Hong-Lin Ren

Colloidal gold probe-based immunochromatographic assay for the rapid detection of brevetoxins in fishery product samples.

One-step immunochromatographic assay using colloidal gold-labeled monoclonal antibody (Mab) probe for the rapid detection of brevetoxins (PbTxs) in fishery product samples was developed. The described assay was based on a competitive format using two antibodies. The primary antibody was conjugated with colloidal gold (detector reagent), the secondary antibody (capture reagent) was immobilized within a defined detection zone (control line) on a diagnostic cellulose nitrate membrane. The toxin in sample compete with immobilized toxin to bind with gold conjugated Mab. The mobile complex (colloidal gold-Mab-toxin) can be captured by the secondary antibody but cannot be captured by BSA-PbTx (test line). The color density of the test line correlated with the concentration of PbTx in sample in the range 10-4000 ng mL(-1). Spiked samples were detected by the assay and the visual detection limit was found to be 20 ng mL(-1). This qualitative test based on the visual evaluation of results did not require any equipment. The assay time for PbTx detection was less than 10 min, suitable for rapid testing on-site.

An enhanced ELISA based on modified colloidal gold nanoparticles for the detection of Pb(II).

A novel probe based on colloidal gold nanoparticles (AuNPs) modified with goat anti-mouse IgG and horseradish peroxidase (HRP) was synthesized and an enhanced enzyme-linked immunosorbent assay (ELISA) based on the probe was developed. In the assay, the synthesized probe is bound with a monoclonal antibody (McAb) which is competitively bound by coated BSA-ITCBE-Pb(II) on plate and Pb(II) in samples. The HRP, used here for signal amplification catalytically oxidize the substrate and generate optical signals that is related to the concentration of Pb(II) and can be measured spectrophotometrically. For the monodisperse AuNPs having high surface areas, it can be conjugated with more amount of HRP than that of IgG. Therefore, compared with traditional ELISA, the signal amplification of catalytically oxidized substrate was enhanced. The detection limit for this novel modified AuNPs probe-based assay was 9 pg mL(-1). The recoveries obtained by standard Pb(II) addition to real samples, including a commercial mineral water, tap water, and lake water were all from 94.9% to 102.9%. And the coefficient of variation (CV) value of all samples was less than 10%. The results indicated that the enhanced assay gave higher sensitivity and reliable reproducibility. It could provide a general detection format for low-molecular weight contaminants.

Monoclonal antibody based inhibition ELISA as a new tool for the analysis of melamine in milk and pet food samples.

Stories of recent cases about melamine misuse to raise the false impression of a high protein content of milk in China emerged in September of 2008, have become an international health event. To meet the need for rapid and reliable monitoring of melamine in milk samples, a monoclonal antibody (mAb) was produced and an inhibition enzyme-linked immunosorbent assay (ELISA) was developed based on the mAb. The standard curve was linear in the range from 0.03 to 9 ng mL(-1) with a detection limit (LOD) of 0.01 ng mL(-1). The sensitivity of the assay was 0.35 ng mL(-1). The average recovery values of melamine in the liquid milk, powder milk, dog food and cat food were 99%, 96%, 9% and 98%, respectively and the coefficient of variation (CV) values of all samples were less than 10%. The obtained results showed a potential method as a tool for the rapid and reliable monitoring of melamine in liquid milk and milk powder samples (158 words).

A competitive immunochromatographic assay based on a novel probe for the detection of mercury (ii) ions in water samples.

