Results Of Competitive Enzyme-linked Immunosorbent Assay Research Articles

Simultaneous rapid detection of glycyrrhizin and sennoside A in Daiokanzoto samples by lateral flow immunoassay.

Glycyrrhizin (GLY) and sennoside A (SA) are characteristic bioactive marker compounds of the Kampo medicine Daiokanzoto. Their accurate detection in blends of Rhei rhizoma and Glycyrrhizae radix of several species (4:1 or 4:2) is essential for quality control and to ensure therapeutic efficacy. A rapid, efficient assay can significantly facilitate their detection. To establish a rapid qualitative assay for GLY and SA detection, a lateral flow immunoassay (LFA) was developed using specific monoclonal antibody (mAb) nanoparticles. This assay harnesses the competitive binding of mAb nanoparticles to the immobilized analytes on test strips and free analytes in the samples. Two conjugates for detecting GLY and SA, GLY-bovine serum albumin and SA-human serum albumin, were separately immobilized on the test zones of LFA strips. The detection mechanism is reliant on the visual detection of color changes in the test zones. When GLY and SA were present in samples, they contended with the immobilized conjugates on the strip to bind with the mAb nanoparticles and produced distinct color patterns in the test zones. The limits of detection of the assay for GLY and SA were both 3.13 μg/mL. The capability of the LFA was substantiated using plant samples and Daiokanzoto, and its alignment with indirect competitive ELISA results was confirmed. The introduced LFA is a groundbreaking procedure that offers a rapid, straightforward, and sensitive method for simultaneously detecting GLY and SA in Daiokanzoto samples. It is instrumental in ensuring product quality.

Hybridoma-derived neutralizing monoclonal antibodies against Beta and Delta variants of SARS-CoV-2 in vivo

Neutralizing monoclonal antibodies (mAb) are a major therapeutic strategy for the treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The continuous emergence of new SARS-CoV-2 variants worldwide has increased the urgency for the development of new mAbs. In this study, we immunized mice with the receptor-binding domain (RBD) of the SARS-CoV-2 prototypic strain (WIV04) and screened 35 RBD-specific mAbs using hybridoma technology. Results of the plaque reduction neutralization test showed that 25 of the mAbs neutralized authentic WIV04 strain infection. The 25 mAbs were divided into three categories based on the competitive enzyme-linked immunosorbent assay results. A representative mAb was selected from each category (RD4, RD10, and RD14) to determine the binding kinetics and median inhibitory concentration (IC50) of WIV04 and two variants of concern (VOC): B.1.351 (Beta) and B.1.617.2 (Delta). RD4 neutralized the B.1.617.2 variant with an IC50 of 2.67 ​ng/mL; however, it completely lost neutralizing activity against the B.1.351 variant. RD10 neutralized both variants with an IC50 exceeding 100 ​ng/mL; whereas RD14 neutralized two variants with a higher IC50 (>1 ​mg/mL). Animal experiments were performed to evaluate the protective effects of RD4 and RD10 against various VOC infections. RD4 could protect Adv-hACE2 transduced mice from B.1.617.2 infection at an antibody concentration of 25 ​mg/kg, while RD10 could protect mice from B.1.351 infection at an antibody concentration of 75 ​mg/kg. These results highlight the potential for future modifications of the mAbs for practical use.

