Xiaofei Hu

Rapid and sensitive detection of β-agonists using a portable fluorescence biosensor based on fluorescent nanosilica and a lateral flow test strip

A portable fluorescence biosensor with rapid and ultrasensitive response for Clenbuterol (CL) has been built up with fluorescent nanosilica and a lateral flow test strip. Quantitative detection of CL was realized by recording the fluorescence intensity of fluorescent nanosilica captured on the test line. The sensing results indicated that the sensitivity of the fluorescent nanosilica-based strip was better than that of conventional colloidal gold-based strips. The visual limit of detection of the strip for qualitative detection was 0.1 ng/mL while the LOD for quantitative detection could down to 0.037 ng/mL by using fluorescence biosensor. The recoveries of test samples were from 89.3% to 97.7%. The assay time for CL detection was less than 8 min, suitable for rapid testing on-site.

Development of an Immunochromatographic Strip Test for the Rapid Detection of Zearalenone in Corn

A rapid immunochromatographic test strip has been developed for the detection of zearalenone (ZEN) residues in corn. For this purpose, a specific anti-ZEN monoclonal antibody (mAb), 4A3-F9, was obtained and identified. ZEN coupled to bovine serum albumin (BSA) via 1,4-butanediol diglycidyl ether was prepared as immunogen. The mAb showed low cross-reactivity with five ZEN analogues. Using an antibody preparation with a titer of ≥1:5.12 × 10(5), the cross-reactivity (CR) of the anti-ZEN monoclonal antibody with four of the analogues was <4%, except for zearalanone, which was 53.121%. The recovery rates of ZEN in spiked corn samples were in the range of 91.30-97.07% with coefficients of variation <5.32%. An immunochromatographic strip was developed using the specific anti-ZEN monoclonal antibody and applied to the screening of corn samples for ZEN residues. The test could be accomplished within 5-10 min. The sensitivity of the test strip in corn sample extract was confirmed to be 20 μg/kg by unaided visual assessment, and the IC50 was calculated as 3.4 ng/mL using a test strip reader. The test strip, analyzed by unaided visual assessment and strip reader, showed very good agreement with competitive indirect ELISA and high-performance liquid chromatography (HPLC) analysis for naturally contaminated corn samples.

Rapid and Sensitive Detection of the Food Allergen Glycinin in Powdered Milk Using a Lateral Flow Colloidal Gold Immunoassay Strip Test

A rapid immunochromatographic lateral flow test strip in a sandwich format was developed with the colloidal gold-labeled mouse antiglycinin monoclonal antibody (mAb) and rabbit antiglycinin polyclonal antibody (pAb) to specifically identify glycinin, a soybean allergen. The test strip is composed of a sample pad, a conjugate reagent pad, an absorbent pad, and a test membrane containing a control line and a test line. This test strip has high sensitivity, and results can be obtained within 10 min without sophisticated procedures. The limit of detection (LOD) of the test strip was calculated to be 0.69 mg/kg using an optical density scanner that measures relative optical density. The assay showed high specificity for glycinin, with no cross-reactions with other soybean proteins or other food allergens. The recoveries of the lateral flow test strip in detecting glycinin in powdered milk samples ranged between 80.5 and 89.9% with relative standard deviations of less than 5.29% (intra-assay) and 6.72% (interassay). Therefore, the test strip is useful as a quantitative, semiquantitative, or qualitative detection method for glycinin in powdered milk. In addition, the test strip can be used to detect glycinin in other processed foods and may be a valuable tool in identifying effective approaches for reducing the impact of glycinin.

