The Improvement Of Detection Method For Trypsin And Chymotrypsin In The Serum And The Milk Of Dairy Cow

Trypsin was mainly used for anti-inflammatory swelling and improving circulation in veterinary clinics,it is beneficial to the local site of inflammation,relieving congestion and drug absorption.Chymotrypsin can decompose arthritic fibrin coagulation and promote wound healing.Combined using of trypsin and chymotrypsin can decompose the protein,make the protein into small molecular peptides and amino acids.Meanwhile,the mixed enzyme by trypsin and chymotrypsin has obvious antibacterial effect on staphylococcus aureus etc.,and can be used in the treatment of cow mastitis mainly caused by gram-positive bacteria including staphylococcus and streptococcus.It is nessessary to establish more accurate detection method to detect trypsin and chymotrypsin in the serum and the milk of dairy cows.The detection principle of trypsin activity is that in the assay trypsin cleaves a substrate to get p-nitroaniline?p-NA?which is detected at X = 405 nm.The detection principle of chymotrypsin activity is also that chymotrypsin cleaves a substrate to get p-nitroaniline?p-NA?which is detected at X,405 nm.To determine the activity of trypsin by Trypsin Activity Assay Kit and chymotrypsin by Chymotrypsin Activity Kit in the serum and the milk of dairy cow respectively,the former detection methods trypsin and chymotrypsin were improved in this study.The results showed that there is good linear relationship between the optical density and standard concentration of trypsin during the range of 0 nmol/?L?0.4 nmol/?L,with regression equation Y = 0.023X₀.0038,R²= 0.9996.The lowest detectable limit of trypsin in the serum was 15 ng/mL.The average recovery in the serum was 85.6%.The RSD of inner-day in the serum was 13.7%,13.2%,8.1%respectively.The RSD of infra-day in the tested serum was 13.8%.The lowest detectable limit of trypsin in the tested milk was 25 ng/mL.The average recovery in the milk was 84.3%.The RSD of inner-day in the milk was 13.9%,10.2%,11.8%respectively.The RSD of intra-day in the milk was 11.0%.The results also showed that in the serum there is good linear relationship between the optical density and standard concentration of chymotrypsin during the range of 1 ?g/mL?6 ?g/mL,with regression equation was Y ?0.0168X-0.0001,R²= 0.9951;There is good linear relationship in the milk between the optical density and standard concentration of chymotrypsin during the range 0.5 ?g/mL?3?g/mL,with regression equation was Y = 0.0295X-0.0036,R² =0.9968;The lowest detectable limit of chymotrypsin in the tested serum was 0.5 ?g/mL.The average recovery in the serum was 72.6%.The RSD of inner-day in the serum was 0.49%,2.4%,1.2%respectively.The RSD of intra-day in the serum was 0.48%.The lowest detectable limit of chymotrypsin in the tested milk was 0.25 ?g/mL.The average recovery in the milk was 88%.The RSD of inner-day in the milk was 9.8%,7.2%,2.3%respectively.The RSD of intra-day in the milk was 7.7%.The improved method was tested to be a high sensitivity,good accuracy and precision.