disease prevention and timely environmental cleaning.MethodsThis research describes a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) that utilizes gold nanoparticles (AuNPs) in a functional, specific antibody labeled probe for the detection of PCV2. Due to their high specific surface area and histocompatibility, AuNPs were used as carriers of HRP labeled anti-PCV2 antibodies to amplify the detection signal.ResultsCompared to conventional sandwich ELISA procedures, this method resulted in higher sensitivity (51-fold) and a shorter assay time with a limit of detection of 195 TCID50/mL. The cross-reactivity assay demonstrated that this assay was PCV2 specific.ConclusionThe amplified Ab (HRP) labeled AuNPs probe provides a sensitive analytical approach for the determination of the traces of the PCV2 antigen in early diagnosis.

Development of a sensitive sandwich enzyme-linked immunosorbent assay test kit for reliable detection of peanut residues in processed food

Development of a sensitive sandwich enzyme-linked immunosorbent assay test kit for reliable detection of peanut residues in processed food

Association between Leptin and Adiponectin in women with Polycystic Ovary Syndrome in North Indian Population

Polycystic ovary syndrome (PCOS) is one of the most common endocrine abnormalities, characterized by biochemical hyperandrogenaemia, chronic anovulation, and polycystic ovaries. The objective of the present study to determine the association of adiponectin and leptin with women with PCOS in North Indian Women. In the hospital-based case-control study 100 women with PCOS and 100 controls were recuited from RAMA Medical College, Kanpur, India. Five ml venous blood sample was collected under aseptic precaution and transferred in the serum separator tubes. Leptin and adiponectin levels were assayed by human sensitive leptin double-antibody sandwich enzyme-linked immunosorbent one-step process assay (QAYEE-BIO life science) according to instructions provided with the kit. In the multivariable logistic regression analysis, leptin was found to be independently associated with 1.14 times higher risk for PCOS (OR 1.14, 95% CI 1.01 to 1.29). Receiver operating curve (ROC) analysis suggested excellent predictive accuracy of the multivariable model with Area under the curve 0.86. In the ROC analysis leptin was associated with 68% area under the curve for predicting PCOS using cut off value of &gt;7.87, (P&lt;0.001, Sensitivity 87.8, Specificity 49.4). Adiponectin level was not significantly associated with predicting PCOS (Area under the curve 0.54, P =0.30, Sensitivity 71.1, specificity 50.5, cut off value &lt;10.2). To conclude we observed that higher leptin levels were independently associated with the risk of PCOS, however adiponectin level was not independently associated with the risk of PCOS.

Utility of procalcitonin as an early diagnostic marker of bacteremia in individuals with periodontitis Stage II and III.

The aim of the present study is to assess the ability of procalcitonin (PCT) to differentiate between periodontal health and Stage II and III periodontitis. We further assessed, if PCT can reflect early bacteremia induced by non-surgical periodontal treatment (NSPT). Sixty-four systemically healthy individuals were divided into Group I, periodontally healthy, and Group II, Stage II and III periodontitis. NSPT was done for both the groups. Standardized serum and salivary samples were obtained and analyzed for PCT levels using highly sensitive double antibody sandwich enzyme-linked immunosorbent assay at baseline and 2 weeks. In addition, the serum levels of PCT were recorded at immediate and 1-hour post-NSPT. Mean PCT levels (saliva=0.03ng/mL and serum=0.05ng/mL) in periodontally healthy group were considerably lower than that in the periodontitis group (saliva=0.22ng/mL and serum=1.85ng/mL) with significant intergroup comparison at P< 0.001. Post NSPT the mean serum PCT values increased from 1.854ng/mL to 1.871ng/mL at the immediate interval and remained at 0.879ng/mL after 2 weeks at P< 0.001. Spearman correlation showed highly significant positive correlation between serum and salivary PCT values to clinical attachment level (CAL) at P< 0.001 and rho=0.78 and 0.75, respectively. Linear regression model showed serum PCT to be a significant predictor for CAL. Screening for serum PCT levels in patients with periodontitis could act not only as a guide to assess the bacterial load and use of antibiotics but also as a predictor for CAL loss in patients with periodontitis.

