Immunoassays

• Romer Labs’ AgraStrip® Gluten G12 is an immunochromatographic lateral flow assay for the
qualitative detection of gluten at thresholds of 5, 10 and 20 ppm in foods, rinse water and
environmental surfaces.
• G12 antibody targets a 33-mer peptide that is resistant to enzymatic digestion and heat
denaturation, as well as being the fragment of the gliadin protein to which Celiac Disease sufferers
react, making it a reliable analytical marker.
• G12 also cross-reacts with barley hordein, rye secalin and oat avenin.
• This study was conducted to validate the AgraStrip® Gluten G12 assay method under the guidance
of the AOAC Performance Tested MethodsSM program against AOAC OMA 2012.01 in rice flour,
bread, incurred bread, cookie, ice cream and chocolate matrices, and stainless steel environmental
surface.
• Matrix studies demonstrated that AgraStrip® Gluten G12 will detect spiked gluten and gliadin at
varying spike levels and assay thresholds in challenging matrices.
• The assay demonstrated no cross-reactivity or interference against 38 additional gluten-free
matrices, and proved its overall suitability as AOAC PTM-certified.

The condensation of 1,1′-ferrocenedimethanol and lipoic acid yields a ferrocene derivative (FcD): 1,1′-ferrocenyl bis(methylene lipoic acid ester), a bipodant linker which was used to aggregate gold nanoparticles (AuNPs). The FcD/AuNP aggregations displayed a very well-resolved signal related to the ferrocene oxidation to ferrocenium. The aggregates were found to form an efficient transducing system for a novel concept of immunosensing based on the shifting of the ferrocene oxidation potential. αHIgG/FcD/AuNP or chicken αGIgG/FcD/AuNP bioconjugates are used along with carbon SPEs as an immunosensing platform for the detection of HIgG or GIgG as model analytes. As shown by CV and DPV, the antigens addition triggers significant anodic potential shifts (up to 75 mV) of the ferrocene oxidation peak as an immunoresponse for the Ag–Ab recognition. The results from selectivity and specificity experiments using bovine serum albumin, ovalbumin and α-lactalbumin as potentially interfering proteins to HIgG Ag showed that these potential shifts would be solely due to the Ag–Ab recognition events with excellent selectivity and specificity in the presence of up to 1000-fold of the competitive proteins.

In recent years, many improvements have been made in foodborne pathogen detection methods to reduce the impact of food contamination. Several rapid methods have been developed with biosensor devices to improve the way of performing pathogen detection. This paper presents an automated endpoint detection system for amplicons generated by loop mediated isothermal amplification (LAMP) on a microfluidic compact disk platform. The developed detection system utilizes a monochromatic ultraviolet (UV) emitter for excitation of fluorescent labeled LAMP amplicons and a color sensor to detect the emitted florescence from target. Then it processes the sensor output and displays the detection results on liquid crystal display (LCD). The sensitivity test has been performed with detection limit up to 2.5 x 10(-3) ng/mu L with different DNA concentrations of Salmonella bacteria. This system allows a rapid and automatic endpoint detection which could lead to the development of a point-of-care diagnosis device for foodborne pathogens detection in a resource-limited environment.

Link to Full-Text Articles :
http://www.mdpi.com/1424-8220/15/3/5376/pdf 
http://www.ncbi.nlm.nih.gov/pubmed/25751077 
http://repository.um.edu.my/98539/1/sensors-15-05376.pdf 
http://eprints.um.edu.my/13409/

Conventional capture (“Sandwich”) ELISAs equally detect denatured inactive and native active botulinum type A toxin. Light chain endoprotease activity assays also fail to distinguish between various inactive molecules including partially denatured and fragmented material still retaining this protease activity. By co-coating microtitre plates with SNAP25 substrate and a monoclonal antibody specific for a conformational epitope of the toxin’s Hc domain, it was possible to develop a highly sensitive (130 Attomolar LoD), precise (1.4% GCV) new assay specific for the biologically active toxin molecule. Capture was performed in phosphate buffer with a fixed optimal concentration of chaotropic agent (e.g. 1.2M urea) to differentially isolate functional toxin molecules. Addition of enzymatically favourable buffer containing zinc and DTT reduced the inter-chain disulphide bond releasing and activating the captured L-chain with subsequent specific cleavage of the SNAP25(1–206) substrate. A neoepitope antibody specific for the newly exposed Q197 epitope was used to quantify the cleaved SNAP25(1–197). The assay’s requirement for the intact toxin molecule was demonstrated with pre-reduced toxin (heavy and light chains), recombinant LHn fragments and stressed samples containing partially or fully denatured material. This is the first known immuno-biochemical assay that correlates with in vivo potency and provides a realistic alternative.