Development of a lateral-flow immunochromatographic test device for the rapid detection of difloxacin residues

Abstract In this study, a rapid competitive immunochromatographic test strip has been developed for specifically testing residues of difloxacin (DIF). 1H10-2B2, one of the three hybridoma lines that were tested and selected by enzyme linked immunosorbent assay, was used in the test strip. The monoclonal antibody has a good sensitivity with an IC50 of 2.2 ng/mL to DIF, 0.24% cross-reactivity towards danofloxacin and no cross-reactivity towards other related compounds and other drugs. The calibration curve of DIF test strip presented a typical sigmoidal curve. In practice, the lower limit of detection using a strip reader was 0.5 ng/ml while 2 ng/ml by unaided visual assessment. Samples of milk were spiked at 2–8 ng/mL, presenting recoveries between 94.5 and 107%, and the coefficient of variation (CV,%) (1.6–9%). The data above suggests that the strip meets the requirements of high sensitivity, good specificity, simplicity and speed, as well as the characteristics of reproducibility and accuracy.

Development of an immunochromatographic test strip for simultaneous qualitative and quantitative detection of ochratoxin A and zearalenone in cereal

BACKGROUND Ochratoxin A (OTA) and zearalenone (ZEN) are natural products of filamentous fungi that are harmful to humans and animals exposed to them even in extremely low concentration. The immunochromatographic test strip has become a popular diagnostic tool for detecting analytes. Its major advantages are that results can be obtained within 5-10 min, all needed reagents are included in the strip and it can be used to detect OTA and ZEN contamination in spots. In this study a colloidal gold-based immunochromatographic test strip of competitive format was developed for the rapid simultaneous qualitative and quantitative detection of OTA and ZEN in corn and other cereals. RESULTS The test strip results with the naked eye showed that the sensitivities were 6 µg kg(-1) OTA and 20 µg kg(-1) ZEN in cereal, while those with a TSR3000 membrane strip reader showed that the IC50 values of OTA and ZEN were 1.7905 and 4.3514 ng mL(-1) and the lower detection limit (LDL) values were 0.7697 and 1.2000 µg kg(-1) respectively. These results were confirmed by high-performance liquid chromatography. CONCLUSION The immunochromatographic test strip developed in this study could be used for the rapid simultaneous, qualitative and quantitative screening of OTA and ZEN in corn samples.

Selection of phage-displayed minotopes of ochratoxin A and its detection in cereal by ELISA

A competitive ELISA (cELISA) for the quantitative detection of ochratoxin A (OTA) was developed that uses a clone selected from a phage random peptide display library. An anti-OTA monoclonal antibody (mAb) was employed as the target for clone selection from a phage random heptapepide library. After four rounds of panning, 22 positive phage clones, in which binding to anti-OTA mAb could be blocked by free OTA, were obtained. The peptide sequences were deduced from DNA sequencing, and the consensual amino acid sequence of the selected peptides is MPLWXDL (X is any amino acid residue). A cELISA for detecting OTA was established using the selected phage clones. In the most sensitive assay, the linear range of the inhibition curve was 125–8000 pg mL−1. The half-inhibitory concentration (IC50) was 481.8 pg mL−1 and the detection limit was 103.2 pg mL−1. The analysis of 15 domestic cereal samples in parallel using this cELISA, a commercial ELISA kit and HPLC demonstrated the effectiveness of the method for detection of OTA in cereal samples.

Development of a colloidal gold-based strip test for the detection of chlorothalonil residues in cucumber

An immunochromatographic lateral flow strip test using the competitive format was developed for the determination of chlorothalonil (CTN) residues in cucumber. The limit of detection was less than 100 ng/mL by visual assessment and was found to be 91.78 ± 0.17 ng/mL for quantitative detection using a test strip reader. The recoveries of test samples were from 89.26% to 102.02% and the coefficient of variation (%) was less than 4.75%. Parallel analysis of cucumber samples with CTN showed comparable results from the strip test and high performance liquid chromatography. The strip test requires 5–8 min to complete and provides a very useful screening method for the quantitative and qualitative detection of CTN residues in cucumber.

