| Objective:To develop a new α1-microglobulin detecting reagent with independent intellectual properties according to the principle of latex turbidimetric immunoassay. Provide an economic and high performance reagent for clinical laboratory, and then estimate the clinical significance of urine and serum α1-microglobulin in patients with diabetic nephropathy(DN) in early stage.Methods:1. Produce a α1-microglobulin reagent kit by the means of latex turbidimetric immunoassay, and determine the optimum conditions of the amount of EDAC, the size of latex particle, the amount of antibody and also reaction time. Develop a new system of α1-microglobulin reagent kit under Hitachi7170. The evaluation tests of methodology include precision test, linear range, stability test, recovery test and interference test. Compare the newly-made reagent kit to Meikang reagent kit that is already used for clinical laboratories.2. The concentration of urine α1-microglobulin measured in150healthy controls is used to evaluate the reference range of normal adults. Grouping81diabetic patients to normoaluminuria, microaluminuria and macroaluminuria according to the amount of urinary albumin. Collect the24h urine and serum of patients to measure serum α1-microglobulin, creatinine,24hour urinary albumin, α1-microglobulin, β2-microglobulin, and NAG. Compare the difference and the correlation between the markers. ROC is used to evaluate the sensitivity, specificity and methodology efficiency of α1-microglobulin and also the other markers for DN.Results:1. The optimal dameter of latex was80nm, and the optimal amount of EDAC was8.0-9.0mg/L. The perfect ratio for antibody and coating buffer is0.08:1. The optimal mixing time was3hours. The within-and between-run imprecision of low, middle and high levels of α1-microglobulin is5.09%,2.68%,2.86%and7.66%,3.30%,3.17%. The linear range were0-92.0mg/L. Reagent keeps stable in3months at2-8℃. The range of recovery rate is96.28%-103.45%, and the average analytical recovery rate is98.92%.≤10g/L serum hemoglobin,<310ummol/L serum bilirubin and≤6.5mmol/L serum triglyceride have no interferences on α1-microglobulin concentration in this assay. However, when the concentration of triglyceride comes to14.75mmol/L, a significant interference on low concentration of α1-microglobulin comes out. The correlation coefficient with Meikang reagent kit is0.991.2. The reference range of urine α1-microglobulin is0-12.8mg/L. According to the concentrations of24h urinary albumin, we divide the diabetic patients into three different groups, one was simple diabetes group(n=39)when the24h urinary albumin is<30mg/d, while the other two are microalbuminia(n=20,24h urinary albumin is30mg/d to300mg/d) and macroalbuminia(n=22,24h urinary albumin>300mg/d). With the increasing of excretion of24h mAlb, serum creatinine, α1-microglobulin and urinaryα1-microglobulin,(32-microglobulin and NAG increase as well. There is a positive correction between urinary/serum α1-microglobulin and mAlb, β2-microglobulin while no correction is found between urinary α1-microglobulin and serum α1-microglobulin(r=0.479). Further study shows that in normoalbuminia and also macroalbuminia group, urinary α1-microglobulin has a positive correlation with urinary β2-microglobulin. Also a positive correlation between urinary α1-microglobulin and β2-microglobulin, serum α1-microglobulin and mAlb, urinary α1-microglobulin is found in macroalbuminia too. ROC shows that the AUC of urinary α1-microglobulin, mAlb, serum α1-microglobulin, urinary β2-microglobulin and NAG is separately0.894,0.816,0.806,0.770and0.680(P<0.05), revealing that urinary and serum α1-microglobulin have great value to estimate the damage of DN.Conclusions:1. Develop a new α1-microglobulin reagent kit according to the principle of latex enhanced turbidimetric immunoassay, providing clinical laboratory a new and economic selection with good quality.2. Urinary α1-microglobulin shows more sensitivity than other kidney damage markers such as creatinine, mAlb and so on. With the help of measuring urinary α1-microglobulin, the impairment of DN with early renal damage is more easily to be detected. |
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