Development of Microplate Immunoenzyme Determination of Nonylphenol with Magnetic Sample Concentration

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Resumo

Nonylphenol is an aromatic organic compound that has an estrogen-like effect and has a negative effect on the human endocrine system. A method has been developed for the competitive determination of nonylphenol using magnetic particles, rabbit antiserum, nonylphenol conjugate with soybean trypsin inhibitor (STI) and biotin. The principle of the analysis is the formation of immune complexes on the surface of magnetite particles due to covalent immobilization of protein G through the oriented immobilization of polyclonal antibodies from rabbit serum during a competitive reaction between the free analyte (nonylphenol) and the bound one (as part of the nonylphenol-STI-biotin conjugate) for the binding sites of specific antibodies. The detection of formed immune complexes is proposed to be carried out using a streptavidin-polyperoxidase conjugate, which makes it possible to achieve a nine-fold gain in the level of the analytical signal. The developed ELISA using magnetite particles allows us to achieve a detection limit of nonylphenol at the level of 3.8 ng/ml, which is 14.5 times lower in comparison with the classical competitive ELISA (55 ng/ml). Based on the results of the experimental work, the optimized volume of the test sample was 500 μl, which makes it possible to concentrate low-contaminated samples by 17 times.

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Sobre autores

A. Berlina

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences

Autor responsável pela correspondência
Email: dzantiev@inbi.ras.ru
Rússia, Moscow

L. Barshevskaya

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences

Email: dzantiev@inbi.ras.ru
Rússia, Moscow

K. Serebrennikova

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences

Email: dzantiev@inbi.ras.ru
Rússia, Moscow

N. Komova

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences

Email: dzantiev@inbi.ras.ru
Rússia, Moscow

A. Zherdev

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences

Email: dzantiev@inbi.ras.ru
Rússia, Moscow

B. Dzantiev

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences

Email: dzantiev@inbi.ras.ru
Rússia, Moscow

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2. Fig. 1. Scheme of ELISA with magnetic preconcentration.

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3. Fig. 2. Absorption spectrum of the NF-SIT conjugate. The cuvette thickness is 1 mm, the concentration of the conjugate in 10 mM FBS is 1.2 mg/ml.

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4. Fig. 3. Characterization of antiserum by ELISA: linear section of the competitive interaction curve (n = 3).

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5. Fig. 4. Testing the preservation of immunochemical activity of the hapten-protein conjugate before and after biotinylation (n = 2).

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6. Fig. 5. Testing of biotin-streptavidin binding in the NF-SIT-biotin preparation and selection of concentrations of the ST-HRP conjugate (A) and ST-rHRP (B) (n = 2).

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7. Fig. 6. Selection of the optimal concentration of the G-IgG protein (based on the concentration of magnetic particles) (n = 3).

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8. Fig. 7. Determination of the optimal concentration of the NF-SIT-biotin conjugate (n = 2). The dotted line indicates the cutoff at optical density 1.0.

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9. Fig. 8. Calibration curve for the determination of NF using the developed system based on MP (n = 3).

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10. Fig. 9. Dependence of the analytical signal in the developed ELISA based on MPs on the volume in which the concentration took place (n = 3).

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