Glycation end-products specific auto-antibodies in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease, which is highly inflammatory. Compared to a healthy control group, SLE patients exhibit a higher concentration of advanced glycation end products (AGEs) and a lower concentration of receptors for AGEs (RAGE) in serum, however, the exact aetiology is still unclear. In the present study, non-enzymatic glycation induced modification of human serum albumin (HSA) has been studied by biophysical techniques. Glycated HSA (G-HSA) was used as an antigen, and serum autoantibody levels were estimated in SLE and normal humans (NH) against it, using direct binding ELISA and competitive inhibition ELISA. Compared to N-HSA, remarkable structural modifications were observed in G-HSA. Modified HSA also showed increased pentosidine fluorescence (213.7 ± 13.4 AU). Glycation of HSA induced a conversion of α-helix and random coil to β-sheet and β-turns. Serum immuno assays results exhibited significantly (p < 0.001) higher binding of G-HSA with serum autoantibodies from SLE patients when compared with native HSA (N-HSA). Furthermore, competitive ELISA results showed significantly (p < 0.001) high percent inhibition of serum IgG from SLE patients with modified antigen. Chronic inflammation with excessive oxidative stress in SLE patients could be a possible reason for structural alterations in blood proteins, generating highly immunogenic unique new-epitopes. These in turn induce the generation of specific autoantibodies against G-HSA that may serve as a potential biomarker for SLE pathogenesis.

An Evaluation of the Mechanisms of Galacto-Oligosaccharide (GOS)-Induced IgE Cross-Linking on Basophils in GOS Allergy.

The prebiotics, galacto-oligosaccharides (GOS), are small carbohydrate molecules with 1–7 galactose units linked to glucose and have been shown to trigger IgE-mediated anaphylaxis in some cases following ingestion. It is still an unresolved question of how GOS cross-links IgE on basophils. In this study, we examined whether human galectins, a class of lectins that bind specifically to β-galactoside carbohydrates, are involved in GOS-induced basophil activation. Basophil activation test to GOS and control allergen, Blomia tropicalis (Blo t) extract were performed in the presence or absence of four sugar-based galectin inhibitors (lactose, thiodigalactoside [TDG], TD139, and GB1107) and one peptide-based inhibitor, G3-C12. Results showed that TD139, GB1107, and G3-C12 did not display a specific inhibitory effect on GOS-induced basophil activation as compared to control allergen. An inhibitory effect of lactose and TDG on GOS-induced basophil activation was observed and varied between subjects with up to 100% inhibition at low doses of GOS. The results of competitive ELISA suggest that the inhibitory effects of high dose lactose and TDG on the basophil activation is likely due to the cross-reactivity of GOS-specific IgE to lactose and TDG. Basophil activation is performed using purified basophils suggested that cell surface receptors on other blood cells were not required to induce basophil activation. In conclusion, our results suggest that GOS, a low molecular weight sugar, is able to cross-link IgE independently.

F1 ATP synthase β subunit is a putative receptor involved in white spot syndrome virus infection in shrimp by binding with viral envelope proteins VP51B and VP150

White spot syndrome virus (WSSV) is highly virulent toward shrimp, and F1 ATP synthase β subunit (ATPsyn-β) has been suggested to be involved in WSSV infection. Therefore, in this study, interactions between Penaeus monodon ATPsyn-β (PmATPsyn-β) and WSSV structural proteins were characterized. Based on the results of yeast two-hybrid, co-immunoprecipitation, and protein pull-down assays, WSSV VP51B and VP150 were identified as being able to interact with PmATPsyn-β. Membrane topology assay results indicated that VP51B and VP150 are envelope proteins with large portions exposed outside the WSSV virion. Cellular localization assay results demonstrated that VP51B and VP150 co-localize with PmATPsyn-β on the membranes of transfected cells. Enzyme-linked immunosorbent assay (ELISA) and competitive ELISA results demonstrated that VP51B and VP150 bound to PmATPsyn-β in a dose-dependent manner, which could be competitively inhibited by the addition of WSSV virions. In vivo neutralization assay results further showed that both recombinant VP51B and VP150 could delay mortality in shrimp challenged with WSSV.

Production of Antigen-Binding Fragment against O,O-Diethyl Organophosphorus Pesticides and Molecular Dynamics Simulations of Antibody Recognition.