Mercury ions (Hg(2+)) are one of the most dangerous pollutants. Even at low concentration, it causes serious environmental and health problems. Current methods for the detection of Hg(2+) in environmental samples are tedious and time consuming because they require sophisticated instrumentation and complicated sample pre-treatment processes. In this work, a novel probe with high selectivity towards Hg(2+) was synthesized and a one step competitive immunochromatographic assay based on the probe for the detection of Hg(2+) was developed and applied for water samples. The detection conjugate was immobilized on one end of the nitrocellulose membrane (detection line) and anti-BSA polyclonal antibody was immobilized on the other end of the membrane (control line). Hg(2+) in samples competed with the probe to bind with immobilized detection conjugate. The visual detection limit of Hg(2+) in spiked water samples was found to be about 1 ppb. The qualitative assay can be performed within 15 min. The advantages of the technique are rapidity, low cost and without the need of any equipment and complicated sample preparation.

A versatile and highly sensitive probe for Hg(II), Pb(II) and Cd(II) detection individually and totally in water samples

The detection of heavy metal ions using enzyme-linked immunosorbent assays (ELISA) has been reported by several research groups. However, highly sensitive and selective detection of total heavy metal ions using ELISA is a major technical limitation. Here we describe the development of a versatile and highly sensitive probe combining goat anti-mice IgG, colloidal gold nanoparticles (AuNPs) and horseradish peroxidase (HRP). We demonstrate the utility of this probe using three kinds of heavy metal complete antigens and three monoclonal antibodies (McAbs) in one ELISA system to establish a high-throughput screening protocol. The procedure was successfully applied to analysis of Hg(II), Pb(II) and Cd(II) individually and totally from different water samples. The sensitivities for the detection of Hg(II), Pb(II) and Cd(II) individually and totally are 27.4, 3.9, 15.8 and 18.2 nM, respectively. And all limit of detection (LODs) are lower than 1.2 nM. The recovery results obtained from the developed technique showed a good correlation (R(2)=0.983) with those from ICP-MS. The major advantage of the probe is the versatility and high sensibility. The probe could be potentially used, upon demand, as a sensitive and versatile detector for a broad range of applications.

A screening lateral flow immunochromatographic assay for on-site detection of okadaic acid in shellfish products

A lateral flow immunochromatographic (LFIC) test strip based on a colloidal gold-monoclonal antibody (McAb) conjugate was developed for on-site rapid detection of okadaic acid (OA) in shellfish. It applies a competitive format using an immobilized toxin conjugate and free toxin present in samples. The McAb against OA was conjugated with 20-nm colloidal gold as detector reagent. The toxin in the sample competed with the immobilized toxin to bind to the gold conjugated with McAb. The colloidal gold/McAb/toxin mobile complex was not captured by OA-bovine serum albumin (BSA) on the test line, but it was captured by goat anti-mouse immunoglobulin G (IgG) on the control line. The color density of the test line correlated with the concentration of toxin in the range of 10-50 ng ml(-1). The qualitative detection limit of 150 μg kg(-1) sample was close to the European Union (EU) regulatory limit (160 μg kg(-1)). Therefore, these strips were able to directly and qualitatively estimate the consuming safety of shellfish. They require no equipment because of available visual results, and they screened numerous samples within 10 min. The results were further confirmed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). As a food safety screening tool, the test strips are convenient and useful to rapidly on-site test the presence of OA in shellfish products.

Development of a novel antibody probe useful for domoic acid detection.

The generation of monoclonal antibody (mAb) against marine toxins can serve as a valuable probe to detect this kind of compounds by immunological methods. However, traditional approaches to mAb generation usually need a comparative large quantity of standard substance (more than 400 microg mouse(-1)), and a comparative long immunization period (more than 6 weeks). Here we report a simple, inexpensive and fast protocol for the generation of monoclonal antibody probe specific for domoic acid (DA). In the method, lymph node cells were harvested from the Balb/C mice of hind footpad injection and fused with murine myeloma cells SP2/0 for hybridoma generation. This method for the preparation of mAb for DA has two main advantages: (a) there is no need for large-scale expensive antigen (only 70 microg antigen for one mouse); (b) immunization protocol can be accomplished within 16 days. Some characteristics of the mAb were studied and a direct competitive ELISA for the detection of DA using the mAb as a probe was developed. The detection limit was 0.41 ng well(-1) in phosphate buffered saline (PBS) and 0.53 ng well(-1) in blue mussel Mytilus edulis. The recoveries of DA from mussel and PBS buffer were from 94.8% to 105.1% and from 96.2% to 103.7%, respectively. Thus, the newly developed direct competitive ELISA using the mAb appears to be a reliable and useful method for monitoring of DA in shellfish (228 words).