Association of LPL Gene Variant and Serum LPL Level with Ischemic Stroke in Iraqi Population

Background: Lipoprotein lipase (LPL) plays an important role in plasma lipoprotein metabolism.An increasing number of studies have suggested an association of LPL gene variants with the risk ofcardiovascular and cerebrovascular diseases.Objective: To analyze whether Hind III polymorphism of LPL gene and serum LPL level are associatedwith ischemic stroke in Iraqi population.Method: Fifty ischemic stroke patients (clinical diagnosis and x-ray CT) and fifty controls were enrolled inthis case–control study. The LPL Hind III polymorphism was determined by PCR-RFLP technique and LPLmass level was estimated using a sensitive sandwich enzyme-linked immunosorbent assay (ELISA).Results: In the present research was not found any association between the Hindlll LPL gene polymorphismand acute ischemic stroke in the population studied; the allele and genotypic frequencies of the studiedpolymorphism was similar in cases and controls and followed the Hardy-Weinberg equilibrium. Therelationship between Hind III genotypes and the LPL mass level was analyzed using ANOVA and furtherconfirmed by Post-hoc analysis. there was no significant difference in LPL mass levels between the genotypegroups H+H+, H+H-, and H-H- (p value&gt;0.05).Conclusions: The Hind III polymorphism of LPLis not a genetic marker for the development of ischemicstroke as well as not determinants of serum LPL level in the Iraqian sample used.

Development of a sensitive monoclonal antibody-based sandwich ELISA to detect Vip3Aa in genetically modified crops

ObjectivesTo develop a sensitive monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) to detect Vip3Aa in genetically modified (GM) crops and their products.ResultsVegetative insecticidal proteins (Vips) are secreted by Bacillus thuringiensis (Bt) and are known to be toxic to Lepidoptera species. Vip3Aa family proteins, Vip3Aa19 and Vip3Aa20, were successfully applied in GM crops to confer an effective and persistent insecticidal resistance. A sensitive monoclonal antibody-based sandwich ELISA was developed to detect Vip3Aa in GM crops and their products. Two monoclonal antibodies were raised against the overexpressed and purified His-Vip3Aa20, were purified from mouse ascites and characterized. A sandwich ELISA method was developed using the 2G3-1D7 monoclonal antibody for capture and the biotin-labeled 1F9-1F5 monoclonal antibody for detection of Vip3Aa20. The linear detection range of the method was found to be approximately 31.25–500 pg/ml, with a sensitivity of 10.24 pg/ml.ConclusionsThe established ELISA was effective for detecting Vip3Aa family proteins other than Vip3Aa8, and was successfully applied in the detection of Vip3Aa20 and Vip3Aa19 expressed in transgenic maize and cotton.

Rationally Designed Synthetic Haptens to Generate Anti-Ciguatoxin Monoclonal Antibodies, and Development of a Practical Sandwich ELISA to Detect Ciguatoxins.

“Ciguatera” fish poisoning (CFP) is one of the well-known food poisoning caused by the ingestion of fish that have accumulated trace amounts of ciguatoxins (CTXs). CFP affects more than 50,000 individuals annually. The difficulty in preventing CFP comes from the lack of reliable methods for analysis of CTXs in contaminated fish, together with the normal appearance, taste, and smell of CTX-contaminated fish. Thus, a sensitive, accurate, routine, and portable analytical method to detect CTXs is urgently required. Monoclonal antibodies (mAbs) specific against either wing of major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) were generated by immunizing mice with rationally designed synthetic haptens-KLH conjugates instead of the CTXs. Haptenic groups with a surface area greater than 400 Å2 are required to produce mAbs that can strongly bind to CTXs. Furthermore, a highly sensitive fluorescence-based sandwich enzyme-linked immunosorbent assay (ELISA) was developed. This protocol can detect and quantify four major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) with a limit of detection (LOD) of less than 1 pg/mL. The LOD determined for this sandwich ELISA is sufficient to detect CTX1B-contaminated fish at the FDA guidance level of 0.01 ppb.

COMBINES-CID: An Efficient Method for De Novo Engineering of Highly Specific Chemically Induced Protein Dimerization Systems.

