•We designed and synthesized Chromotrope FB (Chr FB) hapten with an amino group.•A highly sensitive and specific chemiluminescence immunoassay (CLIA) for Chr FB was developed.•The CLIA method is more sensitive than ELISA and HPLA method.•The method could be successfully used in rapid detection of Chr FB in yoghurt hard candy, vitamin drink and bread.Chromotrope FB (Chr FB) is a synthetic azo dye permitted for use in foods and medicines. An acceptable daily intake (ADI) of Chr FB was 0–0.5 mg/kg in China. In this study, we synthesized a Chr FB hapten with an amino group to prepare its artificial immunogen. Polyclonal antibodies obtained from New Zealand rabbits were applied to develop an indirect competitive chemiluminescent immunoassay (icCLIA) to detect Chr FB in foods. A horseradish peroxidase (HRP)-luminol-H2O2 system was used to yield CL signal with p-iodophenol as an enhancement reagent. The method showed good specificity towards Chr FB and could detect as low as 0.02 ng mL−1 Chr FB in buffer, 0.07 ng g−1 in yoghurt candy, 0.07 ng g−1 in vitamin drink and 0.13 ng g−1 in bread. Compared with HPLC method, the proposed method is more sensitive by two orders of magnitude. The accuracy and precision of this method are acceptable and comparable with HPLC method. Therefore, the proposed method could be used for rapid screening of Chr FB in the mentioned foodstuffs.

Aberrant methylation by DNA transferase is associated with cancer initiation and progression. For high-throughput screening of DNA methyltransferase (MTase) activity and its inhibitors, a novel chemiluminescence immunoassay (CLIA) was established to detect S-adenosylhomocysteine (SAH), the product of S-adenosylmethionine (SAM) transmethylation reactions. We synthesized two kinds of immunogens for SAH and characterized the polyclonal antibodies in each group. The antibody with higher titer was used to develop a competitive CLIA for SAH, in which SAH in samples would compete with SAH coated on microplate in binding with SAH antibodies. Successively, horseradish peroxidase labeled goat antirabbit IgG (HRP-IgG) was conjugated with SAH antibodies on the microplate. In substrate solution containing luminol and H2O2, HRP-IgG catalyzed luminol oxidation by H2O2, generating a high chemiluminescence signal. The method could detect as low as 9.8 ng mL–1 SAH with little cross-reaction (3.8%) to SAM. Since higher DNA MTase activity leads to more production of SAH, a correlation between the chemiluminescence intensity and DNA MTase activity was obtained in the range from 0.1 to 8.0 U/mL of DNA MTase. The inhibition study showed that, in the presence of SAM as methyl donor, Lomeguatrib, 5-Azacytidine, and 5-Aza-2′-deoxycytidine could inhibit the DNA MTase activity with IC50 values of 40.57 nM, 2.26 μM, and 0.48 μM, respectively. These results are consistent with the published studies. The proposed assay does not depend on recognizing methylated cytosines in oligonucleotides (methyl acceptor) and showed the potential as an accessible platform for sensitive detection of DNA MTase activity and screening its inhibitors.

For routine monitoring of the pharmacokinetic behavior of anticancer drug methotrexate (MTX), polyclonal antibodies for MTX were originally produced, and a sensitive chemiluminescence immunoassay (CLIA) was developed for the determination of plasma MTX. Three kinds of coupling reagents (EDC, CDI and isobutyl chloroformate) were utilized to synthesize MTX immunogens. The coupling ratio, titer and sensitivity of polyclonal antibodies for each immunogen were evaluated. Consequently, MTX–EDC–cBSA was found to be the optimal immunogen since it showed the highest coupling ratio and yielded antibodies with the highest sensitivity. Under optimal conditions, the developed CLIA showed a limit of detection (LOD) of 4.3 ng mL−1 in buffer and 9.1 ng mL−1 in plasma with acceptable coefficients of variations (<14.9%). The method exhibited no cross-reaction with the MTX metabolite (7-OH MTX) and structural analogs. When applied in a pharmacokinetic study, the CLIA results were statistically consistent with the HPLC method in measuring key pharmacokinetic parameters (t1/2, Cmax, AUC0–12 and MRT0–12). In conclusion, the CLIA method showed advantages of simple sample preparation, low cost, high sensitivity and good reproducibility. These properties make it a potential tool in the rapid detection of MTX for therapeutic drug monitoring (TDM).

