Detection Of Bovine β-lactoglobulin And Effects Of Tea Polyphenols Upon Immunoreactivity Of β-lactoglobulin

Allergy to cow's milk proteins is a common food allergy. Specific avoidance of cow milkis the only way to prevent milk allergies. However, there is yet no domestic food-labeling lawfor food allergens. It is necessary to establish methods for the detection of milk contaminationin food products.Bovine β-lactoglobulin (βLG) is a major allergen of cow milk and considered to be aspecific milk allergen marker in food products. The current widely used methods to diminishallergenicity of βLG all have limitations. It is crucial to develop new methods that wouldreduce the allergenicity without destroying functional properties of βLG.Our research focused on βLG. A series of detection methods for βLG in food productswere established based on the immunoreactivity of βLG. We prepared βLG-catechincomplexes by binding the protein with epigallocatechingallate (EGCG) and epigallocatechin(EGC), respectively. We explored how EGCG and EGC bound to βLG and the structurechanges of βLG before and after these catechins binding. Then we estimatedimmunoreactivity of these complexes. The present research aimed to perform a mechanisticstudy concerning immunoreactivity changes of βLG before and after catechins binding. Thefollowing six aspects of research are listed below:(1)Eght monoclonal antibodies (mAbs) against βLG allergen were prepared andidentificated. The results demonstrated that these mAbs were pure, specific without anycross-reactivity toward other food allergen sources.(2)Four major IgG epitopes of βLG were synthesized and probed by the mAbs throughindirect ELISA and immuno dot-blotting. The results showed that the epitopes recognized by1G5was located in the region51Glu-64Asp; for1H8,67Arg-88Asn; for3D11and4C3,129Asp-144Pro.(3)Two mAbs (1H8and1G5) with identified epitope regions were paired anddeveloped into detection methods of βLG allergen. The detection methods included doubleantibody sandwich ELISA (S-ELISA), time-resolved fluoroimmunoassay (TRFIA), goldimmunochromatography assay (GICA) and western blotting (WB). The detection limit ofS-ELISA, TRFIA and GICA were1.6ng mL^-1,0.2ng mL^-1and0.2ng mL^-1, respectively.GICA method described here is much simpler and faster than an ELISA assay.(4)The binding interactions between EGCG and EGC which βLG were thoroughlystudied by fluorescence quenching. It indicated that the nature of EGCG quenching was static,while the nature of EGC quenching involved a combined quenching (static and dynamic) process. The thermodynamic parameters of the interaction between EGCG and EGC withβLG indicated that the interactions process was spontaneous. The results suggested thathydrophobic forces play a major role in the binding between EGCG and βLG, Van der Waalsinteractions and hydrogen bonds play major roles between EGC and βLG.(5)The results of multi-spectroscopic experiments revealed that βLG bound by EGCGand EGC, which resulted in slight change of native conformation of βLG without apparentdisruption. It was found that the α-helical and β-sheet content of the protein were slightlyincreased. The conformation of βLG was little more compact after EGCG and EGC binding.The reduction of the binding affinity of βLG-catechins complexes with antibodies may due tothe shielding action of the βLG epitopes induced by the attachment of catechins to βLG.EGCG and EGC in βLG-catechins complex may be attached directly to the epitope(s) orregion(s) in the proximity, thereby preventing anti-βLG antibodies from accessing theantigens. The effect of EGCG was greater than EGC.(6)Thermal treatment on protein would increases binding affinity of EGCG with βLG.EGCG was attached on the preheat βLG,which can reduced the binding activities betweenantigen and antibodies greater than EGCG bingding βLG without preheat.In general, a series of detection methods for βLG in food products were established,which filled up domestic blank. At the same time, we examined the mechanismimmuoreativity changes of βLG before and after tea polypheols binding. We hope thatour research will provide new alternative and theoretic supports for development ofhypoallergenic milk products.