Express detection of nonylphenol in water samples by fluorescence polarization immunoassay

The development of express method for detection of endocrine-disrupting chemicals (EDC) such as alkylphenols is required for ecological monitoring. Several attempts have been made to produce antibodies against 4-nonylphenol (NP) in recent years. This work describes the production of new antibodies against NP and also summarizes the characterization of antibodies obtained earlier. Three approaches used to produce alkylphenol-specific antibodies are compared; these are based on: 1. omega-(4-hydroxyphenyl)nonanoic or omega-(4-hydroxyphenyl)heptanoic acid NP derivatives designed to mimic the linear NP isomer; 2. 4-aminophenol, which potentially mimics various substituted phenolic compounds with different side-chain structures at position 4 of the benzene ring; and 3. a mixture of branched NP isomers, conjugated to the carrier protein via a benzene ring by the Mannich reaction, and expected to be the closest mimic of NP structure by preserving its natural alkyl moiety.Fluorescence polarization immunoassays based on different combinations of antibody and labeled antigen for screening detection of NP were developed and structural aspects of assay sensitivity and specificity were investigated. The assays based on the antisera raised against omega-(4-hydroxyphenyl)nonanoic acid and NP conjugate via Mannich reaction are capable of express detection of NP with detection limit of 7 microg mL(-1 )and assay dynamic range of 18-300 microg mL(-1).     

Pretreatment-free immunochromatographic assay for the detection of streptomycin and its application to the control of milk and dairy products

A rapid pretreatment-free immunochromatographic assay was developed for the control of the streptomycin (STR) content in milk and dairy products. The assay is based on the competition between an immobilized STR–protein conjugate and STR in a sample to be tested for the binding to monoclonal anti-STR antibodies conjugated to colloidal gold during the flow of the sample along a membrane strip with immobilized reactants. It is possible to improve the cut-off level of positive and negative samples distinguished by a change in the molar STR to protein ratio in the immobilized conjugate. The cut-off level (500 ng mL⁻¹) thus achieved corresponds to the stated MRL of STR in milk and dairy products. For STR concentrations in the range of 16–250 ng mL⁻¹ its content can be quantitatively measured based on the degree of binding of a colloidal gold label in the test strip zone with the immobilized STR–protein conjugate. The duration of the assay is 10 min. The selected sizes of membrane pores and colloidal gold particles allow the assay to be carried out at room temperature without additional reactants and pretreatment. The applicability of the assay for milk, whole milk, sour clotted milk, and kefir with different fat content (from 0.5% to 6%) was confirmed. The results of quantitative immunochromatographic assay show good correlation with traditional ELISA (r was equal to 0.935 and 0.940 for the series tested).     

Production of monoclonal antibodies against fullerene C60 and development of a fullerene enzyme immunoassay

The aim of the present study was to produce monoclonal anti-fullerene C(60) antibodies and to develop the enzyme immunoassay for the detection in the first use of free fullerene C(60) both in solutions and in multicomponent biological probes. The immunization of mice with the conjugate of fullerene C(60) carboxylic derivative with thyroglobulin synthesized by carbodiimide activation led to the production of eight clones of anti-fullerene antibodies. The specificity of the antibody-fullerene binding was confirmed. Indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for the determination of water-soluble protein-conjugated fullerene, the fullerene aminocaproic acid, fullerenol and for pristine fullerene in solution. To solubilize extremely hydrophobic free fullerene C(60) a specially selected water-organic mixture compatible with immunoassay was proposed. The detection limit of free fullerene C(60) in solution was 2 μg L(-1). Fullerene C(60) was also detected by ELISA in organ homogenates of rats intraperitoneally or intragastrically administered with fullerene. To reduce the influence of biomatrices on the assay results a technique was developed for the biological sample pretreatment by the extraction of C(60) from bioprobe by toluene followed by the evaporation of toluene and dissolution of the fullerene-containing extract in the selected water-organic media. The ELISA procedure in the first use allowed the detection of fullerene C(60) in different tissues.     

Application of flash photolysis for high threshold chemical reaction stimulation

The application of flash photolysis method for stimulation of high threshold chemical reaction is discussed.     

