Enzyme-Linked Immunosorbent Assay for Detection of Infectious Bronchitis Antibody in Chickens Using Local Isolate of PTS III

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for screening of antibody to avian infectiousbronchitis (IBV). Antigen was prepared from whole virus of infectious bronchitis local isolate PTS-III serotype.Optimum dilution with minimum background for antigen concentration, rabbit anti-chicken conjugate and sera indeveloped ELISA were determined 0.4μg/well, 1:2000 and 1:100, respectively. Correlation optical densities (OD)were compared with a standard commercial ELISA (R2=0.933). The developed ELISA has a better sensitivity to hemagglutinationinhibition (HI) test. The developed local isolate ELISA can be used to detect antibody against infectiousbronchitis virus and it is suitable for sample screening at the diagnostic laboratories

Evaluation of efficacy of some serological tests used for diagnosis of brucellosis in cattle in Egypt using latent class analysis

In this study serum samples were collected from 4 different groups of cattle, Group I (non-vaccinated Brucella infected group), Group II (Vaccinated Brucella infected group), Group III (Non-vaccinated Brucella free group) and Group IV (vaccinated Brucella free group). These samples were subjected to the different serological tests including Rose Bengal plate antigen test, Tube Agglutination test, Rivanol test, Indirect Enzyme Linked Immunosorbent Assay and Competitive Enzyme Linked Immunosorbent Assay. Statistical analysis of the obtained results in different cattle groups was carried out using Latent Class Analysis (Lem model). The prevalence of brucellosis was 6.4%, the sensitivity of RBPT was 96.1% while its specificity was 99.3%, the sensitivity of Rivanol test was 85% while its specificity was 100%, the sensitivity of Indirect Enzyme Linked Immunosorbent assay was 100% while its specificity was 98.3 % and the sensitivity of Competitive Enzyme Linked Immunosorbent assay was 97.1% while its specificity was 100%. The results proved that, the most sensitive test was Indirect Enzyme Linked Immunosorbent assay while the most specific test was Competitive Enzyme Linked Immunosorbent assay. This study therefore, recommends the use of Indirect Enzyme Linked Immunosorbent assay as a screening test and Competitive Enzyme Linked Immunosorbent assay as a confirmatory test. Bacteriological examination was carried out on supramammary lymph nodes and spleen of some slaughtered seropositive cattle, the rate of isolation was 25% from non-vaccinated infected group and 10% from vaccinated infected group. Brucella melitensis biovar3 was recovered only from supramammary lymph nodes.Keywords: Brucellosis, Cattle, Sensitivity, Serology, Specificity, Latent Class Analysi

Enzyme-linked immunosorbent assay for urinary albumin at low concentrations

We describe an enzyme-linked immunosorbent assay (ELISA) for urinary albumin. It requires only commercially available reagents, can detect as little as 16 micrograms of albumin per liter, and analytical recovery ranges from 92 to 116%. The assay is simple, rapid, and inexpensive. Albumin excretion was 6.2 (SD 4.1) mg/24 h in healthy subjects (n = 40), 14.7 (SD 7.2) mg/24 h in albumin-test-strip-negative Type I diabetics (n = 11), and 19.7 (SD 16.2) mg/24 h in patients with essential hypertension (n = 12)

Enzyme-Linked Immunosorbent-Assay for Deoxynivalenol (DON)

Deoxynivalenol (DON), one of the trichothecene mycotoxins, is a worldwide contaminant of wheat and barley, especially when infected by Fusarium graminearum, the causative agent of an epidemic wheat disease called Fusarium Head Blight. Because of the high risk of DON ingestion and the possibility of frequent exposure, it is important to develop a rapid and highly sensitive method for easy identification and quantification of DON in grain samples. In this study, we have developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) to detect DON in wheat. We conjugated 3-O-Hemisuccinyl-DON (3HS-DON) to Bovine serum albumin (BSA) and Ovalbumin (OVA), and obtained DON-specific mice antisera. The indirect competitive ELISA revealed that the optimal concentration of mice serum and the coated antigen was 1/1600 and 1/1500, respectively. The antiserum cross-reacted with the trichothecenes 3-acetyl-DON and T-2 toxin, reaching about 55.2% and 6.3%, respectively, as compared with DON. Results showed that the assay could be performed satisfactorily using an extraction buffer containing less than 15% methanol. Recovery from DON was 82–93% in grains. The linear detection range of DON in grains was between 0.01 and 100 μg/mL

Electrowetting-Based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H 2O 2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.Peer reviewe

