Novel Approaches To The Detection Of Bovine ?-Lactoglobulin By Specific Antibody Against Human IgE Binding Epitopes

Milk is a very popular food because of rich protein content and various nutrient compositions,but as one of eight major allergenic foods,milk allergy has attracted widespread concern.In European Union,America,Japan,Australia and other developed countries,food allergens are need be labeled,including milk and its products.Bovine?-lactoglobulin is one of the major allergens of milk,accounts for10%of total milk proteins and 50%of whey protein.In addition,approximately 82%of cows'milk allergy patients are sensitive to?-lactoglobulin.Moreover,IgE epitopes play a key role in food allergy,while the commercial antibodies used in immunoassays do not specifically recognize?-lactoglobulin IgE epitopes.Therefore,the specific antibody against IgE epitope not only could be used for the more accurate detection of?-lactoglobulin and its potentially allergenic residues in different types of processed foods,but also be used to estimate the allergenic potential of the given food product.The research work was composed of six parts,including 1)development and immunological characterization of polyclonal antibody against recombinant epitope tandem derived from?-lactoglobulin;2)development and immunological characterization of epitope monoclonal antibody against?-lactoglobulin;3)development of sandwich enzyme-linked immunosorbent assay?sELISA?for testing?-lactoglobulin by specific polyclonal antibody against human IgE binding epitopes?pAb-tBLG?and polyclonal antibody against?-lactoglobulin?pAb-BLG?;4)development of platinum nanoparticles probe-based sELISA for detection of?-lactoglobulin by pAb-tBLG;5)development of H₂O₂-sensitive quantum dots-based fluorescent sELISA for detection of?-lactoglobulin by epitope monoclonal antibody?mAb?;6)development of sELISA with covalently bound mAb for detection of?-lactoglobulin.The main methods,results and conclusions were as follows:1.Based on recombinant Escherichia coli,a tandem derived from human IgE linear epitopes of?-lactoglobulin?tBLG?was expressed,and the high purity recombinant tBLG protein was obtained by affinity purification and isolation from the gel.The polyclonal antibody?pAb-tBLG?was developed by immunizing Japanese big-eared rabbits with the purified recombinant tBLG protein.The prepared pAb-tBLG could recognize all the seven IgE epitopes and the blocking peptides,and had good specificity.The specific pAb-tBLG was purified by affinity purification.2.The peptides of five major IgE epitopes were synthesized,and the mAb against the peptides were raised in mouse by hybridoma technology.All the mAbs were identified as IgG with?light chain,and did not contain IgM and IgA.All the mAbs could recognize both the corresponding epitope peptide and its blocking peptide;and had high affinity constant,except for 1P1.3.A sELISA was developed using pAb-BLG as capture antibody and biotinylated pAb-tBLG as detection antibody.The linear range for?-lactoglobulin detection was 31.25–8,000 ng/mL and limit of detection was 1.96 ng/mL.The?-lactoglobulin content in dairy samples?milk,whey and whey powder?was determined,and there was a good agreement between this immunoassay and reversed-phase high-performance liquid chromatography?RP-HPLC?with high recovery?95.17%–104.67%?,indicating the developed sELISA was accuracy and reliable.Additionally,the recovery rate in food samples?yogurt,chocolate and candy?was 98.33%–110.53%.Allergenic residues were also detected in hydrolyzed infant formulas,and?-lactoglobulin allergenic residues were not detected in extensively hydrolyzed formulas,but varied considerably in partially hydrolyzed infant formulas.4.A sELISA was developed based on pAb-tBLG and platinum nanoparticles probe.This sELISA exhibited an ultra-wide linear range of 0.49–1.6×10⁴ ng/mL and with a limit of detection of 0.12 ng/mL,which were 4-fold wilder and 16-fold lower than that of traditional sELISA.The?-lactoglobulin content in food samples detected by the developed sELISA was similar to those tested using conventional sELISA and commercial sELISA kit with high recovery.5.Based on IgE linear epitope mAb 1G9 and H₂O₂-sensitive CdTe quantum dots,a fluorescent sELISA was developed.The fluorescent sELISA with a limit of detection of 0.49 ng/mL,which was 16-folds lower than HRP-based conventional sELISA.Beta-lactoglobulin content in food samples was determined,and the results of fluorescent sELISA showed good performance as conventional sELISA and commercial sELISA kit with high recovery?94.25%–109.83%?.Allergenic residues were also detected in hydrolyzed infant formulas,the results of fluorescent sELISA were similar to that of conventional sELISA,and slight different from commercial sELISA kit.6.A sELISA was developed based on mAb?1G9?-covalently bound plate in sandwich combination with biotinylated pAb-BLG-labeled gold nanoparticles.The linear dynamic range was from 31.25 ng/mL to 64×10³ ng/mL with a limit of detection for?-lactoglobulin of 0.49 ng/mL was obtained,which was 32 times wider and 16 times more sensitive than conventional sELISA.Beta-lactoglobulin in spiked food samples was found with recoveries from 98.29%to 111.84%,and the results of the developed sELISA were in agreement with those of the conventional sELISA and a commercial sELISA kit.Also in partially hydrolyzed infant formulas,the allergenic?-lactoglobulin residues were detected quantitatively,and the results of the developed sELISA were similar to that of conventional sELISA,while slight different from commercial sELISA kit.7.One pAb and five mAbs specific to IgE epitopes of?-lactoglobulin were prepared,which overcome the drawbacks of the traditional commercial antibody which connot specifically recognize IgE epitopes.Four immunoassays based on the epitope antibodies were developed for detection of?-lactoglobulin,which were validated with RP-HPLC or commercial sELISA kit.The results indicated that the methods developed could be practical approachs to determine?-lactoglobulin and its allergenic residues in foods with a high degree of sensitivity,reliability and recovery.