Direct competitive immunosorbent assay for detection of MEHP in human urine

Di-(2-ethylhexyl) phthalate (DEHP) is the most commonly used plasticizer for flexible polyvinyl chloride (PVC), which is also known as one of the environmental endocrine disruptors with the reproductive, developmental and embryonic toxicity after entering human body. Mono-2-ethylhexyl phthalate (MEHP) is one of the most complicate metabolites from DEHP in vivo and responsible for many toxic effects of DEHP. In order to evaluate human exposure to DEHP, a direct competitive enzyme-linked immunosorbent (dcELISA) based on monoclonal antibody (mAb) was developed to detect MEHP. A hybridoma cell line 4B9 secreting mAb against MEHP was prepared, and the horseradish peroxidase (HRP) labeled antigen as a probe in the dcELISA was made. After optimization of ELISA reaction conditions, the standard curve with a linear range from 0.56 to 1000 ng mL(-1) and a detection limit of 0.39 ng mL(-1) was established. The cross-reactivities of anti-MEHP mAb to other ten phthalate esters were less than 5% except for mono-methylphthalate (MME). The average recoveries of MEHP from distilled water and negative human urine were both between 87.4% and 94.72% with coefficient of variation (CV) less than 5%. Here, the ELISA method on detecting MEHP was successfully established and applied to real urine sample analyses and the results were confirmed by HPLC. Furthermore, it was indicated that the immunoassay was reliable and suitable for monitoring MEHP.

A rapid immunomagnetic beads-based immunoassay for the detection of β-casein in bovine milk

An immunomagnetic beads-based enzyme-linked immunosorbent assay (IMBs-ELISA) was developed for the detection of β-casein in bovine milk. Immunomagnetic beads (IMBs) were employed as the solid phase. The anti-β-casein monoclonal antibody (McAb) bound to IMBs was used as capture probe and an anti-β-casein polyclonal antibody (PcAb), labelled with horseradish peroxidase (HRP), was employed as detector probe. Three reaction and two washing steps were needed. Each reaction needed 10 min or less, which significantly shortened detection compared with classic sandwich ELISA. β-Casein in bovine milk was detected across a linear range (2-128 μg mL(-1)). Application results were in accordance with the Kjejdahl method, which suggests the IMBs-ELISA is rapid and reliable for the detection of β-casein in bovine milk.

Enzyme-antibody dual labeled gold nanoparticles probe for ultrasensitive detection of κ-casein in bovine milk samples.

A dual labeled probe was synthesized by coating gold nanoparticles (AuNPs) with anti-κ-CN monoclonal antibody (McAb) and horseradish peroxidase (HRP) enzyme on their surface. The McAb was used as detector and HRP was used as label for signal amplification catalytically oxidize the substrate. AuNPs were used as bridges between the McAb and HRP. Based on the probe, an immunoassay was developed for ultrasensitive detection of κ-CN in bovine milk samples. The assay has a linear response range within 4.2-560 ng mL(-1). The limit of detection (LOD) was 4.2 ng mL(-1) which was 10 times lower than that of traditional McAb-HRP based ELISA. The recoveries of κ-CN from three brand bovine milk samples were from 95.8% to 111.0% that had a good correlation (R(2)=0.998) with those obtained by official standard Kjeldahl method. For higher sensitivity and as simple as the traditional ELISA, the developed immunoassay could provide an alternative approach for ultrasensitive detection of κ-CN in bovine milk sample.