Electrochemical Immunosensing of ST2: A Checkpoint Target in Cancer Diseases

A magnetic beads (MB)-involved amperometric immunosensor for the determination of ST2, a member of the IL1 receptor family, is reported in this work. The method utilizes a sandwich immunoassay and disposable screen-printed carbon electrodes (SPCEs). Magnetic immunoconjugates built on the surface of carboxylic acid-microsized magnetic particles (HOOC-MBs) were used to selectively capture ST2. A biotinylated secondary antibody further conjugated with a streptavidin peroxidase conjugate (Strep-HRP) was used to accomplish the sandwiching of the target protein. The immune platform exhibits great selectivity and a low limit of detection (39.6 pg mL−1) for ST2, allowing the determination of soluble ST2 (sST2) in plasma samples from healthy individuals and patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) in only 45 min once the immunoconjugates have been prepared. The good correlation of the obtained results with those provided by an ELISA kit performed using the same immunoreagents demonstrates the potential of the developed strategy for early diagnosis and/or prognosis of the fatal PDAC disease.

Improvements to the immunoassay for detection of Rathayibacter toxicus in hay

An improved protocol for the previously described enzyme-linked immunosorbent assay for Rathayibacter toxicus in hay is described. The improvements were driven mainly by the export hay industry requirement of same-day turnaround for testing of hay extracts. The preparation of hay extracts was shortened by 8 h. The time for adding samples to the enzyme-linked immunosorbent assay plates was shortened by the use of sample tubes with penetrable stoppers combined with specially designed racks. The monoclonal antibody used in the original protocol was purified and conjugated to horseradish peroxidase. This eliminated the need for a secondary step with an anti-mouse horseradish peroxidase conjugate and thereby shortened the assay by over 1 h. Results with the improved assay protocol showed a very high correlation with results obtained with the original protocol (r = 0.98). The assay is still sensitive enough to detect antigen equivalent to less than 1 average gall per kg of hay. These cost-effective changes have streamlined the testing of large numbers of samples for the presence of R. toxicus, in support of the hay export industry.

Evaluation of Indirect Enzyme-Linked Immunosorbent Assays and IgG Avidity Assays Using a Protein A-Peroxidase Conjugate for Serological Distinction between Brucella abortus S19-Vaccinated and -Infected Cows

ABSTRACT This study aimed to evaluate the use of protein A-peroxidase (horseradish peroxidase [HRPO]) in indirect enzyme-linked immunosorbent assays (iELISAs) and IgG avidity assays for serological distinction between Brucella abortus S19-vaccinated and -infected cows. Four groups were analyzed: GI, 41 nonvaccinated seropositive cows; GII, 79 S19-vaccinated heifers analyzed at 3 months postvaccination; GIII, 105 S19-vaccinated cows analyzed after 24 months of age; and GIV, 278 nonvaccinated seronegative cows. IgG levels and avidity to B. abortus smooth lipopolysaccharide (S-LPS) were determined using anti-bovine IgG-HRPO or protein A-HRPO conjugates. Similar levels of IgG anti-S-LPS were found with GI using both conjugates. Lower IgG levels were detected with GII, GIII, and GIV using protein A-HRPO. Both conjugates showed high performance in discriminating GI from GIII, with high sensitivity (Se; 97.6%) and specificity (Sp; 97.1%). Protein A-HRPO was better in distinguishing GI from GIV (Se, 97.6%; Sp, 94.6%) and GI from GII (Se, 80.5%; Sp, 94.9%). Protein A-HRPO excluded a higher number of positive samples with GII and GIV. IgG avidity showed that protein A-HRPO, but not anti-IgG-HRPO, was able to distinguish nonvaccinated from vaccinated cattle, showing a higher avidity index (AI) with GI than with GII, with 78% of serum samples in GII showing an AI of <50%. Therefore, the iELISA using B. abortus S-LPS antigen and protein A-HRPO conjugate for preferential detection of the IgG2 subclass was shown to be suitable for serological distinction between S19-vaccinated and -infected cows. Also, antibodies generated after vaccination showed lower avidity, suggesting a role for the IgG2 subclass as an antibody of higher-affinity maturation after infection, constituting an additional tool for differentiating vaccinated from infected cattle.

Trace-Level Detection of Atrazine Using Immuno-Chemiluminescence: Dipstick and Automated Flow Injection Analyses Formats

A sensitive chemiluminescence (CL)-based immunoassay technique based on both dipstick and flow injection analytical formats is reported for the detection of atrazine. In the dipstick-based immunoassay technique, antibody (anti-atrazine) was first immobilized on the nitrocellulose membranes. The dipstick was then treated with atrazine and atrazine-horseradish peroxidase conjugate (atra-HRP) to facilitate the competitive binding. The dipstick was further treated with urea-hydrogen peroxide (U-H2O2) and luminol to generate photons. The number of photons generated was inversely proportional to the atrazine concentration. In the flow injection analysis (FIA) format, the antibody was immobilized on protein-A sepharose matrix and packed in a glass capillary column, which functioned as an immunoreactor. Competitive binding of antigen and antibody occurred. The CL signals generated during the biochemical reactions were correlated with atrazine concentrations in the analytical samples. By using dipstick technique, it was possible to detect atrazine concentration down to 0.1 ng/mL; with the FIA format, the detection of atrazine was down to 0.01 ng/mL.