Feng-Guang Pan

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.

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.

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).

Development of a monoclonal antibody-based sandwich-type enzyme-linked immunosorbent assay (ELISA) for detection of abrin in food samples.

Abrin is a plant toxin, which can be easily isolated from the seeds of Abrus precatorius. It may be used as a biological warfare agent. In order to detect abrin in food samples, a two-layer sandwich format enzyme-linked immunosorbent assay based on the monoclonal antibody (mAb) (as capture antibody) and rabbit polyclonal serum (as detecting antibody) was developed and applied for the determination of abrin in some food matrices. The linear range of the mAb was 1-100 μg L(-1) with a detection limit of 0.5 μg L(-1) for abrin in phosphate buffered saline (PBS). The recoveries of abrin from sausage, beer and milk samples ranged 97.5-98.6%, 95.8-98.4% and 94.8-9.6%, respectively, with a coefficient of variation (CV) of 3.7% or less. The newly developed sandwich ELISA using the mAb appears to be a reliable and useful method for detection of abrin in sausage, beer and milk.

The development and optimization of ELISA for the determination of tetrodotoxin

Abstract Objective To optimize the ELISA for the determination of tetrodotoxin. Methods A competitive enzyme-linked immunosorbent assay (ELISA) was used. In the ELISA, 100 μl antigen (1.0 μg/ml) was coated on the microtiter plate for 60 min at 37 C or over night at 4 C. The plate was then washed 3 times with PBS-T for 3-5 s each time. The optimal incubation time for monoclonal antibody (mAb), goat anti-mice IgG peroxidase conjugate and OPD were 30 min. 20 min and 10 min at 37 C, respectively. Results The detection limit is 0.05 ng in each well. The curve was linear for TTX doses between 5-5 000 ng/ml (0.25-250 ng for every assay). The linear regress equation was Y = 0.30 88X — 0.17 41 ( R. = 0.99 01). The average callback for TTX of muscles and gonads were 99.74% and 100.30%, respectively. The sensitivity of optimization ELISA was 5 times than traditional method and the time of 1.8 h were saved. Conclusion The optimized ELISA is an idealmethod for the determination of tetrodotoxin.

Cloning and sequence analysis of major ampullate spidroin-1 partial cDNA from Araneus ventricosus : Cloning and sequence analysis of major ampullate spidroin-1 partial cDNA from Araneus ventricosus

In this paper, we successfully constructed the cDNA library of major ampullate gland of Araneus ventricousus using pUC18 vector and cloned the partial cDNA (AvMaSp1, GenBank accession number AY177203) encoding spider major ampullate gland spidroin-1 by means of picking colony randomly (Bird gun). The partial cDNA sequence of AvMaSp1 was 1 408 bp and its region in encoding was 1 288 bp. The protein deduced from AvMaSp1 contained 429 amino acid residues and molecular weight was 34.07 kDa. The repetitive motif of this cDNA sequence was (GA)nAm(GA)N and the identity with A. diadematus fibroin-1 mRNA (ADF-1, GenBank accession number ADU47853) was 75.0%.