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

Abstract A simple procedure was developed for in vitro synthesis and characterization of aflatoxin B1-lysine adduct using aflatoxin B1, N-α-acetyl lysine and m-chloroperbenzoic acid (MCPBA). At a molar ratio of 1:16 (aflatoxin B1:N-α-cetyl lysine), the recovery of adduct was 62%. Analysis of the adduct by thinlayer chromatography showed a single spot (Rf= 0). Absorption spectra of the adduct showed 2 peaks at 275 and 335 nm. Liquid chromatographic (LC) analysis of th AFB1-lysine adduct showed a relative retention time of 2.1 min. Using the same epoxidation procedure, BSA-AFB1 adduct and ovalbumin-AFB1 adduct were synthesized for production of antibodies and as coating antigen, respectively. Control rat serum, spiked with AFB1-lysine adduct and subjected to LC analysis showed a retention time of 2.1 min, which is similar to that of AFB1-lysine reference standard, synthesized. Further, enzymatically hydrolyzed, control rat serum spiked with BSA-AFB1 adduct showed 2 peaks with retention times of 2.1 and 2.7 min. Based on the LC analysis, recovery of BSA-AFB1 in terms of AFB1-lysine adducts was 67 ± 5%. The major peak (2.1 min) accounted for 72% of the adduct; the second minor peak (2.7 min) accounted for 28% of the total AFB1-lysine adducts formed. Stability studies on the AFB1-lysine adduct synthesized, indicated that it was stable for 1 month. Antibody capture assay showed an absorbance of 0.9 to 1.0 at a dilution of 1:50 000 when ovalbumin-AFB1 was used as a coating antigen. Indirect competitive ELISA showed 50% displacement (IC50) of the antibodies at a concentration of 13 ng AFB1-lysine, whereas the IC50 for AFB1 was 7 ng. The recovery of AFB1-lysine adduct spiked to control rat serum followed by enzymatic hydrolysis and immunoanalysis (indirect ELISA) was 93 ± 6%. The enzyme immunoassay was validated by a rodent model, in which the animals were exposed to aflatoxin B1 (20 μg AFB1/kg body mass/day). The level of AFB1-lysine adduct in the rat serum was 27.3 ± 4.37 μg/mg albumin.

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Enzyme Channelling Immunoassay. A New Homogeneous Enzyme Immunoassay Technique

Enzyme Engineering ◽

10.1007/978-1-4684-3749-2_27 ◽

1980 ◽

pp. 179-186

Author(s):

Edwin F. Ullman ◽

David J. Litman

Keyword(s):

Enzyme Immunoassay ◽

Immunoassay Technique ◽

Homogeneous Enzyme Immunoassay ◽

Enzyme Immunoassay Technique

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Principles of Homogeneous Enzyme-Immunoassay

Enzyme-Immunoassay ◽

10.1201/9781351071840-5 ◽

2018 ◽

pp. 105-134

Author(s):

Edwin F. Ullman ◽

Edward T. Maggio

Keyword(s):

Enzyme Immunoassay ◽

Homogeneous Enzyme Immunoassay

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Monitoring of Therapeutic Serum Concentrations of Antiepileptic Drugs by a Newly Developed Gas Chromatographic Procedure and Enzyme Immunoassay (EMIT): A Comparative Study

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm.1981.19.5.249 ◽

1981 ◽

Vol 19 (5) ◽

Author(s):

W. R. Külpmann ◽

M. Oellerich

Keyword(s):

Comparative Study ◽

Enzyme Immunoassay ◽

Antiepileptic Drugs ◽

Serum Concentrations ◽

Chromatographic Procedure

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A comparative study on magnetic and non-magnetic particles in nanofluid propagating over a wedge

Canadian Journal of Physics ◽

10.1139/cjp-2018-0159 ◽

2019 ◽

Vol 97 (3) ◽

pp. 277-285 ◽

Cited By ~ 36

Author(s):

Mohsan Hassan ◽

Rahmat Ellahi ◽

Muhammad Mubashir Bhatti ◽

Ahmed Zeeshan

Keyword(s):

Comparative Study ◽

Magnetic Particles

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Enzyme Immunoassay for Measuring Aflatoxin B1 in Legal Cannabis

Toxins ◽

10.3390/toxins12040265 ◽

2020 ◽

Vol 12 (4) ◽

pp. 265 ◽

Cited By ~ 1

Author(s):

Fabio Di Nardo ◽

Simone Cavalera ◽

Claudio Baggiani ◽

Matteo Chiarello ◽

Marco Pazzi ◽

...