An original technique for obtaining stable conjugates using colloid carbon particles as indicator labels has been developed. The reliability and stability of the diagnostic reagents obtained is provided by covalent binding of various affine compounds on the surface of carbon particles. The stability of the reagents has been studied under various storage conditions for 3-10 years. It was shown that even when storage conditions were outside the optimal range, some conjugates preserved their analytical characteristics for 10 years. A number of systems for analytical detection of various ligands have been designed based on the carbon-protein conjugates synthesized. The major characteristics of these systems are their high sensitivity and specificity, reliability, and reproducibility, simplicity in operation, and quick results.

Immunosensors are affinity ligand-based biosensor solid-state devices in which the immunochemical reaction is coupled to a transducer. The fundamental basis of all immunosensors is the specificity of the molecular recognition of antigens by antibodies to form a stable complex. This is similar to the immunoassay methodology.Immunosensors can be categorized based on the detection principle applied. The main developments are electrochemical, optical, and microgravimetric immunosensors. In contrast to immunoassay, modern transducer technology enables the label-free detection and quantification of the immune complex. The analysis of trace substances in environmental science, pharmaceutical and food industries is a challenge since many of these applications demand a continuous monitoring mode. The use of immunosensors in these applications is most appropriate. Food chemists should take advantage of immunosensors in food and clinical diagnostics. There are many recent developments in the i...

The enzyme linked immunosorbent assay (ELISA) is a potential tool for the determination of dextran. In this study, dextran–BSA antigens were prepared by Reductive amination method, and were confirmed by SDS-PAGE and free amino detection. The effects of coupled reaction conditions such as different oxidation degree of dextran, the reaction time were investigated and the immunity of the resulting dextran-BSA neoglycoprotein antigens were evaluated through the interaction with standard dextran antibody. The immunogen was immunized with white rabbits to obtained polyclonal antibody respectively. A general and broad class-specific Elisa detection method was developed according to Elisa theory. The method was put to use for quantitative analysis of dextran in practical saccharose samples.
Keywords: Dextran; Neoglycoprotein Antigen; Preparation; Polyclonal Antibody; ELISA.

The enzyme linked immunosorbent assay (ELISA) is a potential tool for the determination of dextran. In this study, dextran–BSA antigens were prepared by Reductive amination method, and were confirmed by SDS-PAGE and free amino detection. The effects of coupled reaction conditions such as different oxidation degree of dextran, the reaction time were investigated and the immunity of the resulting dextranBSA neoglycoprotein antigens were evaluated through the interaction with standard dextran antibody. The immunogen was immunized with white rabbits to obtained polyclonal antibody respectively. A general and broad class-specific Elisa detection method was developed according to Elisa theory. The method was put to use for quantitative analysis of dextran in practical saccharose samples.
Keywords: Dextran; Neoglycoprotein Antigen; Preparation; Polyclonal Antibody; ELISA.

in which the immunochemical reaction is coupled to a transducer. The fundamental basis of all immunosensors is the specificity of the molecular recognition of antigens by antibodies to form a stable complex. This is similar to the immunoassay methodology. Immunosensors can be categorized based on the detection principle applied. The main developments are electrochemical, optical, and microgravimetric immunosensors. In contrast to immunoassay, modern transducer technology enables the label-free detection and quantification of the immune complex. The analysis of trace substances in environmental science, pharmaceutical and food industries is a challenge since many of these applications demand a continuous monitoring mode. The use of immunosensors in these applications is most appropriate. Food chemists should take advantage of immunosensors in food and clinical diagnostics. There are many recent developments in the immunosensor field which have potential impacts. The future role of th...

