Development of a Novel Immunoaffinity Column for Aflatoxin Analysis Using an Organic Solvent-Tolerant Monoclonal Antibody

Abstract

An organic solvent-tolerant monoclonal antibody specific to aflatoxins B(1), B(2), G(1), G(2), and M(1) (AFB(1), AFB(2), AFG(1), AFG(2), and AFM(1)) was prepared. In an indirect competitive enzyme-linked immunosorbent assay, the half maximal inhibitory concentration (IC(50)) values were 1.9, 2.1, 2.1, 2.4, and 2.8 ng/mL for AFB(1), AFB(2), AFG(1), AFG(2), and AFM(1), respectively. Antibody reactivity was retained at 40% methanol concentration or at acetonitrile concentrations up to 40%. An immunoaffinity column (IAC) was prepared using agarose gel beads with bound antibody. The IAC retained the tested AFs that were 89, 90, 95, 90, and 89% for AFB(1), AFB(2), AFG(1), AFG(2), and AFM(1) at 20% acetonitrile concentrations or that were 81, 87, 79, and 83% for AFB(1), AFB(2), AFG(1), and AFG(2) at 60% methanol concentrations. Roasted peanuts and seven kinds of spices were spiked with 8.0, 1.0, 6.0, and 1.0 ng for AFB(1), AFB(2), AFG(1), and AFG(2) per 1 g sample and extracted with 90% acetonitrile. The roasted peanuts and cayenne pepper out of the spices were also extracted with 70% methanol. The extracts were diluted 5-fold with phosphate-buffered saline and applied to the IAC. The spiked aflatoxins were recovered with satisfactory rates: 78 (RSD, 2.1%) to 127% (RSD, 1.7%). The developed IAC was used for the analysis of aflatoxins in naturally contaminated samples of roasted peanuts and cayenne pepper. The newly developed IAC showed substantially organic solvent tolerance at the concentration that could not be used for existing IACs, and the column showed good ability to clean up samples for food analysis.

Determination of sterigmatocystin in grain using gas chromatography-mass spectrometry with an on-column injector

“…According to Uchigashima et al (2009), the binding capacity of the IAC was at least 400 ng (0.2 mL gel/column) for total aflatoxins (AFB 1 , AFB 2 , AFG 1 , and AFG 2 : 100 ng each). Recently, Sasaki et al (2014) reported the use of an IAC for STC cleanup because it shows cross-reactivity with aflatoxins and reported that the binding capacity of the IAC for STC was at least 200 ng/column and recovery exceeded 95 %.…”

OPTIMIZATION AND EFFICIENCY IMPROVEMENT OF IN‐HOUSE IMMUNOAFFINITY COLUMN KU-AF02 FOR AFLATOXIN DETECTION

“…For instance, in developing IAC for zearalenone and ochratoxin clean-up, some research found that 1 mg antibody coupled with 1 mL of CNBr-activated sepharose 4B, as supporting material, provide 40 -50% stability toward high concentration of acetonitrile and methanol (Uchigashima et al 2009;Uchigashima et al 2012). The ratio used was obviously lower than the recommended ratio, but still the recovery percentage were satisfactorily ranging from 80 to 100%.…”

Sampling Techniques for the Determination of Mycotoxins in Food Matrices

“…68,69 A further improvement of the IAC technique was recently proposed by using organic solvent-tolerant monoclonal antibody for the production of IAC columns which have been applied to check for contamination of different spices: in particular, tolerance and good cleanup ability in the presence of acetonitrile and up to 60% methanol were observed. 70 Also, multifunctional column cleanup was applied to the detection of aflatoxins and ochratoxin in spices: for aflatoxins after extraction with acetonitrile:water (9:1), cleanup was performed using MultiSep 228 column for LC-FLD analysis, obtaining good recovery values (80-85%); for ochratoxin A, Mycosep 229 was used. 71 A simplified method was recently proposed for simultaneous determination of aflatoxins, dyes, and pesticides in spices, using acetonitrile for extraction via ultrasonic bath and, after dilution of the extract with pure water, direct injection in the LC-MS/MS system.…”