Zhi-Li Xiao

Simultaneous determination of malachite green, brilliant green and crystal violet in grass carp tissues by a broad-specificity indirect competitive enzyme-linked immunosorbent assay.

An immunizing hapten (4-(carboxymethoxy)phenyl)bis(4-(diethylamino)phenyl)methylium for brilliant green (BG), a triphenylmethane dye with a potential illegal use in fish feeding, was synthesized and used to produce polyclonal antibody (PcAb) against BG. Unexpectedly, the obtained PcAb showed high cross-reactivity (CR) to malachite green (MG) and crystal violet (CV) in an indirect competitive enzyme-linked immunosorbent assay (icELISA). After screening against three heterologous coating antigens, the icELISA exhibited good sensitivity and uniform response to BG (IC(50) of 1.98 ng mL(-1) and CR of 100%), MG (IC(50) of 1.61 ng mL(-1) and CR of 105%) and CV (IC(50) of 1.34 ng mL(-1) and CR of 142%) when using (4-(carboxymethoxy)phenyl)bis(4-(dimethylamino)phenyl)methylium as the coating hapten. Therefore, a broad-specificity icELISA for simultaneous determination of BG, MG and CV was developed. The recoveries of single analyte and mixture of three analytes from spiked grass carp tissues were estimated ranging from 74.94% to 110.39%. A statistically significant correlation of results was obtained between the developed icELISA and previously established HPLC approaches with the food-relevant three triphenylmethane dyes concentration range 1.83-200 ng mL(-1) (R(2)=0.9224), indicating good accuracy of the icELISA and suitability for the broad-specific detection of the three triphenylmethane dyes in grass carp tissues.

Enhanced sensitive immunoassay: noncompetitive phage anti-immune complex assay for the determination of malachite green and leucomalachite green.

To develop a more sensitive immunoassay for malachite green (MG) and leucomalachite green (LMG), we identified the immunocomplex binding phage-borne peptides for use in the noncompetitive phage anti-immunocomplex assay (PHAIA). An anti-LMG monoclonal antibody (mAb) was used to select immunocomplex binding peptides from a circular random eight-amino-acid phage-displayed library. After three rounds of panning-elution, five peptides that bound the LMG–mAb immunocomplex were obtained. One of the phage-borne peptide clones that resulted in an assay with the highest sensitivity was chosen for further research. The concentration of LMG producing 50% of the saturated signal and the limit of detection of the assay were 7.02 and 0.55 ng/mL, respectively, with a linear range of 1.35 to 21.56 ng/mL. The PHAIA based on the same antibody was 16 times more sensitive compared to the competitive immunoassay. PHAIA was used to analyze LMG, MG, and two mixtures of spiked fish samples, with validation by high-performance liquid chromatography (HPLC) with fluorescence detector. Results showed a good correlation (R2LMG = 0.9841; R2MG = 0.993; R2Mixture = 0.9903) between the data of PHAIA and HPLC, thus the assay was an efficient method for monitoring food safety.

Synthesis of novel haptens and development of an enzyme-linked immunosorbent assay for quantification of histamine in foods.

Novel haptens were designed and synthesized to prepare antibodies against free histamine, but none resulted in producing suitable antibodies for developing an enzyme-linked immunosorbent assay (ELISA). However, an antiserum was obtained having high specificity and affinity to p-nitrobenzoylated histamine (NPHA), which can be easily formed from reaction between histamine and p-nitrobenzoic acid N-hydroxysuccinimide ester (PNBA-OSu) under mild conditions. Based on rabbit polyclonal antibodies, a competitive indirect ELISA (ciELISA) for histamine determination in foods was developed. After ciELISA and derivatization optimization, the assay showed good sensitivity, with limits of detection of 1.8 mg/kg, 93.6 μg/L, and 93.6 μg/kg in fish, red wine, and yoghurt, respectively, with negligible cross-reactivity with related biogenic amines and amino acids. Average recovery of histamine in fortified food samples ranged from 80.9% to 110.1% with coefficients of variation below 16.3%. Good correlation between the ciELISA and liquid chromatography-tandem mass spectrometry was obtained for spiked food samples.

Hapten synthesis and development of a competitive indirect enzyme-linked immunosorbent assay for acrylamide in food samples.