•Two kinds of immunogens were synthesized to produce antibodies of nateglinide.•Effect of coating antigen, antibody dilution and reaction time was studied.•A highly sensitive and specific immunoassay for nateglinide was developed.•The method could be used in rapid detection of nateglinide in tablets and serum.In this study, we prepared polyclonal antibodies against anti-diabetic drug nateglinide (NTG), and established a sensitive chemiluminescent immunoassay (CLIA) to detect NTG in tablets and serum. Two kinds of immunogens were synthesized using ethylcarbodiimide (EDC)/hydroxysuccinimide (NHS) and carbonyldiimidazole (CDI)/4-dimethylaminopyridine (DMAP) as coupling reagents respectively. When activated by EDC/NHS, more molecules of NTG coupled with carrier protein in immunogens. A horseradish peroxidase (HRP)-luminol-H2O2 system with p-iodophenol enhancement was applied in the CLIA analysis. The antibodies in EDC/NHS group showed higher titer, sensitivity and wider detection linear range than those in CDI/DMAP group, and were chosen for next studies. The developed CLIA assay exhibited good selectivity towards NTG among structually similar analogs. The method could detect as low as 0.35 ng mL−1 NTG in buffer, 2.1 ng mL−1 NTG in serum and 0.84 ng mL−1 NTG in tablets. The CLIA method provided consistent results with HPLC method (r=0.9986) in determination of NTG from 5.0 to 400 µg mL−1. The CLIA method could detect 78 samples in one assay, and the samples need only dilution in pretreatment. As a summary, this research offers a sensitive assay for high-throughout screening of NTG in formulation control and pharmacokinetic studies.

As a synthetic food dye, erythrosine is associated with serious toxicity in inducing thyroid tumors, and the use of erythrosine is strictly regulated in most counties including China. In this study, a direct enzyme-linked immunosorbent assay (ELISA) has been developed for analysis of erythrosine in food products. A highly specific monoclonal antibody (MAb) for erythrosine was produced using erythrosine–bovine serum albumin (BSA) conjugate as an artificial antigen, and horseradish peroxidase (HRP)-labeled MAb for erythrosine was utilized as the detection antibody. Under the optimized condition, the UV absorbance in microplate related well with erythrosine concentration in the range of 0.1–10.0 μg/g. The proposed method could be applied to determine erythrosine in beverage and cookie, with good recoveries (80 –103 %) for the three spiked levels (30, 50, and 100 μg/g), and the relative standard deviations of detected amount were <12.3 %.

An indirect immunoassay for the determination of vitamin B2 in food samples and vitamin tablets was developed. A carbodiimide-modified active ester method was used to synthesize the immunogen for vitamin B2. The coupling ratio of vitamin B2 to carrier protein in immunogen was 19.98:1. The titer of the polyclonal antibody was 1:64000, and the antibody showed high specificity in the presence of vitamin B2 photolytic products and other B group vitamins. The immunoassay showed detection limits (LODs) of 1.07 ng/mL in PBS, 24.6 ng/g in vitamin drink, and 0.50 mg/kg in milk powder. Recovery was 99.58–110.91% in milk powder and 70.20–100.5% in vitamin drink. Vitamin B2 samples were analyzed by high-pressure liquid chromatography (HPLC) and the immunoassay, and results showed good agreement. Finally, this method was applied to detect vitamin B2 in commercial milk powder and vitamin tablets, and the detected amount correlated well with the labeled amount.