Detection of antibodies to Sendai virus by enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Sendai virus, a paramyxovirus, is described. The assay was found to be about 20-fold more sensitive than the hemagglutination inhibition assay. Differentiation between virus specific IgG and IgM is possible. The test appears to be especially useful in the study of early events in antibody formation in vivo as well as in vitro. Abbreviations: BSA, bovine serum albumin; CF, chorioallantoic fluid; ChHC, chicken host components; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; HAU, hemagglutinating units; HAIU, hemagglutination inhibition units; HIA, hemagglutination inhibition assay; HRPO, horse radish peroxidase; i.n., intranasal; i.p., intraperitoneal; i.v., intravenous; M, molar; A, absorbance; PBS, phosphate buffered saline; RAM/Ig, rabbit anti-mouse-Ig; RAM/IgG, rabbit anti-mouse-IgG; RAM/IgM, rabbit anti-mouse-IgM (Fc); RIA, radioimmunoassay; T-cells, thymus derived cells; TCID50, 50% tissue culture infective doses; s.c., subcutaneous; SV, Sendai virus; w/v, weight per volum

Effect of Spacer and the Enzyme-Linked Immunosorbent Assay

The effect of spacers and the enzyme-linked immunosorbent assay (ELISA) formats on the functional parameters of assays such as lower detection limit, inhibitory concentration at 50 per cent (IC50), and specificity were studied. Enzyme conjugates having hydrophobic and hydrophilic spacers were prepared using O-isopropyl methylphosphonic acid (IMPA) and horseradish peroxidase (HRP) as an enzyme label. Comparison was made with reference to enzyme conjugate without any spacer. The present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate significantly improves the sensitivity of assays. An enhanced IC50 value achieved was 0.01 μg mL−1 for free antigen detection by direct immunoassay using hydrophilic spacers and precoating of ELISA plates by secondary antibody. The use of a hydrophilic spacer might have helped in projecting the hapten in the aqueous phase, leading to enhanced antibody binding signal and improved sensitivity of the assay

A serological assay to detect SARS-CoV-2 seroconversion in humans

Development of an enzyme-linked immunosorbent assay to detect antibodies to the SARS-CoV-2 spike protein in human sera and plasma. Here, we describe a serological enzyme-linked immunosorbent assay for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the handling of infectious virus, can be adjusted to detect different antibody types in serum and plasma and is amenable to scaling. Serological assays are of critical importance to help define previous exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy and investigate correlates of protection.Peer reviewe

Dynamics of Mycobacterium tuberculosis Ag85B Revealed by a Sensitive Enzyme-Linked Immunosorbent Assay.

Secretion of specific proteins contributes to pathogenesis and immune responses in tuberculosis and other bacterial infections, yet the kinetics of protein secretion and fate of secreted proteins in vivo are poorly understood. We generated new monoclonal antibodies that recognize the Mycobacterium tuberculosis secreted protein Ag85B and used them to establish and characterize a sensitive enzyme-linked immunosorbent assay (ELISA) to quantitate Ag85B in samples generated in vitro and in vivo We found that nutritional or culture conditions had little impact on the secretion of Ag85B and that there is considerable variation in Ag85B secretion by distinct strains in the M. tuberculosis complex: compared with the commonly used H37Rv strain (lineage 4), Mycobacterium africanum (lineage 6) secretes less Ag85B, and two strains from lineage 2 secrete more Ag85B. We also used the ELISA to determine that the rate of secretion of Ag85B is 10- to 100-fold lower than that of proteins secreted by Gram-negative and Gram-positive bacteria, respectively. ELISA quantitation of Ag85B in lung homogenates of M. tuberculosis H37Rv-infected mice revealed that although Ag85B accumulates in the lungs as the bacterial population expands, the amount of Ag85B per bacterium decreases nearly 10,000-fold at later stages of infection, coincident with the development of T cell responses and arrest of bacterial population growth. These results indicate that bacterial protein secretion in vivo is dynamic and regulated, and quantitation of secreted bacterial proteins can contribute to the understanding of pathogenesis and immunity in tuberculosis and other infections.IMPORTANCE Bacterial protein secretion contributes to host-pathogen interactions, yet the process and consequences of bacterial protein secretion during infection are poorly understood. We developed a sensitive ELISA to quantitate a protein (termed Ag85B) secreted by M. tuberculosis and used it to find that Ag85B secretion occurs with slower kinetics than for proteins secreted by Gram-positive and Gram-negative bacteria and that accumulation of Ag85B in the lungs is markedly regulated as a function of the bacterial population density. Our results demonstrate that quantitation of bacterial proteins during infection can reveal novel insights into host-pathogen interactions