Limin Cao

Broad-Specific Antibodies for a Generic Immunoassay of Quinolone: Development of a Molecular Model for Selection of Haptens Based on Molecular Field-Overlapping

A new molecular model for quinolone haptens was developed based on molecular field-overlapping. The quanlitive modeling of 3-D conformations showed that the conformation difference among quinolones is caused mainly by the different substitutes at the 1 and 7 positions. The 8-substitute also showed some effect by its inter-reaction with the 1-substitute. The conformational similarity of 27 quinolones to each other was for the first time calculated and exploited for a selection of haptens according to desired broad specificity of corresponding antibodies. The developed model was preliminarily validated with antibodies against different quinolones. A significant positive correlation (R = 0.7793) was observed between calculated overlapping coefficients of haptens and the cross-reactivity of corresponding polyclonal antibodies (Pabs), which confirmed the overall accuracy of the developed model and its application in quantitative structure-activity relationship analysis. On the basis of molecular modeling results, the strategy for the production of broad specific antibodies against quinolones was suggested and the potentiality of several candidates was predicted.

Antibacterial activity of egg yolk antibody (IgY) against Listeria monocytogenes and preliminary evaluation of its potential for food preservation.

BACKGROUNDnEgg yolk antibody (IgY) is a unique type of immunoglobulin found in egg yolks, and many reports have described its ability to inhibit corresponding antigen bacteria. The objective of this study was to evaluate the antibacterial activity of IgY specific to Listeria monocytogenes, an important food pathogen to both humans and animals, as well as its potential use for food preservation.nnnRESULTSnSpecific IgY was generated by immunising Leghorn chickens with whole cells of L. monocytogenes, and its inhibitory effect on bacterial growth was tested in liquid medium and food samples. After 8 h of incubation with specific IgY, there was a significant decrease in the growth (absorbance at 600 nm) of L. monocytogenes in comparison with controls. IgY also inhibited the growth of L. monocytogenes inoculated onto fresh or smoked salmon samples. Compared with those of blanks, numbers of L. monocytogenes were reduced by more than 2 log units after 15 days of storage at 6 ± 1 °C in the presence of specific IgY.nnnCONCLUSIONnThe results suggest the potential application of specific IgY as a natural antimicrobial agent for food preservation.

Direct competitive enzyme-linked immunosorbent assay (ELISA) for rapid screening of anisakid larvae in seafood

BACKGROUNDnAnisakid larvae are one of the most important pathogenic parasites in marine products; however, simple and rapid analytical techniques for them are still very limited. In this research, based on specific rabbit polyclonal antibodies which were raised against crude extracts of Anisakis larvae, purified by protein A affinity chromatography and labeled with horseradish peroxidase, a direct competitive enzyme-linked immunosorbent assay (ELISA) was developed and validated for detection of anisakid larvae in seafood.nnnRESULTSnThe established method exhibited a broad selectivity to Anisakis larvae and Pseudoterranova larvae, and the lowest detection limit to them was estimated to be about 5 parasites kg(-1) in food matrix. Using Pseudopleuronectes yokohamae, Scomberomorus niphonius and Ommastrephes bartrami as samples and within spiking concentrations from 20 to 100 larvae kg(-1), the determination recovery for Anisakis larvae and Pseudoterranova larvae ranged from 77.8% to 107.0%, with relative standard deviations all less than 20%.nnnCONCLUSIONnThe results allowed us to suggest the established direct competitive ELISA as an effective analytical tool for fast screening of anisakid larvae in sea foods.

Effects of specific egg yolk antibody (IgY) on the quality and shelf life of refrigerated Paralichthys olivaceus

BACKGROUNDnThe spoilage of fishery food has been attributed to limited types of microorganisms called specific spoilage organisms (SSO). Unlike traditional food-preserving techniques which usually exploit broad-spectrum antimicrobial agents, here, based on the specific antimicrobial activity of egg yolk antibodies (IgY) against two SSO in refrigerated fish (Shewanella putrefaciens and Pseudomonas fluorescens), a novel strategy for fish preservation was suggested and evaluated.nnnRESULTSnDuring storage of Paralichthys olivaceus fillets at 4 ± 1 °C, the bacteria growth (including total microorganisms and the two SSO) in test groups was significantly inhibited in comparison to that of controls (P < 0.05). This antibacterial activity of the specific IgY was also confirmed by chemical analysis (pH, total volatile base nitrogen and 2-thiobarbituric acid value) and sensory evaluation, and the shelf life of samples was extended approximately from 9 days to 12-15 days in the presence of the specific IgY.nnnCONCLUSIONnThese results indicated a significant antimicrobial activity of the anti-SSO IgY for refrigerated fish products, which allowed us to suggest its potential as a bio-preservative for seafood.

The effect of fish matrix on the enzyme-linked immunosorbent assay of antibiotics

BACKGROUNDnThe matrix effect is considered to be a problem in the immunoassay of foodstuffs. However, information on the interference from aquatic products, as well as the mechanism involved, is very limited. In this study, using three flatfishes (Scophthalmus maximus, Paralichthys olivaceus and Cymoglossus robustus) as samples, the effect of the fish matrix on the competitive indirect enzyme-linked immunosorbent assay (ci-ELISA) of antibiotic (norfloxacin) residues was investigated. The mechanism of the observed matrix effect is also preliminarily discussed.nnnRESULTSnWithin the working range of the calibration curves, a significant (P = 0.05) but irregular variation in the inhibition ratio was observed in the presence of fish extracts. Further experiments revealed that such a matrix effect could be caused by some water-soluble fish proteins with a wide range of molecular weight (from below 14.4 kDa to about 116.0 kDa), and the ions from fish muscles may also contribute to the interference. The results of western blotting indicated that some fish protein components might effectively bind with antibody reagents used.nnnCONCLUSIONnSignificant interference in the immunoassay of norfloxacin was observed in the presence of fish matrix. Some proteins and ions were demonstrated to contribute to the matrix effect investigated. Although the detailed mechanism is still unclear, the non-specific interaction between fish proteins and immunoglobulin G (IgG) or horseradish peroxidase (HRP) labelled IgG was assumed to be an important source of the matrix effect in immunoassays.

Dot-immunogold filtration assay for rapid screening of three fluoroquinolones

Abstract A dot-immunogold filtration assay (DIGFA) was described for simultaneous screening of three fluoroquinolones (FQs): enrofloxacin (ENR), ciprofloxacin (CIP) and norfloxacin (NOR). Colloidal gold particles were used as visible labels, and anti-ENR polyclonal antibodies from mice and rabbits were exploited for gold labelling and membrane coating, respectively. Several parameters affecting DIGFA were optimised, and the operation was validated with pure buffer matrix and spiked eel samples, respectively. The detection limits in eel samples were estimated to be about 20 µg kg−1 for ENR and CIP, and 50 µg kg−1 for NOR. With pretreated nitrocellulose membranes, the determination could be completed within 30 minutes, and the results could be read just by the naked eye without the help of any equipment.