Enhanced surface plasmon resonance immunoassay for human complement factor 4

Using an enhanced surface plasmon resonance (SPR) immunosensor, we have determined the concentration of human complement factor 4 (C4). Antibody protein was concentrated into a carboxymethyldextran-modified gold surface by electrostatic attraction force and a simultaneous covalent immobilization of antibody based on amine coupling reaction took place. The sandwich method was applied to enhance the response signal and the specificity of antigen binding assay. The antibody immobilized surface had good response to C4 in the range of 0.02-20 mug/ml by this enhanced immunoassay. The regeneration effect by pH 2 glycine-HC1 buffer was also investigated. The same antibody immobilized surface could be used more than 80 cycles of C4 binding and regeneration. In addition, the ability to determinate C4 directly from serum sample without any purification was investigated. The sensitivity, specificity and reproducibility of the enhanced immunoassay are satisfactory. The results clearly demonstrate the advantages of the enhanced SPR technique for C4 immunoassay.

Real-time immunoassay of antibody activity in serum by surface plasmon resonance biosensor

A surface plasmon resonance biosensor has been used to determine antibody activity in serum. As a model system, the interaction of mouse IgG and sheep anti-mouse IgG polyclonal antibody was investigated in real time. The factors, including pH value, ionic strength, protein concentration, influencing electrostatic adsorption of mouse IgG protein onto carboxylated dextran-coated sensor chip surface, were studied. The procedures of mouse IgG protein immobilization and immune reaction were monitored in real time. The regeneration effect using the different elution reagents was also investigated. The same mouse IgG immobilized surface can be used for 100 cycles of binding and elution with only 0.38% loss per regeneration in reactivity. The results show that the surface plasmon resonance biosensor is a rapid, simple, sensitive, accurate and reliable detection technique for real-time immunoassay of antibody activity. The assay allows antibodies to be detected and studied in their native form without any purification. (C) 2000 Elsevier Science B.V. All rights reserved.

Synthesis and properties of two novel Ru(phen)(3)(2+)-based probes for electrochemiluminescent immunoassay

Two novel Ru(phen)(3)(2+)-based probes for electrochemiluminescent immunoassay, Ru(phen)(2)(phen-NHCOCH2CH2COOH)(PF6)(2) and Ru(phen)(2) (phen-NHCOCH2CH2CH2COOH) (PF6)(2), were designed, synthesized and characterized. Electrochemistry, Fluorescence, and electrochemiluminescence of the two complexed are also reported.

Development and Single-Laboratory Validation of a Pseudofunctional Biosensor Immunoassay for the Detection of the Okadaic Acid Group of Toxins

A rapid analytical optical biosensor-based immunoassay was developed and validated for the detection of okadaic acid (OA) and its structurally related toxins from shellfish matrix. The assay utilizes a monoclonal antibody which binds to the OA group of toxins in order of their toxicities, resulting in a pseudofunctional assay. Single-laboratory validation of the assay for quantitative detection of OA determined that it has an action limit of 120 mu g/kg, a limit of detection of 31 mu g/kg, and a working range of 31-174 mu g/kg. The midpoint on the standard matrix calibration curve is 80 mu g/kg, half the current regulatory limit. Inter- and intra-assay studies of negative mussel samples spiked with various OA concentrations produced average coefficient of variation (CV) and standard deviation (SD) values of 7.9 and 10.1, respectively. The assay was also validated to confirm the ability to accurately codetect and quantify dinophysistoxin-1 (DTX-1), DTX-2, and DTX-3 from shellfish matrix. Alkaline hydrolysis was not required for the detection of DTX-3 from matrix. Excellent correlations with the data generated by the biosensor method and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were obtained using a certified reference material (R-2 = 0.99), laboratory reference material, and naturally contaminated mussel samples (R-2 = 0.97). This new procedure could be used as a rapid screening procedure replacing animal-based tests for DSP toxins.

Rapid Surface Plasmon Resonance Immunoassay for the Determination of Deoxynivalenol in Wheat, Wheat Products, and Maize-Based Baby Food

A rapid screening assay (9 min/sample) has been developed and validated for the detection of deoxynivalenol in durum wheat, wheat products, and maize-based baby foods using an SPA biosensor. Through a single laboratory validation, the limits of detection (LOD) for wheat, wheat-based breakfast cereal, and maize-based baby food were 57, 9, and 6 mu g/kg, respectively. Intra-assay and interassay precisions were calculated for each matrix at the maximum and half-maximum European Union regulatory limits and expressed as the coefficient of variation (CV). All CVs fell below 10% with the exception of the between-run CV for breakfast cereal. Recoveries at the concentrations tested ranged from 92 to 115% for all matrices. Action limits of 161, 348, and 1378 mu g/kg were calculated for baby food, wheat-based breakfast cereal, and wheat, respectively, and the linear range of the assay was determined as 250-2000 mu g/kg.

