| BackgroundDiabetes mellitus which caused by multiple risk factors is a complex metabolic diseasewith high morbidity and may leads serious damage to health. Current guidelines for thediagnosis and treatment of diabetes mellitus recommend distinguish Type1diabetes fromType2diabetes, which are the two main subclass of diabetes, because of their differentpathogenesis and treatment measures. Most Type1diabetes is due to the absolute insulindeficiency leaded by autoimmune destruction of pancreatic β cells. One of itsimmunological characteristics is that autoantibodies to islet β cell-associated molecules canbe found in peripheral blood and these autoantibodies as detection markers provide aneffective solution for diagnostic classification. But autoantibodies commonly used inclinical, such as insulin autoantibody (IAA), glutamic acid decarboxylase autoantibody(GADA), protein tyrosine phosphatase autoantibody (IA-2A) have their own defects ofpredictive or diagnostic value because of the inconsistency of appearance, duration andintensity in type1diabetes. In recent years, newly discovered zinc transporter8autoantibody (ZnT8A) have good sensitivity and specificity for the detection of type1diabetes and combined detection of IAA, GADA, IA-2A and ZnT8A can improve thedetective performance and get better predictive and diagnostic value due to de integration ofadvantages of different autoantibodies. However the common radioligand binding assay andenzyme-linked immuonsorbant assay are mainly designed to determine single item, andthere are inter palte variation and inter well variation because of micropalte as reactioncarrier, so it is difficult to simultaneously detect four indicators under the same conditions.These inconvenience makes combination detection costly, time-consuming, complicated tooperate and hard to control the variation, resulting the testing work restricted in clinic. ObjectiveTo develop a dot immunogold filtration assay to detect diabetes autoantibodies basedon the solid phase membrane immune response principle and build a serological diagnostickit for combined detection of multiple diabetes autoantibodies including IAA, GADA,IA-2A and ZnT8A, making the four indicators can be detected under the same conditionsimultaneously and finally provide a quick, convenient, accurate and inexpensive detection way.MethodPrepared colloidal gold and conjugated it with streptavidin as stained probe, coatedrecombinant human insulin, glutamic acid decarboxylase (GAD), tyrosine phosphatase2(IA-2), zinc transporter8(ZnT8) antigen onto nitrocellulose membrane, built abiotin-streptavidin signal amplification reaction system and determined its optimal testingconditions to develop dot immunogold filtration assay for detecting diabetes autoantibodies,then assembled immune infiltration devices and reagents into immune infiltrationcombination detection kit, preliminarily use it to detect clinical cases and healthy controlsamples to evaluate its performance.Results1. The18nm colloidal gold prepared had been proved to have good dispersion andhomogeneity by the identification of transmission electron microscopy and was successfullyconjugate to streptavidin to form stained probe which was purified by high-speedcentrifugation at last.2. The optimal reaction conditions to detect IAA, GADA, IA-2A, ZnT8A weredetermined and the immune infiltration combination detection kit had been built with itspreparation process optimized.3. Sensitivity of the kit was66.7％in42T1DM patients and specificity was97%in100healthy individuals. There was no significant difference in comparison with thedetecting results in T1DM patients by using ELISA kit (P>0.05). Test results wereconsistent with3batches of the kits and stable performance was exhibited after10weekslater stored at4°C.ConclusionAn immune infiltration combination detection kit which can simultaneously detectIAA, GADA, IA-2A and ZnT8A was successfully built and proved to have a good detectionperformance and practical value. |
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