Immunoassay for pesticides is an emerging analytical method since it is rapid, efficient, sensitive, and inexpensive. In this study, a recombinant antigen-binding fragment (Fab) against a broad set of O,O-diethyl organophosphorus pesticides (DOPs) was produced and characterized. The κ chain and Fd fragment were amplified via PCR and inserted into the vector pComb3XSS and the soluble Fab on phagemid pComb3XSS was induced by isopropyl β-d-thiogalactoside in E. coli TOP 10F’. SDS-PAGE, Western blotting, and indirect competitive ELISA results indicated that Fab maintained the good characteristics of the parental mAb. To better understand antibody recognition, the three-dimensional (3D) model of Fab was built via homologous modeling and the interaction between Fab and DOPs was studied via molecular docking and dynamics simulations. The model clearly explained the interaction manner of Fab and DOPs, and showed that the Arg-L96 and Arg-H52 were mainly responsible for antibody binding. This work provided a foundation for further mutagenesis of Fab to improve its characteristics.

Preparation of Monoclonal Antibody for Brevetoxin 1 and Development of Ic-ELISA and Colloidal Gold Strip to Detect Brevetoxin 1.

Brevetoxin-1 (BTX-1), a marine toxin mostly produced by the dinoflagellatae Karenia brevis, has caused the death of marine organisms and has had numerous toxicological effects on human health. Hence, it is very necessary to develop a rapid, economical, and reliable immunoassay method for BTX-1 detection. In this study, two kinds of complete antigen were synthesized using the succinic anhydride and isobutyl chloroformate two-step methods. Conjugate BTX-1-OVA was used as an antigen for mice immunization, and BTX-1-BSA for measuring the titer of the produced antibodies. A hybridoma cell line 6C6 stably secreting monoclonal antibody (mAb) against BTX-1 was obtained by fusing SP2/0 myeloma cells with the spleen cells from the immunized mouse. The hybridoma 6C6 was injected into the abdomen of BALB/c mice to obtain ascites, and the anti-BTX-1 mAb was harvested from ascites by precipitation with caprylic acid/ammonium sulfate (CA-AS). The anti-BTX-1 mAb was identified as an IgG1 subtype, and the cross-reactivity results showed that anti-BTX-1 mAb was highly specific to BTX-1 with the affinity of 1.06 × 108 L/mol. The indirect competitive ELISA results indicated that the linear range for BTX-1 detection was 14–263 ng/mL with IC50 of 60 ng/mL, and a detection limit of 14 ng/mL. The average recovery rate from the spiked samples was 88 ± 2% in intra-assay and 89 ± 2% in inter-assay. The limit of detection (LOD) using the colloidal gold strip was 200 ng/mL with high specificity. Therefore, the anti-BTX-1 mAb can be used to detect BTX-1 in shellfish and other related samples.

Simple One-step Purification of Hepatitis B Core Antigen in Escherichia coli

Hepatitis B core antigen (HBcAg) is one of the most important diagnostic reagents in hepatitis B virus (HBV) infection. The purifi- cation of HBcAg using conventional methods, such as sucrose density gradient ultracentrifugation, is time and cost consuming. To overcome this diculty , we aimed at developing an ecient method for producing a soluble form of HBcAg for diagnostic applica- tion. The HBcAg construct was secreted into periplasm space of E. coli, which facilitates purification by selective disruption of the outer membrane. The His-tagged HBcAg was purified from prepared periplasmic fraction using Ni-NTA anity chromatography with an average yield of 20 mg/L culture. The results of competitive ELISA indicated that the antigenicity of periplasmic HBcAg is comparable with commercial E. coli-derived antigen. These results demonstrate the periplasmic system is suitable for the rapid and simple purification of bioactive and soluble HBcAg.