Keyword(s):

Enzyme Immunoassay ◽

Aflatoxin B1 ◽

Analytical Methods ◽

Limit Of Detection ◽

Direct Analysis ◽

Aflatoxin Contamination ◽

Throughput Capacity ◽

Human Consumption ◽

The European Union ◽

Coefficients Of Variation

The diffusion of the legalization of cannabis for recreational, medicinal and nutraceutical uses requires the development of adequate analytical methods to assure the safety and security of such products. In particular, aflatoxins are considered to pose a major risk for the health of cannabis consumers. Among analytical methods that allows for adequate monitoring of food safety, immunoassays play a major role thanks to their cost-effectiveness, high-throughput capacity, simplicity and limited requirement for equipment and skilled operators. Therefore, a rapid and sensitive enzyme immunoassay has been adapted to measure the most hazardous aflatoxin B1 in cannabis products. The assay was acceptably accurate (recovery rate: 78–136%), reproducible (intra- and inter-assay means coefficients of variation 11.8% and 13.8%, respectively), and sensitive (limit of detection and range of quantification: 0.35 ng mL−1 and 0.4–2 ng mL−1, respectively corresponding to 7 ng g−1 and 8–40 ng g−1 ng g−1 in the plant) and provided results which agreed with a HPLC-MS/MS method for the direct analysis of aflatoxin B1 in cannabis inflorescence and leaves. In addition, the carcinogenic aflatoxin B1 was detected in 50% of the cannabis products analyzed (14 samples collected from small retails) at levels exceeding those admitted by the European Union in commodities intended for direct human consumption, thus envisaging the need for effective surveillance of aflatoxin contamination in legal cannabis.

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Homogeneous enzyme immunoassay for tobramycin evaluated and compared with a radioimmunoassay.

Clinical Chemistry ◽

10.1093/clinchem/28.6.1370 ◽

1982 ◽

Vol 28 (6) ◽

pp. 1370-1374 ◽

Cited By ~ 5

Author(s):

J Woo ◽

M A Longley ◽

D C Cannon

Keyword(s):

Enzyme Immunoassay ◽

Room Temperature ◽

Correlation Study ◽

Stability Study ◽

Data Handling ◽

Handling System ◽

Analytical Recovery ◽

Assay Precision ◽

Absorbance Data ◽

Homogeneous Enzyme Immunoassay

Abstract We evaluated a commercially available homogeneous enzyme immunoassay (EMIT, Syva Co.) for tobramycin against a reference radioimmunoassay (RIA) method. Between-assay precision (CV) was 2.9% at 6.2 mg/L and 3.0% for values in the range of 1.0-7.6 mg/L. Accuracy based on a recovery experiment (1.0-13.0 mg/L) yielded an analytical recovery of 88-112%. A correlation study with 75 sera from patients on tobramycin therapy showed that EMIT = 0.984 RIA - 0.0808, r = 0.993. Neither the EMIT nor the RIA procedure was affected by the presence of gentamicin, amikacin, and vancomycin. Absorbance data from the EMIT system calculated with the conventional RIA logit-log algorithm correlate well with results generated by the Syva data-handling system (logit-log = 1.077 Syva - 0.318, r = 0.998). A reagent stability study indicated that the EMIT reagents, once reconstituted, remain stable for at least 17 days when stored at refrigerated temperatures, or 11 days if stored at room temperature, thus enabling frequent "stat" assays without the need to prepare a calibration curve each time.

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