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.

Carboxyl-Functionalized, Europium Nanoparticle-Based Fluorescent Immunochromatographic Assay for Sensitive Detection of Citrinin in Monascus Fermented Food

“…CIT must be coupled to a protein carrier to exhibit this immunogenicity because it is a kind of small molecular weight mycotoxin, which is non-immunogenic. Therefore, we prepared artificial antigens to obtain specific monoclonal antibodies by linking CIT to BSA and HSA by the formaldehyde method [37]. The high sensitivity of the fluorescence immunoassay depends on the quality and classification of probes formed by monoclonal antibodies and fluorescent materials.…”

A Sensitive Two-Analyte Immunochromatographic Strip for Simultaneously Detecting Aflatoxin M1 and Chloramphenicol in Milk

“…To further adsorb antibodies and serve as probes in immunostrips, 7 μg of AFM1 or 18 μg of CAP antibody in 0.1 mL of borax buffer (pH 6.0) were added dropwise to 2 mL of gold nanoparticle solution with constant stirring at room temperature for 1 h. Subsequently, 200 μL of BSA solution was added to stop the reaction for 30 min, centrifuged at 13,000× g rpm for 30 min at 4 °C, and then the supernatant was discarded. The pellets containing AFM1 Ab–gold or CAP Ab–gold nanoparticles were reconstituted in 0.2 mL of 20 mM Tris-buffer saline and stored at 4 °C for further usage [ 29 ].…”

Sensitive enzyme‐linked immunosorbent assay and gold nanoparticle immunochromatocgraphic strip for rapid detecting chloramphenicol in food

“…The 15-20 nm of gold nanoparticles were prepared (Wu, Yu, Liu, & Yu, 2018). The hydrogen tetrachloroaurate (III) trihydrate solution (19.9 mg of hydrogen tetrachloroaurate [III] trihydrate in 50 ml deionized water) was heated to boiling with steady stirring, 5 ml of 1% trisodium citrate was added and the color of the solution would change from yellow to transparent then black finally red with 5 min stirring.…”

“…The protocol of conjugation was referred to previously published (Wu et al, 2018). The 0.4 ml of gold nanoparticle solution was diluted for fivefold with boric acid-borax buffer (pH 6.0) and conjugated with CAP antibody (0.018 mg).…”