Effect of hapten structures on specific and sensitive enzyme-linked immunosorbent assays for N-methylcarbamate insecticide metolcarb

Five different haptens of the N-methylcarbamate insecticide metolcarb were designed and synthesized. All of the haptens were conjugated with ovalbumin (OVA) for the coating antigen, and one hapten containing all of the structure of metolcarb was conjugated with bovine serum albumin (BSA) for the immunogen. Two polyclonal antisera were raised against the BSA conjugate, and ten antibody/coating conjugate combinations were selected for studies of assay sensitivity and specificity for metolcarb. A class-specific combination was found, with the I 50 of the assay ranged from 0.64 to 20.98 μg mL −1 for seven tested N-methylcarbamate insecticides except for pirimicarb. Considering titer, I 50 and cross-reactivity of all combinations of antibody/coating conjugate, a competitive indirect enzyme-linked immunosorbent assay (ELISA) in a homologous system, whose limit of detection (LoD) reached 1.4 ng mL −1, was presented. The results of competitive ELISAs indicated that coating hapten structure can significantly affect not only assay sensitivity but also its specificity.

Alzheimer’s-disease-associated conformation of intrinsically disordered tau protein studied by intrinsically disordered protein liquid-phase competitive enzyme-linked immunosorbent assay

Tau protein, the major constituent of paired helical filaments in Alzheimer’s disease, belongs to the intrinsically disordered proteins (IDPs). IDPs are an emerging group in the protein kingdom characterized by the absence of a rigid three-dimensional structure. Disordered proteins usually acquire a “functional fold” upon binding to their interaction partner(s). This property of IDPs implies the need for innovative approaches to measure their binding affinity. We have mapped and measured the Alzheimer’s-disease-associated epitope on intrinsically disordered tau protein with a novel two-step sandwich competitive enzyme-linked immunosorbent assay (ELISA). This approach allowed us to determine the binding affinity of disordered tau protein in liquid phase without any disturbance to the competitive equilibrium and without any need for covalent or noncovalent modification of tau protein. Furthermore, the global fitting method, used for the reconstruction of tau binding curves, significantly improved the assay readout. The proposed novel competitive ELISA allowed us to determine the changes in the standard Gibbs energy of binding, thus enabling measurement of tau protein conformation in the core of paired helical filaments. IDP competitive ELISA results showed, for the first time, that the tau protein C terminus of the Alzheimer’s-disease-derived paired helical filaments core subunit adopts β-turn type I′ fold and is accessible from solution.

A novel BLyS antagonist peptide designed based on the 3-D complex structure of BCMA and BLyS

B lymphocyte stimulator (BLyS) is a member of tumor necrosis factor (TNF) family. Because of its roles in autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjogren syndrome (SS), BLyS antagonists have been tested to treat SLE- and RA-like symptoms in mice and obtained optimistic results. So far, reported BLyS antagonists were mostly decoyed BLyS receptors or anti-BLyS antibodies. In this study, a novel BLyS antagonist peptide, PT, was designed based on the modeling 3-D complex structure of BCMA and BLyS. The interaction mode of PT with BLyS was analyzed theoretically. The results of competitive ELISA demonstrated that PT could inhibit the binding of BCMA-Fc and anti-BLyS antibody to BLyS in vitro. In addition, PT could partly block the proliferating activity of BLyS on mice splenocytes. The BLyS antagonizing activity of PT was significant ( p < 0.05). This study highlights the possibility of using BLyS antagonist peptide to neutralize BLyS activity. Further optimization of PT with computer-guided molecular design method to enhance its biopotency may be useful in developing new BLyS antagonists to treat BLyS-related autoimmune diseases.

Production and Characterization of DDT Antibodies and Its Application to Enzyme Immunoassay: Relation of Response and Affinity to Coating Ligand