Chemically induced dimerization (CID) systems, in which two proteins dimerize only in the presence of a small molecule ligand, offer versatile tools for small molecule sensing and actuation. However, only a handful of CID systems exist and creating one with the desired sensitivity and specificity for any given ligand is an unsolved problem. Here, we developed a combinatorial binders-enabled selection of CID (COMBINES-CID) method broadly applicable to different ligands. We demonstrated a proof-of-principle by generating nanobody-based heterodimerization systems induced by cannabidiol with high ligand selectivity. We applied the CID system to a sensitive sandwich enzyme-linked immunosorbent assay-like assay of cannabidiol in body fluids with a detection limit of ∼0.25 ng/mL. COMBINES-CID provides an efficient, cost-effective solution for expanding the biosensor toolkit for small molecule detection.

Generation and Application of Rat Monoclonal Antibodies Specific for a Human Blood Coagulation Protein: von Willebrand Factor.

von Willebrand factor (VWF) is a glycoprotein that plays a central role in the initiation of blood coagulation. VWF performs two important functions: it acts as a molecular bridge between platelets and as a carrier for coagulation factor VIII (FVIII). von Willebrand disease (VWD) and acquired von Willebrand syndrome (AVWS) are caused by the absence of and/or abnormality in VWF. The pathophysiology of VWD and AVWS is complex and, therefore, it is difficult to diagnose them by conducting the same laboratory tests in all patients. To develop useful monoclonal antibodies (mAbs) for the diagnosis of VWD and AVWS, rat mAbs against human VWF were generated. Immunoblotting analysis revealed that mAbs recognized the reduced and nonreduced VWF protein and endogenous VWF in normal human plasma. Furthermore, we developed a highly sensitive monoclonal antibody-based sandwich enzyme-linked immunosorbent assay technique. In conclusion, the development of VWF-specific mAbs would be useful in the diagnosis of VWD and AVWS.

A Highly Sensitive Sandwich ELISA to Detect CSF Progranulin: A Potential Biomarker for CNS Disorders.

Progranulin (PGRN) plays critical roles in inflammation, tumorigenesis, and neurodegeneration. PGRN levels in blood and cerebrospinal fluid (CSF) are being increasingly investigated as potential biomarkers for these disorders. However, the value of CSF PGRN as a biomarker has been limited because currently available commercial enzyme-linked immunosorbent assay (ELISA) kits have suboptimal sensitivity for detecting CSF PGRN. In this study, pairs of monoclonal antibodies (MAbs) were first screened from eleven monoclonal antiPGRN antibodies using indirect ELISA, then a sandwich ELISA was established using the 2 optimized MAbs. This system displayed high sensitivity, with a lower limit of detection of 60.0 pg/mL and a lower limit of quantification of 150 pg/mL. By using this ELISA system, we showed varied CSF PGRN levels in different brain disorders. For example, as compared with the normal controls, patients with Alzheimer disease or multiple sclerosis showed mildly increased CSF PGRN; those with aseptic encephalitis or neuropsychiatric systemic lupus erythematosus showed moderately increased CSF PGRN; those with bacterial leptomeningitis showed severely increased CSF PGRN. Additionally, determining CSF PGRN levels could monitor CNS metastasis and CSF seeding of carcinomas. These results indicate that this system can be valuable in studying the diagnostic and prognostic value of CSF PGRN in brain disorders.

Type IV chitinase quantification in four different grape cultivars (Vitis vinifera) in northeast of Iran by an indirect sandwich enzyme-linked immunosorbent assay

ABSTRACTThe incidence of grape (Vitis vinifera) allergy in the northeast of Iran is second to melon allergy. Type IV chitinase is one of the major grape allergens. The current study investigates the level of type IV chitinase in four grape variants for the first time in Khorasan Razavi Province using a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA). This assay was developed using a polyclonal antibody as a capture antibody and monoclonal antibody as a secondary one. Finally, the amount of type IV chitinase was measured by the validated ELISA test. The sensitivity of the developed sandwich ELISA is 16 ± 0.05 ng/ml, and its mean coefficients of intraday and interday variations are <5% and <15%, respectively. The recovery of the designed ELISA is 64 ± 0.9 %. The assessments showed that the highest level of type IV chitinase was 39.7 ± 2.3 μg/g in Peykani grape, whereas in the Sultana cultivar, it was 1.76 ± 0.1 μg/g. According to the data, the level of type IV chitinase is variable in different cultivars, and hence, it will be helpful for clinicians to recommend a less allergenic variety to the patient.