To detect a biomarker for small cell lung carcinoma, neuron specific enolase (NSE), a sensitive and specific chemiluminescence enzyme immunoassay was developed. Fluorescein isothiocyanate (FITC) labeled NSE capture antibody connected with NSE and alkaline phosphatase (ALP) labeled NSE detection antibody in a sandwich-type detection manner. This immune complex was further reacted with anti-FITC coated magnetic beads. In a magnetic field, the complex was enriched, and the sensitivity was thus enhanced. The limit of detection (LOD) of this method was <0.2 ng mL−1. The proposed immunoassay was highly selective, and not interfered by hook effect. The recovery was >83.0% and the coefficient of variation was <10.0%. Human sera from 120 patients were tested with the presented and traditional chemiluminescence enzyme immunoassay. An excellent linear relationship was obtained between two techniques. Overall, this immunoassay offers a promising alternative for NSE detection than traditional clinical examinations.Graphical abstractHighlights► The FITC labeled NSE capture antibody and ALP labeled NSE detection antibody were prepared to develop a sandwich detection format. ► The immune complex formed in aqueous solution and then bound with anti-FITC immobilized magnetic beads for detection of NSE by chemiluminescence intensity. ► The presented method showed high sensitivity and satisfactory recovery and coefficient of variation. ► A linear relationship was obtained for detection results of 120 patients’ sera by the proposed method and traditional chemiluminescence immunoassay.

Trenbolone (TRE) is a steroid used by veterinarians on livestock to increase appetite and body weight. The use of TRE has been restricted because of its harmful side effect for consumers. To effectively control TRE residue in food and food product, a rapid and convenient immunoassay was developed by preparing an anti-TRE monoclonal antibody. The immunogen and coating antigen were prepared by coupling TRE hapten with carrier proteins via 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC) method. The optimized method gave an average IC50 value of 0.323 ng mL−1 towards TRE and an average detection limit (LOD) of 0.06 ng mL−1, which is much lower than the maximum residue levels (2.0 ng g−1) accepted by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). The specificity of the antibody was evaluated by measuring cross-reactivity of six structurally related compounds, including 19-nortestosterone (9.7%), testosterone (0.13%), methyltestosterone (<0.01%), methandrostenolone (<0.01%), (+)-dehydroisoandrosterone (<0.001%) and β-estradiol (<0.001%). The recovery rates of the test in detection of TRE-fortified animal tissue, urine and animal feed samples were in the range of 81.3–89.4%, while the intra- and inter-assay coefficients of variation were less than 12.0%.

Sudan dyes are banned to be used in food additives because of the carcinogenicity of their metabolites. A rapid and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the residues of Sudan dyes. Novel immunogen and coating antigen were synthesized via glutaraldehyde linking. The hapten-bovine serum albumin (BSA) was applied as immunogen and the hapten-ovalbumin (OVA) was served as coating antigen. The monoclonal antibody obtained showed high sensitivity to Sudan I with an IC50 value of 1.7 μg L−1 in buffer and was suitable to detect the residues of Sudan red in food products. The specificity of the assay was studied by measuring cross-reactivity of the antibody with the structurally related compounds of Sudan II (<1%), Sudan IV (<1%) and para red (120%). Chilli jam and chilli oil samples spiked with Sudan dyes were analyzed by the method. The detection limit (LOD) of the ELISA method applied in chilli jam and chilli oil was 9.0 μg L−1 and 19.6 μg L−1, respectively. The recovery rates of Sudan-I in chilli oil and chilli jam were in the range of 80%–110% with coefficients of variation <25%. The intra-assay variation and inter-assay variation in buffer were both <9%.

Melamine (MEL) has been involved in several food recalls after the discovery of severe kidney damages in children and pets poisoned by melamine-adulterated food. To detect MEL residue in foods and animal feeds, an indirect competitive ELISA (cELISA) method was developed in this study based on preparation of monoclonal antibodies (MAbs) to MEL. The immunogen was prepared by linking MEL hapten with carrier protein via carbodiimide method. The method is applicable in the range of 5.0−135.0 μg L−1 MEL in buffer solution, with an IC50 value of 22.6 ± 1.9 μg L−1. The MAbs showed high specificity with low cross-reactivity (≤1%) toward cyanurate, ammelide, and ammeline. The method was utilized in the detection of MEL in raw milk, milk powder, and animal feeds, with detection limits of 0.1 mg L−1 for milk, 0.2 mg kg−1 for milk powder, and 0.5 mg L−1 for feeds. The recovery ratio was 79−110% for all matrices. The intra-assay and interassay coefficients of variation were <12.0 and <13.0%, respectively. Finally, the application of the cELISA in quantity evaluation of MEL in various feeds from local markets was evaluated and discussed.