The high level of acrylamide in widely consumed processed foods poses a potentially significant risk to human health, which has led to an increasing demand for rapid, simple, and selective analytical methods. In the present work, several haptens for acrylamide were designed in an attempt to prepare antibodies with acrylamide affinity, but they failed their purpose. However, a polyclonal antibody was produced against 4-mercaptophenylacetic acid (4-MPA)-derivatized acrylamide, which showed high binding affinity to the derivative. As acrylamide easily reacted with 4-MPA at high derivation yield, a competitive indirect enzyme-linked immunosorbent assay (ciELISA) for acrylamide via a preanalysis derivatization was developed. The derivatization and ELISA conditions were fully optimized to produce a method for acrylamide assay that exhibited an IC50 of 2.86 μg/kg, limit of detection at 0.036 μg/kg, and linear range of 0.25-24.15 μg/kg. The results of preanalysis recovery tests of acrylamide-spiked food samples and screening of blind food samples by both ciELISA and HPLC-MS/MS indicated the proposed ciELISAs good accuracy and reliability. This method was thus deemed suitable for routine acrylamide screening in food samples at low cost.

Development of a Monoclonal Antibody-Based Competitive Indirect Enzyme-Linked Immunosorbent Assay for Furaltadone Metabolite AMOZ in Fish and Shrimp Samples

A monoclonal antibody-based competitive indirect enzyme-linked immunosorbent assay (ELISA) with improved sensitivity and specificity for the determination of furaltadone metabolite 5-methylamorpholino-3-amino-2-oxazolidone (AMOZ) was described. AMOZ was derivatized with 2-(3-formylphenoxy)acetic acid and coupled with bovine serum albumin to form a novel immunogen. BABL/c mice were immunized and monoclonal antibody specific to the nitrophenyl derivative of AMOZ (NP-AMOZ) was produced and characterized. Four other haptens with different heterology to the immunizing hapten were synthesized and coupled to ovalbumin as coating antigens to study the effect of heterologous coating on assay sensitivity. Under the optimized heterologous coating format, the competitive indirect ELISA showed very high sensitivity to NP-AMOZ, with an IC(50) of 0.14 μg/L and limit of detection of 0.01 μg/L. The assay showed high specificity toward NP-AMOZ, and negligible cross-reactivity with analogous compounds was observed. The average recoveries of AMOZ from spiked fish and shrimp samples were estimated to range from 81.0 to 104.0%, with coefficients of variation below 20%. Good correlation was obtained between the results of ELISA analysis and of standard liquid chromatography-tandem mass spectrometry analysis. These results indicated that the proposed ELISA is ideally suited as a monitoring method for AMOZ residues at trace level.

Design and synthesis of immunoconjugates and development of an indirect ELISA for rapid detection of 3, 5-dinitrosalicyclic Acid hydrazide.

In this study novel immunoconjugates were designed, synthesized and then used to develop a rapid, specific and sensitive indirect ELISA method to directly detect residues of 3,5-dinitrosalicyclic acid hydrazide (DNSH), a toxic metabolite of nifursol present in chicken tissues. The hapten DNSHA was first designed and used to covalently couple to BSA to form an immunogen which was immunized to rabbits to produce a polyclonal antibody against DNSH. Furthermore, a novel 3,5-dinitrosalicylic acid-ovalbumin (DNSA-OVA) immunoconjugate structurally different from DNSHA-OVA was designed and used as a “substructural coating antigen” to improve the sensitivity of an indirect ELISA analysis for a direct DNSH detection. Based on the “substructural coating antigen” concept, an optimized indirect ELISA method was established that exhibited good specificity and high sensitivity for detecting DNSH, with a cross-reactivity of less than 0.1% (excluding the parent compound nifursol), IC50 of 0.217 nmol/mL and detection limit of 0.018 nmol/mL. Finally, a simple and efficient analysis of DNSH samples in chicken tissues showed that the average recovery rate of the indirect ELISA analysis was 82.3%, with the average coefficient of variation 15.9%. Thus, the developed indirect ELISA method exhibited the potential for a rapid detection of DNSH residues in tissue.

