Production of Monoclonal Antibodies for Determination of Egg Yolk Cholesterol from Japanese Quail
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This research aimed at producing monoclonal antibody against cholesterol for measurement of cholesterol level in egg yolk of Japanese quail by Enzyme-Linked Immunosorbent Assay (ELISA). Cholesterol and BSA conjugated (Cholesterol-3-BSA) antigen was created for stimulation antibody against cholesterol. Balb/c mice at 6 weeks of ages were used in this study. Production of monoclonal antibody was initiated by injecting 100 μg of Cholesterol-3-BSA together with Freund's complete adjuvant as a stimulant. This subcutaneous injection has been done 3 times every 2 weeks. Spleenocytes from the mice that had produced antibody against the cholesterol were collected for fusion with myeloma cells. These fused cells were cultured in 96-wells microplates. Types of monoclonal antibodies were classified by direct ELISA method. The monoclonal antibody obtained was analyzed to quantify cholesterol via competitive ELISA and was compared to Zak’s method (1957). It was discovered that Balb/c mice produced antibody against cholesterol on the 4th week after immunization. Ten percent of wells (35/352) showed clonal growth. Within those 35 wells, 20 wells produced antibody against cholesterol. Clone was separated by limiting dilution into 12 individual clones which were all antibody typed Immunoglobulin G. One of these 12 clones was able to grow further, while the rest (11 clones) were dead. From ELISA experiment, the cholesterol at 50% binding was found concentration valued 7.2 pg/ml. By comparing with the Zak's method (1957), which concentration at mid point of standard curve was 325.0 μg/ml. The cholesterol level resulted from ELISA and Zak (1957) in averaged±S.E.(n)were 645.3±140.9 (40) and 656.2±40.8 (40) mg/100 g egg yolk, respectively, (p> 0.05). In conclusion, we could established ELISA method for analyzing cholesterol content as small as 7.2 pg/ml, which useable for detection in egg yolk of Japanese quail. The sensivity of the ELISA method was 45.14 x 106 times higher than the Zak method.
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