To development an immunodetection method for DDT, 1,1,1-trichloro-2,2-bis(4-chorophenyl)ethane (p,p'-DDT) and its metabolites (p,p'-DDA, p,p'-DDE, p,p'-DDD), five derivatives of DDT haptens have been synthesised and characterized as coating ligands for antibody evaluation. The appropriate lengths of linkers were introduced to investigate a matching pair of coating ligand and antibody. Among these hapten derivatives, 2,2-bis(4-chlorophenyl)acetic acid (DDA), 5,5-bis(4-chlorophenyl)-5-hydroxypentanoic acid (DDHP) and 5,5-bis(4-chlorophenyl)-5-chloropentanoic acid (DDCP) were conjugated with keyhole limpet hemocyanin (KLH) for its use as an immunogen. The bovine serum albumin (BSA) conjugates of these derivatives were prepared as a coating ligand for monoclonal antibody screening. Fifteen monoclonal antibody clones were screened using these probes. 6,6-Bis(4-chlorophenyl)-6-hydroxyhexanoic acid (DDHH) and 3-[6,6-Bis(4-chlorophenyl)-6-hydroxyhexanoylamino]propanoic acid (DDHHAP), in addition to the above hapten derivatives, were conjugated to ovualbumin (OVA) and bovine serum albumin (BSA) for their use as coating ligands to measure the titration level of the antibody and the displacement of free analytes. The indirect competitive ELISA results indicate that the titration level and free analyte displacement were greatly influenced by the DDT derivatives and carrier proteins used. Three matching pairs of monoclonal antibodies and coating ligands were selected for the DDT immunoassay: antibody clone 1A3 and coating ligand DDA-OVA, 1A1 and DDHHAP-BSA, and 1A4 and DDHP-OVA.

Fine mapping of bovine herpesvirus 1 (BHV-1) glycoprotein C neutralizing epitopes by type-specific monoclonal antibodies and synthetic peptides

Bovine herpesvirus glycoprotein C (gC) functions as a major virus attachment protein. Here, two BHV-1 gC-specific epitopes that are specified by complement-dependent neutralizing MAbs are mapped. The BHV-1 gC-specific peptides and MAbs were used to specifically localize continuous epitopes by direct binding to the MAbs and by blocking the Mab reactivity (competitive ELISA) to authentic viral antigen. The results of competitive ELISA indicated that the complement-dependent neutralizing epitopes recognized by MAbs F2 and 24 were located between BHV-1 gC amino acids (aa) 47–69 and (aa) 109–119, respectively.

Competitive enzyme-linked immunosorbent assay using monoclonal antibodies to the Brucella melitensis BP26 protein to evaluate antibody responses in infected and B. melitensis Rev.1 vaccinated sheep

Competitive enzyme-linked immunosorbent assay (C-ELISA) was performed using 15 monoclonal antibodies (MAbs), specific for Brucella BP26 (previously also called CP28), a periplasmic protein antigen, to investigate antibody responses in naturally and B. melitensis H38 experimentally infected and B. melitensis Rev.1 vaccinated sheep. The antigen preparation consisted of cytosoluble protein extract (CPE) of B. melitensis B115. By combining the C-ELISA results of several MAbs, a high percentage of naturally infected animals were detected which showed different status in the current conventional diagnostic tests. Indeed, 90% of sheep which were positive in the conventional bacteriological and serological tests were positive in C-ELISA. 72% of the bacteriologically negative but serologically and delayed type hypersensitivity positive sheep were also found positive in the C-ELISA. Moreover, 79% of the bacteriologically and serologically negative sheep but delayed type hypersensitivity positive were also detected by C-ELISA. Thus, these results confirmed the importance of BP26 as a frequently recognized target of the humoral immune response of infected sheep. The 8 B. melitensis H38 experimentally infected sheep showed various degrees of antibody responses at the 90th day after infection, which was delayed in comparison to that against O-polysaccharide (O-PS). Of the 15 MAbs tested, only one MAb was weakly inhibited (20 to 35% inhibition) by 56% of negative control sera. Furthermore, no antibody response against BP26 was detected in B. melitensis Rev.1 vaccinated sheep. Results of the C-ELISA with the 15 MAbs showed individual variability of the antibody responses against BP26. Thus, it is suggested that several epitopes of BP26 are of interest for diagnosis of B. melitensis infection in sheep.

