The Establishment Of The Reference Method For The Measurement Of The Catalytic Concentration Of Cholinesterase

Objective: To investigate the affecting factors and establish the reference method for themeasurement of the catalytic concentration of cholinesterase.Methods:⑴The measurands of cholinesterase were divided into measuring the proteincontent and measuring the catalytic concentration, according to the measurement methodsat home and abroad over the past20years. The catalytic concentration measuring wasselected according to the feasibility of measurement automation, the standardization of themeasurement, the accessibility and the cost of reagent.⑵The cholinesterase assay kitwere selected according to the superiority of measurement principle, the market share, theaccessibility, and its performance was evaluated.⑶The affecting factors and its optimalmeasuring conditions of the cholinesterase measurement were obtained according to thebasic theory, the research status of reference method, the principle and characteristics ofenzymatic measurement.⑷The reagents and its optimal measuring conditions ofcholinesterase measurement were selected using the one-way-variant method.⑸The keyfactors of the measuring conditions were optimized using the response surfacemethodology.⑹The measuring conditions of the factors involved were verifiedby experiment.Results: The catalytic concentration was selected as the measurand of the referen-ce method.⑵The measurement principle of the reference method was selected: b-utyrylthiocholine/5,5’-Dithiobis (2-nitrobenzoic acid)(DTNB).⑶The affecting fact-ors and its optimal measuring conditions: the measuring wavelength was405nm, themolar extinction coefficient was13300LM-1cm-1, the bandwidth was2nm, the sam-ple volume ratio was1:251(0.0040), the delay time was60s, the measuring time w-as90s.⑷The reagents and its optimal measuring conditions: the concentration ofbutyrylthiocholine was1.48mmol/L, the concentration of DTNB was0.30mmol/L, t-he buffer pH was8.0, buffer ionic strength,0.30mmol/L.⑸The key factors werethe buffer pH(A), the concentration of butyrylthiocholine(B) and the concentration of DTNB(C). The multiple regression formula was obtained using the response surf-ace methodology:Y=8476.13‐73.29A20.00B‐4.56C‐126.86A232.85B271.48C2‐55.47AB‐1.06AC‐16.34BC⑹The results of the verified experiment: the measured value was8755U/L,up to95%of the theoretical maximum value.Conclusion: The reference method of the cholinesterase measurement is establishedusing the catalytic concentration method. By combining the one-way-variants method withthe response surface methodology, the affecting factors and optimal measurementconditions are obtained successfully.