Hapten synthesis and antibody production for the development of a melamine immunoassay

The incorporation of melamine into food products is banned but its misuse has been widely reported in both animal feeds and food. The development of a rapid screening immunoassay for monitoring of the substance is an urgent requirement. Two haptens of melamine were synthesized by introducing spacer arms of different lengths and structures on the triazine ring of the analyte molecular structure. 6-Aminocaproic acid and 3-mercaptopropionic acid were reacted with 2-chloro-4,6-diamino-1,3,5-triazine (CAAT) to produce hapten 1[3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylamino) hexanoic acid] and hapten 2[3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylthio) propanoic acid]. respectively. The molecular structures of the two haptens were identified by I H nuclear magnetic resonance spectrometry, mass spectrometry and infrared spectrometry. An immunogen was prepared by coupling hapten 1 to bovine serum albumin (BSA). Two plate coating antigens were prepared by coupling both haptens to egg ovalbumin (OVA). A competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed to evaluate homogeneous and heterogeneous assay formats. The results showed that polyclonal antibodies with high titers were obtained, and the heterogeneous immunoassay format demonstrated a better performance with an IC50 of 70.6 ng mL(-1), a LOD of 2.6 ng mL(-1) and a LOQ of 7.6 ng mL(-1). Except for cyromazine, no obvious cross-reactivity to common compounds was found. The data showed that the hapten synthesis was successful and the resultant antisera could be used in an immunoassay for the rapid and sensitive detection of this banned chemical. (C) 2010 Elsevier B.V. All rights reserved.

A rapid optical immunoassay for the screening of T-2 and HT-2 toxin in cereals and maize-based baby food

A rapid surface plasmon resonance (SPR) screening assay has been developed for the combined detection of T-2 and HT-2 toxins in naturally contaminated cereals using a sensor chip coated with an HT-2 toxin derivative and a monoclonal antibody. The antibody raised against HT-2 displayed high cross-reactivity with T-2 toxin while there was no cross-reaction observed with other commonly occurring trichothecenes. A simple extraction procedure using 40% methanol was applied to baby food, breakfast cereal, and wheat samples prior to biosensor analysis. Limits of detection (LOD) for each matrix were determined as 25 mu g kg(-1) for baby food and breakfast cereal and 26 mu g kg(-1) for wheat. Intra-assay precision (n = 6) was calculated for each matrix. The results were expressed as the relative standard deviation and determined as 2.8% (100 mu g kg(-1)) and 1.8% (200 mu g kg(-1)) in breakfast cereal, 4.6% (50 mu g kg(-1)) and 3.6% (100 mu g kg(-1)) in wheat and 0.97% (25 mu g kg(-1)) and 6.3% (50 mu g kg(-1)) in baby food. Between run precision (n = 3) performed at the same levels yielded relative standard deviations of 6.7% and 3.9% for breakfast cereals, 3.3% and 1.6% for wheat and 6.8% and 0.08% for baby food, respectively. (C) 2010 Elsevier B.V. All rights reserved.

Comparison of porcine urine and bile as matrices to screen for the residues of two sulfonamides using a semi-automated enzyme immunoassay.

Porcine urine enzyme immunoassays for sulfamethazine and sulfadiazine have previously been employed as screening tests to predict the concentrations of the drugs in the corresponding tissues (kidneys), If a urine was found positive (> 800 ng ml(-1)) the corresponding kidney was then analysed by an enzyme immunoassay and, if found positive, a confirmatory analysis by HPLC was performed. Urine was chosen as the screening matrix since sulfonamides are mainly eliminated through this body fluid, However, after obtaining a number of false positive predictions, an investigation was carried out to assess the possibility of using an alternative body fluid which would act as a superior indicator of the presence of sulfonamides in porcine kidney, An initial study indicated that serum, plasma and bile could all be used as screening matrices. From these, bile was chosen as the preferred sample matrix and an extensive study followed to compare the efficiencies of sulfonamide positive bile and urine at predicting sulphonamide positive kidneys, Bile was found to be 17 times more efficient than urine at predicting a sulfamethazine positive kidney and 11 times more efficient at predicting a sulfadiazine positive kidney, With this enhanced performance of the initial screening test, the need for the costly and time consuming kidney enzyme immunoassay, prior to HPLC analysis, was eliminated