Comparison of two express immunotechniques with polyelectrolyte carriers, ELISA and FIIAA, for the analysis of atrazine

Conventional immunoassays on microtiterplates are very useful analytical tools in environmental analysis, but the long assay times, usually in the range of hours, are a drawback. To overcome this disadvantage, the development of fast (express) assay formats is described, which use polyelectrolytes as carriers. Two semi-homogeneous immunochemical methods, namely the polyelectrolyte-ELISA (enzyme-linked immunosorbent assay) and the express-FIIAA (flow injection immunoaffinity analysis) for the analysis of the herbicide atrazine were set-up. Using polyclonal antisera for atrazine, the following results were obtained. Standard curves for atrazine showed a linear range from 3 to 100 μg l⁻¹ in polyelectrolyte-ELISA and 0.3-100 μg l⁻¹ in express-FIIAA. The test midpoints in polyelectrolyte-ELISA and express-FIIAA were 12 and 5 μg l⁻¹, respectively. The duration time of the immunochemical reaction was in both assays 15 min, but the total assay time differed (30 min (polyelectrolyte-ELISA) and 18 min (express-FIIAA)). A significant difference between the formats could be observed in the number of samples that can be determined per day. The polyelectrolyte-ELISA can handle samples in parallel on a microtiterplate (usually 20/plate), whereas in the express-FIIAA the samples are automatically analysed one after another. This first demonstration of these techniques shows the potential of these methods, but also their limitations.     

Comparative study of strategies for antibody immobilization onto the surface of magnetic particles in pseudo-homogeneous enzyme immunoassay of aflatoxin B1

Pseudo-homogeneous enzyme immunoassay (EIA) of aflatoxin B1 (AFB1) was accomplished using anti-AFM1 monoclonal antibodies conjugated with magnetic particles (MPs). The assay includes the concentration of AFB1 from the test sample on the surface of the MP-antibody conjugate, the binding of the AFB1-peroxidase conjugate to free sites of the antibodies, the separation of the complexes that formed from unreacted components by means of magnetic field, and the evaluation of the enzymatic activity of MP-bound peroxidase. A comparative study of antibody conjugates, which were prepared by three different methods, namely, by physical adsorption on native MP and covalent binding to oleic acidor polystyrene-coated MPs, was performed. For these conjugates, the detection limits of EIA for AFB1 are 2.6, 0.4, and 0.6 ng/mL, respectively. The advantages of the pseudo-homogeneous EIA format as a tool for the highly sensitive control of toxic contaminants in food are the shorter time of incubation of immunoassay reagents (5 min; the total assay time is 20 min) and the possibility of concentrating the analyte from the test samples.     

Factors influencing the detection limit of the lateral-flow sandwich immunoassay: a case study with potato virus X

Key factors influencing the analyte detection limit of the sandwich immunochromatographic assay (ICA), namely, the size of gold nanoparticles, the antibody concentration, the conjugation pH, and characteristics of membranes, are discussed. The impacts of these factors were quantitatively characterized and compared for the first time using the same antigen (potato virus X). The antibody-colloidal gold conjugates synthesized at pH 9.0-9.5 (the pH was examined in the range from 7.5 to 10.0) and at an antibody concentration of 15 μg/mL (the concentration was tested from 10 to 100 μg/mL) demonstrated maximum binding with the analyte. The relationship between the size of gold nanoparticles and the ICA detection limit was determined. The detection limit decreases from 80 to 3 ng/mL (for antibodies with K (D) = 1.0 × 10(-9) M, data were obtained using a BIAcore X instrument) for a series of particles with a diameter from 6.4 to 33.4 nm (electron microscopy and dynamic light scattering data). In the case of larger particles (52 nm in diameter), the detection limit increases and reaches 9 ng/mL. A 10 mM phosphate buffer, pH 8, and a 50 mM phosphate buffer, pH 7, were the conditions of choice for the deposition of reactants. Taking into account these facts, we developed a lateral-flow test system for the rapid (10 min) detection of potato virus X in plant leaves. The ICA provided a visual detection limit of 3 ng/mL. In the case of the instrumental processing, potato virus X can be determined in the concentration range from 3 to 300 ng/mL with a detection limit 2 ng/mL.     

Development of a rapid, specific fluorescence polarization immunoassay for the herbicide chlorsulfuron

A strategy for design of a derivative of chlorsulfuron, which mimics half of the herbicide molecule, was proposed. The 1-[(2-chloro)phenylsulfonyl]monoamidosuccinic acid was synthesized as a derivative of chlorsulfuron for conjugation to carrier proteins. Rabbits were immunized and the resulting polyclonal antibodies were assessed by the fluorescence polarization technique. The antibodies were highly specific to chlorsulfuron. Cross-reactivity to the structurally similar sulfonylurea and urea herbicides chlorbromuron, amidosulfuron, chlortoluron, isoproturon, diuron and linuron was less than 0.1%. A rapid fluorescence polarization immunoassay (FPIA) for chlorsulfuron detection in water samples was developed and optimized. The detection limit of chlorsulfuron in 50 μl of sample was 10 ng ml⁻¹. Total time for the measurement of 10 samples is 7 min. The proposed FPIA is suitable for rapid testing for pesticide contamination where the highest sensitivity is not critical or in combination with pre-concentration techniques.