Research On Detecting The IgG Bioactive Content In Infant Formula Milk Powder

The infant formula milk powder should show the same or similar physiological function as the breast milk, not only in the macroscopic composition and the content, but also in the microcosmic component. Therefore, it is important to study the protective nutrient substance for achieving the human infant formula milk powder. The protective immune active substance is crucial for the approaching of the infant formula milk powder to the breast milk, and represents the new trend in infant formula milk powder. The protective immune active substances include immunoglobulin G--IgG. At present, there is no accurate and reliable measurement because the IgG content in infant formula milk is lower than in other products.The objective of this research is to study the measurement of the IgG bioactive content in infant formula milk powder with ELISA kit, meanwhile comparing the effect of homemade ELISA kit with HPLC. The results not only provide reference to detecting the IgG bioactive content in infant formula milk powder, but also create a new way to measure the IgG bioactive content in infant formula milk powder--double antibody sandwich method. The major conclusions are as follows:(1)The standard method of measuring the bovine IgG bioactive content: Plates were coated with rabbit anti-bovine IgG 3 hours at 37℃and overnight at 4℃. Dilution of enzyme-conjugated second antibody was 1:250 and incubated 1 hour at 37℃.Substrate/chromogen solution was prepared by adding 10 mL phophate-citrate buffer, 150μL TMB solution and 30μL H2O2 and incubated 20 minutes. After stopping, plates were read at 450 nm on a Titertek Multiskan MCC plate reader. The calibration curve of bovine IgG by ELISA kit is y = 225.45x2 - 21.237x - 1.4713, linear correlation and average recovery were 0.9997, 0.93~1.06, respectively.(2)By selecting the centrifugal time and the solvent, the pretreatment conditions of the milk powder sample were established: when the milk powder samples were dissolved in PBS solution, adjusted pH value between 4.5 and 4.6, centrifuged 15min by 4000r/min, filtered and took clear liquid to pH 7.4 again in 100mL. This pretrement is simple and the separation effect is good. (3)The double antibody sandwich method established here has the recovery rate of 93.06~100.12% and the variation coefficient of 3.09%~1.200%. The variation coefficients in the same plate and different plates were also less than 5%. This method is stable and shows highly accurate data in the quality control chart of the SPSS software.(4)The ELISA kit was preserved 60 days at 4℃. According to the quality control chart of SPSS software, the ELISA kit is stable and data is accurate and reliable. The kit can not only save test time (measure directly, no need to preserve overnight), but also simplify the test process. The HPLC method which shows higher data and less accurate is compared to the ELISA kit.