Brucella melitensis cell envelope protein and lipopolysaccharide epitopes involved in humoral immune responses of naturally and experimentally infected sheep.

Cell envelope fraction (CEF) of Brucella melitensis B115 was used to investigate antibody responses of B. melitensis naturally and strain H38 experimentally infected sheep by immunoblotting, indirect enzyme-linked immunosorbent assay (ELISA) (I-ELISA), and competitive ELISA (C-ELISA) with monoclonal antibodies (MAbs). MAbs used were directed to outer membrane proteins with molecular masses of 10, 16.5, 19, 25 to 27, 31 to 34, 36 to 38, and 89 kDa; to the heat shock protein DnaK, to O-polysaccharide (O-PS) common (C) and M epitopes; and to rough lipopolysaccharide (R-LPS) epitopes. In immunoblotting, all infected sheep sera tested recognized a large number of protein bands, including the above-cited proteins and other proteins for which MAbs have not been defined. The antibody response pattern was different from one animal to another, even within the experimentally infected sheep which were infected under the same experimental conditions. A number of protein bands were recognized by the sheep sera prior to experimental infection and by other uninfected sheep sera. The antibody reactivity to these antigens and others might explain the nonspecific antibody reactivity of sera in I-ELISA with CEF. C-ELISA confirmed also the individual variability of the antibody responses of infected sheep to protein antigens. Antibody responses to O-PS C and M epitopes were detected in all experimentally infected sheep and in half of the naturally infected sheep, but these responses were also heterogeneous in relation to their intensities. Antibody responses to R-LPS epitopes detected by use of C-ELISA with the anti-R-LPS MAbs were low or negative in most of the infected animals. Despite antibody response heterogeneity for CEF antigens, immunoblot and C-ELISA results indicated that, among the CEF antigens, the O-PS epitopes (C and M epitopes) and epitopes of the major 25- to 27- and 31- to 34-kDa outer membrane proteins seem to be the most promising for detecting B. melitensis infection in sheep.

Monoclonal antibodies to turbot ( Scophthalmus maximus) immunoglobulins: characterization and applicability in immunoassays

Five monoclonal antibodies (mAbs) to immunoglobulins (Igs) of the turbot Scophthalmus maximus were produced and characterized. All the mAbs (denominated UR1, UR3, UR4, UR6 and UR7) are of isotype IgG1/κ and show good anti-turbot Ig reactivity in enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Results of competitive ELISA and immunoblotting analysis indicate that these five mAbs react with at least three different epitopes on the turbot Ig H chain. Except in the case of UR1, reactivity with periodate-treated purified turbot Ig was much lower than with the untreated Ig, suggesting that carbohydrate residues are involved in epitope recognition. All the mAbs showed reactivity with sera from the closely related species Scophthalmus rhombus but not with sera from species of other flatfish genera. One of these mAbs (UR3) has been successfully applied for the detection of antibodies against Vibrio anguillarum in ELISA.

Group and Strain-Specific Neutralization Sites of Infectious Bursal Disease Virus Defined with Monoclonal Antibodies

Two somatic cell hybridizations were performed utilizing splenocytes from mice immunized with one or more strains of infectious bursal disease virus (IBDV). Supernatants from hybridoma cell lines were initially screened by the enzyme-linked immunosorbent assay (ELISA) against multiple strains of IBDV. Cell lines that secreted antibodies with ELISA reactivity patterns of interest were cloned, and their monoclonal antibodies (MCAs) were subsequently tested in cross-virus-neutralization tests. Two of the nine MCAs selected exhibited strong neutralizing activity and precipitated IBDV antigens in agar gel precipitin tests as well. MCA B69 significantly neutralized only the cloned D78 strain of IBDV, whereas MCA R63 neutralized all IBDV strains (representing both serotype I and II viruses) against which it was tested. Results of competitive ELISAs that used the R63 and B69 MCAs showed that the two neutralization sites on the D78 strain were not overlapping.