Preparation Of Monoclonal Antibodies Against β-casein And Development Of Two-type ELISAs For β-casein

Total milk proteins include caseins （80%） and whey proteins （20%）. Four major caseins canbe found in milk, namely αs1-, α_s2-, β-, and κ-casein. And the concentration of β-casein is steady,accounting36%of the casein content in milk. So the quantity of β-casein in bovine milk couldbe used to detect milk sample adulteration. In this study, the female BALB/C mice，6-8weeksold, were immunized with β-casein by subcutaneous injection in a hind footpad. After twoprotocols of immunization were carried out, the sera were tested by indirect ELISA, and the miceshowing the highest serum reactivity were selected for hybridoma production; Cell fusion wascarried out with PEG, and positive hybridoma cells were screened by indirect ELISA, thepositive hybridoma cells were subcloned three times by limiting dilution. A hybidoma cell,named4H1, steadily secreting monoclonal antibody （McAb） against β-casein was obtained afterthe third clone. The isotype of the McAb was IgG_2a. The hybridoma cells were injected into theperitoneal cavity of Balb/c mice to produce large quantities of the ascites, which were purified bycaprylic acid,（NH₄）₂SO₄solution and protein G affinity chromatography, ascites titer purifiedusing these two methods can reach1.0×10⁶, the affinity constant of monoclonal antibody is5.29×10⁸mol L^-1. Two-type ELISAs were established and developed using purified ascites.One type ELISA is based on miscroplates. A standard curve was obtained withmiscroplate-sandwich ELISA. The regression equation of β-casein was y=1.2314x-0.4135with correlation coefficient R2=0.9951. The linear detection range was from0.1to10.0μg mL-1.The other type is an immunomagnetic bead-based sandwich ELISA method. A standard curvewas obtained with immunomagnetic bead-based sandwich ELISA. The equation of linearregression of β-casein in this method was y=4.3782x-0.7154, and the correlation coefficientR²=0.9706with a linear detection range from2to128μg mL^-1. Immunomagnetic beads havinghigh surface areas, which can be immobilized with more amount of McAbs compared with themicrotiter plate. It needs only10min to fulfill each reaction step of this method, so the detection of β-casein in milk was achieved within30min. Thus, using this immunomagnetic bead-basedsandwich ELISA, the assay time of detection was significantly shortened compared to thetraditional miscroplate-sandwich ELISA and the efficiency of detection was improved.