Application Of Response Surface Methodology In The Optimization Of Measurement Conditions For Enzymatic Kits

Objective:According to the characteristics of enzymatic catalytic activity measurements,select three enzymatic items,and use response surface methodology(RSM)to obtain the best reagent conditions,measurement conditions and interactions between important influence factors under fewer experiment times.To get the best results in the development of enzymatic kits.Methods:1.Choose three routine enzymatic items with higher frequency of clinical testing: alanine aminotransferase(ALT)and γ-glutamy transferase(GGT)and alkaline phosphatase(ALP).Methodology based on enzymatic reference methods which are recommended by JCTLM;Serum samples were collected from the laboratory of Beijing Aerospace General Hospital,reference laboratory external quality control(RELA)samples were from IFCC,numbered 2016Rela-A and 2016Rela-B;ALT and GGT select European reference material(ERM)from Institute for Reference Materials and Measurements(IRMM),ALP select certified reference material(CRM)from Japanese Clinical Laboratory Standards Committee(JCCLS).2.Single-factor analysis method was used to validate the important influence factors affecting the measurement results of each enzymatic item,and determine the experimental area,ie,the range of value change after quantification of each influence factor.3.According to the results of single-factor analysis method,the important influence factors were validated,and the different experimental conditions and/or combinations of measurement conditions were determined by using the Box-Behnken design method which is commonly used in RSM.4.On the Agilent CARY100 UV-Vis spectrophotometer,combine the experimental conditions and/or measurement conditions which were obtained by the Box-Behnken design with the appropriate reagents and set the measurement parameters to measure the serum samples.5.Minitab16 software was used for analysis.A response surface model was established to obtain regression equations between measurement results and important influence factors of each enzymatic item,and the theoretical optimal value of important influence factors were predicted.6.Fix other experimental conditions except important influencing factors,formulate reagents and set parameters according to the theoretically optimal values of important influence factors,and measure RELA samples,if their measurement results meet IFCC requirements(Permissible relative bias ±5.25%).The response surface model is validated.Otherwise,the experimental area is adjusted according to the verification results and the experiment is repeated.7.After the model verification is passed,set the parameters on the Hitachi automatic biochemical analyzer 7180 to verify the trueness,if the measurement results meet the MU requirements of reference materials,indicating that the verification is successful.Results:1.Single-factor analysis of variance showed that there was a statistically significant difference among the different concentration groups of ALT(P<0.01);there was a statistically significant difference among the different concentration groups of GGT(P<0.01);there was a statistically significant difference among the different concentration groups of ALP(P<0.01),the main influence factors were ALT: p H of the reaction solution,L(+)-alanine concentration,and β-nicotinamide adenine dinucleotide Disodium salt(β-NADH)concentration;GGT: p H of the reaction solution,glycylglycine concentration,L-γ-glutamyl-3-carboxy-4-nitroaniline concentration;ALP: p H of the reaction solution,,p-nitrophenol phosphate(4-NPP)concentration,2-amino-2-methyl-1-propanol(AMP)concentration.The identified experimental areas were ALT: p H of the reaction solution(7.05-7.25),L(+)-alanine concentration(450 mmol/l-550 mmol/l),and β-NADH concentration(0.13mmol/l-0.23mmol/l),GGT: p H of the reaction solution(7.6-7.8),glycylglycine concentration(140mmol/l-160mmol/l),L-γ-glutamyl-3-carboxy-4-nitroaniline concentration(4.5mmol/l-5.5mmol/l);ALP: p H of the reaction solution(10.0-10.4),4-NPP concentration(14mmol/l-18mmol/l),AMP concentration(700mmol/l-800mmol/l).2.Set the important influence factors of each enzymatic item as independent variables A,B,and C,and set the measurement result as the dependent variable Y to establish the RSM model.ALT:Y=126.600+2.799A+0.956B+1.048C-6.951A2-3.169B2-3.556C2+0.215AB+0.062AC-0.887BC;GGT: Y=161.607+3.574A+1.221B+1.337C-8.873A2-4.046B2-4.539C2+0.275AB+0.079AC-1.132BC;ALP: Y=404.582+8.947A+3.056B+3.348C-22.212A2-10.128B2-11.365C2+0.688AB+0.198 A C-2.834 BC.3.The best theoretical values of the important influence factors for each enzymatic item were ALT: p H of the reaction solution 7.17,L(+)-alanine concentration 505.69 mmol/l,β-NADH concentration 0.17 mmol/l,GGT: p H of the reaction solution 7.72,Glycylglycine concentration 146.80 mmol/l,L-γ-glutamyl-3-carboxy-4-nitroaniline concentration 5.06 mmol/l;ALP: p H of the reaction solution 10.24,4-NPP concentration 15.83 mmol/l,AMP concentration 747.28 mmol /l.4.The RSM model validation results were ALT: 2016Rela-A’s bias is +0.48%,2016Rela-B’s bias is +0.92%;GGT: 2016Rela-A’s bias is +0.30%,2016Rela-B’s bias is +0.85%;ALP: 2016Rela-A’s bias is +1.70%,2016Rela-B’s bias is-0.41%;each item ’s bias can fulfil the requirement of IFCC(± 5.25%).5.The relative bias between the verification results and the reference materials’ indicated values on the Hitachi 7180 automatic biochemical analyzer were ALT:-0.81%,meets the requirement of ±2.50% of the reference material’s MU,GGT:-1.19%,meets the requirement of ±2.10% of the reference reference material’s MU,and ALP:-2.41%,meets the requirement of ± 3.06% of the reference material’s MU.Conclusions:This study successfully established RSM models for the three enzymatic items of ALT,GGT and ALP,which effectively compensated for the deficiencies of the traditional single-factor analysis method and obtained the best measurement conditions with fewer experiment times.This research has greatly improved the experimental efficiency,laid the foundation for advancement of standardization and harmonization of laboratory medicine,and also provided new ideas and references for the optimization of experimental conditions in the development of other in vitro diagnostic test kits.