Evaluation of enzyme immunoassays in the diagnosis of camel (Camelus dromedarius) trypanosomiasis:a preliminary investigation

Three enzyme immunoassays were used for the serodiagnosis of Trypanosoma evansi in camels in the Sudan in order to evaluate their ability to discriminate between infected and non-infected animals. Two assays were used for the detection of trypanosomal antibodies, one using specific anti-camel IgG conjugate and another using a non-specific Protein A conjugate. The third assay detected the presence of trypanosomal antigens using anti-T. evansi antibodies in a double antibody sandwich assay. Inspection of the frequency distribution of assay results suggested that the ELISA for circulating trypanosomal antibodies using specific antisera and the ELISA for circulating antigens can distinguish between non-infected camels and infected camels exhibiting patent infections or not. The ELISA using Protein A conjugate to bind non-specifically to camel immunoglobulin did not appear to discriminate between infected and non-infected animals

Synthesis of 6-Aminopenicillanic Acid-Protein Conjugates for Development of Enzyme Immunoassay for B-Lactam Antibiotics

An enzyme immunoassay specific for several B-lactam antibiotics rather than individual antibiotics was investigated. The goal to develop an enzyme immunoassay for analysis of a whole class of compounds at one time is different than the goal of most enzyme immunoassays which desire specificity for drugs or hormone levels. Detection of the presence of all B-lactam antibiotics is wanted and identification of specific antibiotics is not needed. 6-Aminopenicillanic acid, the common structural moiety of B-lactam antibiotics was used tin this investigation. Methods of preparation of 6-aminopencillanic ac id conjugates 2nd antibodies needed for enzyme immunoassays have been developed. 6-Aminopenicillanic acid was conjugated to ovalbumin and bovine gamma globulin for production of antibodies with specificity to 6-aminopenicillanic acid. 6-Aminopenicillanic acid was also linked to the enzyme· horseradish peroxidase for future use in enzyme immunoassays. Antibodies produced against 6-aminopenicillanic acid are antigenic towards the thiazolidine ring of penicillins as shown by their affinity to ampicillin and penicillin. Anti-6-aminopenicillanic acid antibodies should therefore be antigenic towards other semisynthetic penicillins because 6-aminopenicillanic acid is usually used in their synthesis

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)

Fv antibodies to aflatoxin B1 derived from a pre-immunized antibody phage display library system

The production and characterization of recombinant antibodies to aflatoxin B[SUB1] (AFB[SUB1]), a potent mycotoxin and carcinogen is described. The antibody fragments produced were then applied for use in a surface plasmon resonance-based biosensor (BIAcore), which measures biomolecular interactions in 'real-time'. Single chain Fv (scFv) antibodies were generated to aflatoxin B1 from an established phage display system, which incorporated a range of different plasmids for efficient scFv expression. The scFv's were used in the development of a competitive ELISA, and also for the development of surface plasmon resonance (SPR)-based inhibition immunoassays. They were found to be suitable for the detection of AFB[SUB1], in this format, with the assays being sensitive and reproducible

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

Comparison of enzyme-linked immunosorbent assay, surface plasmon resonance and biolayer interferometry for screening of deoxynivalenol in wheat and wheat dust

A sample preparation method was developed for the screening of deoxynivalenol (DON) in wheat and wheat dust. Extraction was carried out with water and was successful due to the polar character of DON. For detection, an enzyme-linked immunosorbent assay (ELISA) was compared to the sensor-based techniques of surface plasmon resonance (SPR) and biolayer interferometry (BLI) in terms of sensitivity, affinity and matrix effect. The matrix effects from wheat and wheat dust using SPR were too high to further use this screenings method. The preferred ELISA and BLI methods were validated according to the criteria established in Commission Regulation 519/2014/EC and Commission Decision 2002/657/EC. A small survey was executed on 16 wheat lots and their corresponding dust samples using the validated ELISA method. A linear correlation (r = 0.889) was found for the DON concentration in dust versus the DON concentration in wheat (LOD wheat: 233 g/kg, LOD wheat dust: 458 g/kg)

Expansion of space station diagnostic capability to include serological identification of viral and bacterial infections

It is necessary that an adequate microbiology capability be provided as part of the Health Maintenance Facility (HMF) to support expected microbial disease events during long periods of space flight. The applications of morphological and biochemical studies to confirm the presence of certain bacterial and fungal disease agents are currently available and under consideration. This confirmation would be greatly facilitated through employment of serological methods to aid in the identification for not only bacterial and fungal agents, but viruses as well. A number of serological approached were considered, particularly the use of Enzyme Linked Immunosorbent Assays (ELISAs), which could be utilized during space flight conditions. A solid phase, membrane supported ELISA for the detection of Bordetella pertussis was developed to show a potential model system that would meet the HMF requirements and specifications for the future space station. A second model system for the detection of Legionella pneumophilia, an expected bacterial disease agent, is currently under investigation

Heterologous screening of hybridomas for the development of broad-specific monoclonal antibodies against deoxynivalenol and its analogues

Hapten heterology was introduced into the steps of hybridoma selection for the development of monoclonal antibodies (MAbs) against deoxynivalenol (DON). Firstly, a novel heterologous DON hapten was synthesised and covalently coupled to proteins (i.e. bovine serum albumin (BSA), ovalbumin and horseradish peroxidase) using the linkage of cyanuric chloride (CC). After immunisation, antisera from different DON immunogens were checked for the presence of useful antibodies. Next, both homologous and heterologous enzyme-linked immunosorbent assays were conducted to screen for hybridomas. It was found that heterologous screening could significantly reduce the proportion of false positives and appeared to be an efficient approach for selecting hybridomas of interest. This strategy resulted in two kinds of broad-selective MAbs against DON and its analogues. They were quite distinct from other reported DON-antibodies in their cross-reactivity profiles. A unique MAb 13H1 derived from DON-CC-BSA immunogen could recognise DON and its analogues in the order of HT-2 toxin > 15-acetyl-DON > DON > nivalenol, with IC50 ranging from 1.14 to 7.69 mu g/ml. Another preferable MAb 10H10 generated from DON-BSA immunogen manifested relatively similar affinity to DON, 3-acetyl-DON and 15-acetyl-DON, with IC50 values of 22, 15 and 34 ng/ml, respectively. This is the first broad-specific MAb against DON and its two acetylated forms and thus it can be used for simultaneous detection of the three mycotoxins