Yaning Sun

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 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 an immunochromatographic strip for antibody detection of pseudorabies virus in swine

An immunochromatographic strip was developed for the serological detection of pseudorabies virus (PRV) in swine. In the strip, the expressed protein of gB, one of the glycoproteins of PRV, labeled with colloidal gold, was used as the detector; staphylococcal protein A and swine anti–pseudorabies virus antibody were blotted on nitrocellulose membrane for the test and control lines, respectively. The specificity of the strip was 98.1%, and the sensitivity of the strip with reference anti-PRV serum was 96.0%. Swine serum samples (296) were collected to evaluate the characteristics of the strip in comparison with an existing commercial kit. The agreement was 93.6%. Furthermore, the dipstick assay based on the strip is rapid (5 min) and easy to perform with no requirement of professional skills, reagents, or equipment. This suggests that the immunochromatographic strip is an acceptable alternative for use in clinical laboratories lacking specialized equipment and for field diagnosis.

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.

Nanoparticle orientationally displayed antigen epitopes improve neutralizing antibody level in a model of porcine circovirus type 2

Recent advancements in biotechnology have enabled the rapid identification and subsequent expression of pathogenic microbial major antigens that induce protective immune responses. However, subunit vaccines have not been successfully commercialized mainly due to the lack of sufficient levels of neutralizing antibodies (NAs). High levels of NA rely on the efficient recognition and cross-linking of multiple neutralizing epitopes with B-cell receptors (BCRs). Nanoparticles are able to display coupled antigenic arrays at high density and provide multiple binding molecular scenarios with BCRs. The high-resolution antigenic structure makes it possible to accurately display stable neutralizing epitopes. Therefore, the development of a nanovaccine that orientationally displays neutralizing epitopes is a feasible strategy. To address this hypothesis, the capsid (Cap) protein of porcine circovirus type 2 as model antigen was conjugated to gold nanoparticles (AuNPs) through direct reaction of the mercapto group of the unique cysteines with AuNPs, rendering Cap-AuNPs to have neutralizing epitopes on outer surface and an immunodominant epitope buried within the inner surface. In vitro studies showed that AuNPs promoted the phagocytosis of Cap protein and NA levels were significantly improved, meanwhile antibody levels against the immunodominant epitope was significantly reduced. In mouse studies, Cap-AuNP-immunized mice displayed a high production of interleukin (IL)-4, IL-10, and interferon-γ, suggesting that Cap-AuNPs can effectively activate CD4+ and CD8+ T cells and balance Th1 and Th2 cellular responses. This study presents a new vaccine design strategy based on antigen structure, where nanoparticles are coupled to antigens in well-ordered arrays and orientationally display neutralizing epitopes to enhance NA levels.

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 immunochromatographic lateral flow strip for the simultaneous detection of aminoglycoside residues in milk

A colloidal gold-based immunochromatographic strip with a competitive format has been developed for the rapid, simultaneous, semi-quantitative and quantitative detection of several aminoglycoside residues in milk, including gentamicin sulfate (GM), neomycin sulfate (NEO) and kanamycin sulfate (KN). Three monoclonal antibodies against the three corresponding aminoglycosides were conjugated to colloidal gold particles and applied to the conjugate pads of the strip. The competitors [GM-bovine serum albumin (GM-BSA), NEO-BSA and KN-BSA conjugates] of GM, NEO and KN were immobilized onto a nitrocellulose (NC) membrane at three detection zones, T1, T2, and T3, respectively. The minimal cut-off values of the strip were 10 ng mL−1 for GM, and 100 ng mL−1 for NEO and KN, which are lower than the maximum residue levels (MRLs) established for aminoglycosides. The IC50 values of the strip were 0.737 ng mL−1, 8.971 ng mL−1 and 11.110 ng mL−1 for GM, NEO and KN respectively. In conclusion, the immunochromatographic lateral flow strip could be used for rapid, simultaneous, semi-quantitative and quantitative detection of GM, NEO and KN residues in milk.

Utilization of a lateral flow colloidal gold immunoassay strip based on surface-enhanced Raman spectroscopy for ultrasensitive detection of antibiotics in milk

An ultrasensitive method for the detection of antibiotics in milk is developed based on inexpensive, simple, rapid and portable lateral flow immunoassay (LFI) strip, in combination with high sensitivity surface-enhanced Raman spectroscopy (SERS). In our strategy, an immunoprobe was prepared from colloidal gold (AuNPs) conjugated with both a monoclonal antibody against neomycin (NEO-mAb) and a Raman probe molecule 4-aminothiophenol (PATP). The competitive interaction with immunoprobe between free NEO and the coated antigen (NEO-OVA) resulted in the change of the amount of the immobilized immunoprobe on the paper substrate. The LFI procedure was completed within 15min. The Raman intensity of PATP on the test line of the LFI strip was measured for the quantitative determination of NEO. The IC50 and the limit of detection (LOD) of this assay are 0.04ng/mL and 0.216pg/mL of NEO, respectively. There is no cross-reactivity (CR) of the assay with other compounds, showing high specificity of the assay. The recoveries for milk samples with added NEO are in the range of 89.7%-105.6% with the relative standard deviations (RSD) of 2.4%-5.3% (n=3). The result reveals that this method possesses high specificity, sensitivity, reproducibility and stability, and can be used to detect a variety of antibiotic residues in milk samples.