Colloidal gold˗McAb probe˗based rapid immunoassay strip for simultaneous detection of fumonisins in maize

BACKGROUND Fumonisins are a kind of toxic and carcinogenic mycotoxin. A rapid immunochromatographic test strip has been developed for simultaneous detection of fumonisin B1 , B2 and B3 (FB1 , FB2 and FB3 ) in maize based on colloidal gold-labelled monoclonal antibody (McAb) against FB1 probe. RESULTS The anti-FB1 McAb (2E11-H3) was produced through immunisation and cell fusion, and identified as high affinity, specificity and sensitivity. The cross-reaction ratios with fumonisin B2 and B3 were accordingly 385% and 72.4%, while none with other analogues. The colloid gold-labelled anti-FB1 McAb probe was successfully prepared and used for establishing the immunochromatographic strip. The test strip showed high sensitivity and specificity, the IC50 for FB1 was 58.08 ng mL-1 , LOD was 11.24 ng mL-1 , calculated from standard curve. Moreover, the test strip exhibited high cross-reactivity with FB2 and FB3 , and could be applied to the simultaneous detection of FBs (FB1 :FB2 :FB3 = 12:4:1) in maize sample with high accuracy and precision. The average recoveries of FBs in maize ranged from 90.42% to 95.29%, and CVs were 1.25-3.77%. The results of the test strip for FBs samples showed good correlation with high-performance liquid chromatography analysis. CONCLUSION The immunochromatographic test strip could be employed in the rapid simultaneous detection of FB1 , FB2 and FB3 in maize samples on-site.

Eu3+‐labeled IgG‐based time‐resolved fluoroimmunoassay for highly sensitive detection of aflatoxin B1 in feed

BACKGROUND Aflatoxin B1 (AFB1 ) is a kind of toxic and carcinogenic mycotoxin. A time-resolved fluoroimmunoassay (TRFIA) was established for quantitative detection of AFB1 in feed using Eu3+ -labeled IgG as tracer. RESULTS Monoclonal antibody (McAb) against AFB1 (9B11-D7) was prepared through immunization and cell fusion and was identified as high affinity, specificity and sensibility by enzyme-linked immunosorbent assay (ELISA). The 50% inhibition value (IC50 ) was 0.81 ng mL-1 , the limit of detection (LOD) was 0.10 ng mL-1 and detection range was 0.10-3.94 ng mL-1 . Goat anti-mouse immunoglobulin G (IgG) was modified by Eu3+ -DATT, generating Eu3+ -labeled IgG. Under optimal assay conditions, TRFIA was shown to be highly sensitive and specific in detection of AFB1 . The IC50 and LOD were 94.73 pg mL-1 and 3.55 pg mL-1 , respectively, and detection range was 3.55-1.11 × 103  pg mL-1 . Cross-reactivity with AFM1 , AFB2 , AFG1 and AFG2 was 31.26%, 37.6%, 127.46% and 35.74%, respectively, but zero with other analogues. In determination of AFB1 spiked in feed sample, TRFIA showed high accuracy and precision. The average recoveries ranged from 93.71% to 97.80%, and coefficient of variation was 1.25-3.73%. Good correlation between TRFIA and HPLC was demonstrated for determination of AFB1 in feeds, confirming the reliability of the developed method. CONCLUSION The developed TRFIA exhibited good potential for employment in the ultrasensitive detection of AFB1 in feed and could be used to determine total aflatoxins.

Development of an ELISA for detection of Sudan I in food samples using monoclonal antibody

A highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been developed using a new monoclonal antibody for detecting the food colourant Sudan I. The half-maximum inhibition concentrations (IC50) and the limit of detection (calculated as IC20) of ELISA for Sudan I were 2 and 0.01 ng mL−1, respectively. The study showed little cross-activity with Sudan I structural analogues (below 0.01%). The average recoveries in intra- and interassays for Sudan I from fortified fresh tomato and chilli samples by ELISA were in ranges of 82–94% and 79–91%, respectively. The coefficients of variation of intra- and interassays were 6–8% and 6–10%, respectively. The IC50 was approximately 2.0 ng mL−1 after the Sudan I-Kit had been kept for 180 days.