A sensitivity-enhanced heterologous immunochromatographic assay based on a monoclonal antibody for the rapid detection of histamine in saury samples

Histamine (HA) is an essential test item for fishery samples. However, the fast and effective determination of HA is difficult due to its simple structure and small molecular size. In this study, a sensitive and specific monoclonal antibody against p-nitrobenzoylated histamine (NPHA), which can be easily obtained from the reaction of HA and p-nitrobenzoic acid N-hydroxysuccinimide ester (PNBA-OSu) under mild conditions, was generated for the first time. Based on this mAb, an immunochromato-graphic assay strip (ICA strip) using a colloidal gold nano particles-antibody (GNPs) probe for rapid detection of HA in saury samples was established. After screening the coating antigens and optimization of analytical parameters, a heterologous coating based ICA strip exhibited the most excellent detection ability with a visual detection limit (VDL) of 6.0 mg kg−1 in qualitative experiments and a detection limit (by a strip reader) of 1.0 mg kg−1 in semi-quantitative experiments for saury samples, with no cross-reactivity with HA analogs. Good correlation between the ICA strip with liquid chromatography-tandem mass spectrometry was achieved for spiked and naturally contaminated saury samples. Overall, this method is suitable for screening of HA residue for large scale fish samples in a quick, simple and low-cost manner.

Production and identification of high affinity monoclonal antibodies against pesticide carbofuran

Abstract To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for peoples health and environmental protection, the hapten 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl]-amino]-butanoic acid (BFNB) of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier (BFNB-bovine serum albumin, BFNB-BSA) conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay (ELISA), based on BFNB-ovoalbumin conjugates (BFNB-OVA). Purified monoclonal antibody (McAb) was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line (5D3) secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1:2.048 × 10 3 and 1:1.024 × 10 6 , respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 10 9 L mol −1 , with an IC 50 value of 1.18 ng mL −1 and a detection limit of 0.01 ng mL −1 . Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized (4.59 × 10 −4 % for 2,3-dihydro-2,2-dimethyl-7-benzofuranol and less than 3.0 × 10 −4 % for others). The prepared McAb had a very high affinity and specificity, and it could be used to develop ELISA for rapid determination of carbofuran.

Novel haptens synthesis and development of a monoclonal antibody-based enzyme-linked immunosorbent assay for leuco-malachite green in fish

ABSTRACT To produce specific antibodies against leuco-malachite green (LMG), 15 haptens were synthesized and characterized, and conjugated to carrier protein for immunization. One antigen with excellent reactogenicity and immunogenicity was discovered. Specific monoclonal antibody with high sensitivity for LMG in competitive indirect enzyme-linked immunosorbent assay (ciELISA) was screened and selected. After the screening of a series of heterologous coating antigen and optimization of working conditions, the proposed ciELISA showed the 50% inhibition value (IC50) of 1.16 ng mL−1 and the limit of detection of 0.06 ng mL−1 for LMG. The average recoveries of LMG from spiked fish samples ranged from 78.0% to 101.0%, with coefficients of variation below 15%. Good correlation (R2 = 0.9977) was obtained between the results of ciELISA analysis and those of standard liquid chromatography–tandem mass spectrometry analysis. The proposed ciELISA is ideal for the rapid and sensitive detection of LMG with a low cost and high throughput.

Bispecific Monoclonal Antibody-based Multi-analyte ELISA for Furaltadone Metabolite, Malachite Green and Leucomalachite Green in Aquatic Products

A new multianalyte immunoassay was designed to screen furaltadone metabolite 5-morpholinomethyl-3-amino-2-oxazolidone (AMOZ), malachite green (MG), and leucomalachite green (LMG) in aquatic products using a bispecific monoclonal antibody (BsMAb). Gradient drug mutagenesis methods were separately used to prepare an anti-3-nitrobenzaldehyde-derivatized AMOZ (3-NPAMOZ) hybridoma cell line that was hypoxanthine-guanine-phosphoribosyltransferase (HGRPT) deficient and an anti-LMG hybridoma cell line that was thymidine kinase (TK) deficient. BsMAb recognizing 3-NPAMOZ and LMG was generated using hybrid-hybridomas of HGRPT and TK deficient cell lines. For AMOZ and LMG, respectively, the BsMAb-based indirect competitive ELSIA (ic-ELISA) values of 1.7 ng/mL and 45.3 ng/mL and detection limits of 0.2 ng/mL and 4.8 ng/mL. To establish the ic-ELISA, 3-NPAMOZ derivatized from AMOZ with 3-nitrobenzaldehyde and LMG reduced from MG by potassium borohydride was recognized by BsMAb. Recoveries of AMOZ, MG, and LMG in aquatic products were satisfactory and correlated with HPLC analysis. Thus, the multianalyte ic-ELISA is suitable for rapid quantification of AMOZ, MG, and LMG in aquatic products.