The aim of the current study was to describe the use of a pool of different antisera raised against pregnancy-associated glycoproteins (PAGs; purified from both ovine and caprine placentas) for early pregnancy diagnosis in ovine species. Sixty-three pluriparous Sarda ewes (Ovis aries) were synchronized. Blood samples were withdrawn on Days 18, 24, 26, 28, 30, and 50 after mating. These samples were assayed for progesterone (radioimmunoassay [RIA] including an extraction step) and for pregnancy-associated glycoproteins (RIA-706 and RIA-srPool). Progesterone concentrations were under 1.0 ng/mL in all nonpregnant Sarda ewes. In pregnant ewes, mean progesterone concentrations ranged from 2.4 ng/mL (Day 24, single pregnancies) to 4.4 ng/mL (Day 28, multiple pregnancies). During all periods of examination, PAGs remained lower than 0.8 ng/mL in nonpregnant ewes. On Day 18 of pregnancy, PAG concentrations could be detected in 26 of 43 (60.5%) and in 41 of 43 (95.3%) pregnant ewes using the ...

The soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, pain, cancer, and other diseases. However, there is not a simple, inexpensive, and reliable method to estimate levels of active sEH in tissues. Toward developing such an assay, a polyclonal variable domain of heavy chain antibody (VHH) sandwich immunoassay was developed. Ten VHHs, which are highly selective for native human sEH, were isolated from a phage-displayed library. The ten VHHs have no significant cross-reactivity with human microsomal epoxide hydrolase, rat and mouse sEH, and denatured human sEH. There is a high correlation between protein levels of the sEH determined by the enzyme-linked immunosorbent assay (ELISA) and the catalytic activity of the enzyme in S9 fractions of human tissues (liver, kidney, and lung). The VHH-based ELISA appears to be a new reliable method for monitoring the sEH and may be useful as a diagnostic tool for diseases influenced by sEH. This study also demonstrates the broad utility of VHH in biochemical and pharmacological research.

Immunosensors are affinity ligand-based biosensor solid-state devices in which the immunochemical reaction is coupled to a transducer. The fundamental basis of all immunosensors is the specificity of the molecular recognition of antigens by antibodies to form a stable complex. This is similar to the immunoassay methodology. Immunosensors can be categorized based on the detection principle applied. The main developments are electrochemical, optical, and
microgravimetric immunosensors. In contrast to immunoassay, modern transducer technology enables the label-free detection and quantification of the immune complex. The analysis of trace substances in environmental science, pharmaceutical and food industries is a challenge since many of these applications demand a continuous monitoring mode. The use of immunosensors in these applications is most appropriate. Food chemists should take advantage of immunosensors in food and clinical diagnostics. There are many recent developments in the immunosensor field which have potential impacts. The future role of this technique in intra-laboratory, as well as
bedside testing, will become even more important as the food laboratory is faced with increasing pressure to contain costs. Objective of this paper is to give a general overview of the possible application of immunosensors to the food analysis field.

Botulinum neurotoxins contain proteases that cleave specific intra-neural proteins essential for neurotransmitter release. Toxin types A, C1 and E intra-cellularly cleave SNAP25 and/or Syntaxin (type C1 only) resulting in a flaccid paralysis. Although highly sensitive, robust in vitro endopeptidase immunoassays have been developed for some serotypes, an endopeptidase immunoassay for type C1 has not previously been described. The current studies utilised solid phase synthesized SNAP25(137-206) peptide substrate, and a new specific antibody to the SNAP25(191-198) octapeptide epitope that becomes exposed following cleavage by type C1 toxin. The highly specific nature of the detecting antibody was illustrated by the failure of anti-SNAP25(191-198) to recognise the type A cleavage product which differs by just one amino acid residue. Conversely, anti-SNAP25(190-197), which recognises the type A cleavage product, fails to cross react with the type C1 toxin cleavage product. Utilising Syntaxin(232-266) peptide substrate, and a specific antibody to the cleavage product epitope, Syntaxin(254-261), it was also possible to develop an endopeptidase immunoassay. Assay sensitivities allowed the detection of less than 0.1 LD(50)/ml (25 pg/ml) of type C1 haemagglutinin-complexed toxin. The assay failed to detect toxin serotypes A, B, D, E, F or G and therefore also provides an alternative highly specific in vitro identity test. In the absence of trypsin inhibitors, the assay is also capable of detecting 2 pg/ml of trypsin activity, or trypsin like contaminants. These new immunoassays will therefore provide highly specific tools for monitoring botulinum toxin light chain endopeptidase activity and serotype identity.

"The introduction of computers to immunoassay laboratories has resulted in considerable improvement in the management of immunoassay data. The RIASTAT software has been very valuable in processing of thyroid hormone radioimmunoassay data. In this article, the various concepts underpinning cubic spline, logit-log, and five parameters models are reviewed, as they apply to the computation of precision profiles, assay sensitivities, and working ranges of standard curves. The usefulness of these models in assay optimization and in thyroid function assessment is highlighted.

