Development of a Sensitive Enzyme-Linked Immunosorbent Assay and Rapid Gold Nanoparticle Immunochromatographic Strip for Detecting Citrinin in Monascus Fermented Food

Abstract

Antibodies against citrinin (CTN) were generated from rabbits, which were injected with CTN-keyhole limpet hemocyanin (KLH). This work involved the development of a sensitive competitive direct enzyme-linked immunosorbent assay (cdELISA) and a rapid gold nanoparticle immunochromatographic strip (immunostrip) method for analyzing CTN in Monascus-fermented food. CTN at a concentration of 5.0 ng/mL caused 50% inhibition (IC₅₀) of CTN-horseradish peroxidase (CTN-HRP) binding to the antibodies in the cdELISA. The capable on-site detection of CTN was accomplished by a rapid antibody-gold nanoparticle immunostrip with a detection limit of 20 ng/mL and that was completed within 15 min. A close inspection of 19 Monascus-fermented foods by cdELISA confirmed that 14 were contaminated with citrinin at levels from 28.6⁻9454 ng/g. Further analysis with the immunostrip is consistent with those results obtained using cdELISA. Both means are sensitive enough for the rapid examination of CTN in Monascus-fermented food products.

Keywords: Citrinin; ELISA; Monascus fermented food; gold nanoparticle immunochromatographic strip.

Figure 1

(A). The standard curve of citrinin (●) in a ciELISA. (B). Cross-reactivity of the CTN polyclonal antibody with CTN (●), ochratoxin A (■), and 1-hydroxy-2-naphthoic acid (▲) as determined by a cdELISA. Data were calculated by the average of three sets of experiments. The absorbance of the control, A0, with no toxin present was 1.5.

Figure 2

A graphic description of the immunostrip. Ab-gold nanoparticle; C, control line (Goat anti-rabbit IgG); T, test line (CTN-OVA); released pad.

Figure 3

The visual detection limit of the immunostrip for CTN. Different concentrations (0–100 ng/mL) of a CTN certified standard was dissolved in PBS. (A) The visual detection limit is 20 ng/mL using 8 μL of Ab-gold nanoparticle conjugates were loaded into the sample solution; (B) the visual detection limit is approximately 20~50 ng/mL when 8 µL of conjugates were absorbed on the release pad. (C) The standard curve of the T line color density value without the release pad; when the value is less than 25 it indicates a positive result. (D) The standard curve for the T line color density value with the release pad; when the value is less than 25 it indicates a positive result. Each concentration was tested three repeats.

Figure 4

Analysis of CTN with the immunostrip in 19 red yeast fermented samples and one control rice sample. Samples 1–6 showed that the red line vanished in the test zone, which verified that they are CTN positive. Samples 7–19 containing CTN less than 20 ng/mL displayed two red lines indicating that they are negative. Each sample was tested three repeats.

Figure 5

Two different sizes of gold nanoparticles were used for conjugation with antibodies. The competitiveness of the antigen on the test line with antibody-gold nanoparticles of 15 nm (A) is better than that of the 40 nm particles (B).