Development of a double-antibody sandwich ELISA for rapid detection to C-peptide in human urine

C-peptide level is recognized as an important indicator of diabetes diagnosis. A sensitive and specific double-antibody sandwich enzyme-linked immunosorbent assay for the detection of C-peptide based on double antibody sandwich method was studied in this paper. The rabbit and hen were innunized with PLL-C-peptide and BSA-C-peptide respectively to obtain specific Yolk antibody (IgY) and polyclonal antibody used to construct the sandwich ELISA for the measurement of C-peptide. The limit of detection was 0.51 μg/mL and the half maximal inhibitory concentration (IC50) was 3.26 μg/mL. The method developed in the study showed no evident cross-reactivity with other similar analogs. The detection standard curve of C-peptide exhibited a good linearity (R2 = 0.9896, n = 15). 17 types of the urine of diabetes patients on c-peptide levels compared with the hospital type of diabetes information, with a conclusion of a high consistent rate. Therefore, the methods could be selectively used for rapid screening of C-peptide in human urine, and the type of diabetes has some referential significance.

Development of a tree shrew-specific interferon-gamma assay

ABSTRACTTree shrews (Tupaia belangeri) are small squirrel-like mammals closely related to primates. Due to their susceptibility to several human viruses, tree shrews have been proposed as potential animal models for the study of human viral infections. However, there are no standardized assays currently available for the detection of tree shrew-specific interferon (IFN)-γ, a major cytokine secreted during the antiviral immune response. Herein, we developed a novel enzyme-linked immunosorbent assay (ELISA) for the quantification of IFN-γ in tree shrew serum samples. Tree shrew-specific IFN-γ was expressed in Escherichia coli via fusion with glutathione S-transferase (GST-TS-IFN-γ) to obtain recombinant IFN-γ. To generate anti-IFN-γ monoclonal antibodies, mice were immunized with the GST-TS-IFN-γ recombinant fusion protein, and hybridoma cell lines were established. Similarly, anti-IFN-γ polyclonal antibodies were obtained from immunized rabbits, purified, and conjugated to horseradish peroxidase (HRP). Based on the results obtained from the antibody matching test, we optimized the monoclonal antibody (1:2000) and the HRP-conjugated polyclonal antibody (1:8000) as coating and detection antibodies, respectively. Titration curves were generated with recombinant IFN-γ to develop a sensitive sandwich ELISA; the lowest detection limit of the assay was 20 ng/mL. We also tested mitogen-stimulated tree shrew blood samples in this ELISA, and found significantly higher levels of IFN-γ in the stimulated versus the unstimulated samples. Most importantly, our ELISA system detected native IFN-γ in serum samples from 50 healthy tree shrews. We have thus developed a novel ELISA, and have demonstrated the first ELISA-based measurement of IFN-γ in tree shrew serum samples.

Molecular forms of AlpA and AlpB lytic endopeptidases from Lysobacter sp. Xl1: immunochemical determination of their intra- and extracellular localization

Homologous endopeptidases AlpA and AlpB are components of the secreted complex of lytic enzymes of the Gram-negative bacterium Lysobacter sp. ХL1. These enzymes are synthesized as precursors that consist of a signal peptide, propeptide, and proteolytically active mature part. To understand the topogenetic features of these proteins, bacterial cell fractions were investigated by a sensitive sandwich enzymelinked immunosorbent assay and immunoblot analysis with the use of monoclonal antibodies recognizing unique epitopes of proteins’ mature forms and their propeptides. Only mature forms of the enzymes, without propeptides, were shown to be released outside the cell into the environment. AlpA significantly exceeds AlpB in the production level at the early stationary growth stage. The AlpB precursor was revealed in the cytoplasmic and periplasmic fractions, and the AlpA precursor was found only in the cytoplasmic fraction. The periplasmic fraction was also found to contain the mature forms of both enzymes and their propeptides. These results indicate that AlpA and AlpB are released into the environment through different mechanisms. AlpA is translocated across the cell envelope without being interrupted in the periplasm. The homologous AlpB enzyme, on the contrary, accumulates in the periplasmic space and is captured by outer membrane vesicles in the process of their formation.