KEY WORD: computers, mathematical models, precision profile, RIA, thyroid function."

BACKGROUND:
The emergence of drug resistant typhoid fever is a major public health problem, especially in Asia. An oral single dose typhoid vaccine would have major advantages. M01ZH09 is a live oral single dose candidate typhoid vaccine containing Salmonella enterica serovar Typhi (Ty2 aroC(-)ssaV(-)) ZH9 with two independently attenuating deletions. Studies in healthy adults demonstrated immunogenicity and an acceptable safety profile.
OBJECTIVES:
We conducted a randomised placebo controlled, single-blind trial to evaluate the safety and immunogenicity of M01ZH09 in healthy Vietnamese children aged 5 to 14 years.
METHODS:
Subjects were randomly assigned to receive either a nominal dose of 5x10(9) CFU of M01ZH09 or placebo and were followed up for 28 days. The primary safety outcome was the proportion of subjects with any adverse event attributed to M01ZH09. The primary immunogenicity endpoint was the proportion of subjects who showed a positive immune response to M01ZH09 in the Salmonella Typhi lipopolysaccharide (LPS) specific serum IgA and IgG ELISA.
PRINCIPAL FINDINGS:
One hundred and fifty-one children were enrolled, 101 subjects received M01ZH09 and 50 subjects received placebo. An intention to treat analysis was conducted. There were no serious adverse events and no bacteraemias. In the M01ZH09 group, 26 (26%; 95% CI, 18-5%) of 101 subjects experienced adverse events compared to 11 (22%; 95% CI, 12-36%) of 50 subjects in the placebo group (odds ratio (OR) [95%CI] = 1.23 [0.550-2.747]; p = 0.691). Faecal shedding of S. Typhi (Ty2 aroC(-)ssaV(-)) ZH9 was detected in 51 (51%; 95% CI, 41-61%) of 100 M01ZH09 subjects. No shedding was detected beyond day 3. A positive immune response, defined as 70% increase (1.7 fold change) in LPS specific serum IgG (day 14 or 28) and/or 50% increase (1.5 fold change) in LPS specific serum IgA (day 7 or 14) from baseline was detected in 98 (97%; 95% CI, 92-99%) of 101 M01ZH09 recipients and 8 (16%; 95% CI, 7-29%) of 50 placebo recipients. Twenty-eight (100%; 95% CI, 88-100%) of 28 vaccine recipients who were evaluated in the LPS specific IgA ELISPOT assay showed a positive response compared to none of the 14 placebo recipients tested.
CONCLUSIONS:
This was the first phase II trial of a novel oral candidate typhoid vaccine in children in an endemic country. M01ZH09 had an appropriate safety profile and was immunogenic in children.
TRIAL REGISTRATION:
Controlled-trials.com ISRCTN91111837.

Clostridium botulinum type A toxin is the most prevalent cause of naturally occurring outbreaks of human botulism in the world. The active dichain neurotoxin molecule is composed of a heavy chain (H-chain) of ~100 kDa with the Carboxy-terminal end consisting of a receptor binding (HC) domain, while the Amino-terminal (HN) domain is linked by a critical disulfide bond to a light chain (L-chain) of ~50 kDa. Although the mouse bioassay (MBA) is traditionally used to confirm the presence of toxin in serum or food, its sensitivity is insufficient to detect low toxin levels in approximately 30 to 60% of botulism patients. A novel FDC (Functional Dual Coating) microtitre plate immuno-biochemical assay, which quantitates botulinum toxicity by measuring the HC (receptor binding) domain linked with L-chain endopeptidase activity, was modified to allow human serum (lysed or unlysed) to be tested without interference from the matrix, with toxin detection down to 0.03 mouse LD50 ml-1 serum or 0.13 pg ml-1 using just 100 µl of clinical samples. The assay was specific for type A toxin and could additionally be applied to whole blood and food samples. Low levels of 1 to 2 mouse LD50 ml-1 serum of type A toxin were quantitated for the first time using the modified FDC assay in two severely intoxicated UK patients that required mechanical ventilation and antitoxin. Toxin levels in recovered food sample extracts were also detected and one MBA negative sample found to contain 0.32 LD50 ml-1 extract. The FDC assay provides a real alternative for Public Health Laboratories to unambiguously confirm all cases of type A botulism and, due to its sensitivity, a promising new tool in toxin pharmacokinetic studies.