# Efficient Detection of Pre-Proinsulin by Double Antibody Sandwich ELISA
## 4 Discussion  
Diabetes is a chronic and metabolic disease that occurs when the pancreas does not produce enough insulin or when the body is unable to use the insulin produced effectively [17]. When diabetes is left uncontrolled or poorly controlled over time, it can lead to increased atherosclerosis throughout the body, resulting in atherosclerotic disease. At present, there is no eradication of diabetes, but there are various treatments that can be used to help control it. Medication is divided into two categories: the first is oral medication, including sulphonylurea, biguanides, alpha glucosidase inhibitors, insulin sensitizers and glargine insulin promoters; the second is insulin therapy. Insulin preparations include animal insulin, human insulin and insulin analogues [18]. The disadvantages of drug treatment, however, are the development of resistance and weight gain. In addition to the use of medication, exercise therapy is more recommended and can increase physical activity to improve the body's sensitivity to insulin. In addition, diet is the basis of treatment for all types of diabetes, and some patients can control mild diabetes with diet alone [19]. When patients with uropathy are unable to control their blood sugar through diet, exercise and other medications, or when medications cannot be used, then insulin therapy is necessary. Insulin precursors are produced in the synthesis of recombinant insulin and the precursor proteins inevitably trigger immunogenic interference with the effectiveness of insulin therapy. Therefore, the detection and quality control of insulin precursors is necessary and important. To address this issue, we have developed an ELISA method for the rapid and highly sensitive detection of insulin precursors. In all of European pharmacopeia, US pharmacopeia and Chinese pharmacopeia, to control the production process of insulin precursors was proposed, indicating that detecting insulin precursors has become an indispensable link in process control and final product release. The process control is mostly performed by a proper HPLC method [20]. However, because of the low content of precursor material in the final product, and the high sensitivity of the detection method, detecting insulin precursors in the product release control is difficult for each enterprise. Both insulin and insulin analogues are recombinantly expressed as insulin precursors and are obtained by enzymatic purification. For the determination of insulin precursors, the US Pharmacopoeia sets a limit of detection (10 ng/mg) but does not give a specific method, whereas European Pharmacopoeia recommends the use of radioimmunoassay (10 ppm), but this method requires high instrumentation and radioactive contamination. [PERSON] et al. used the RP-HPLC method for the determination of insulin precursors, but the low sensitivity clearly does not meet the requirements of the relevant [21]. Enzyme-linked immunosorbent assays have high sensitivity, but they are mostly used to detect natural pro-insulin in serum [22; 23]. There are many kinds of intermediate products and similar molecular structures when the precursor substances in insulin are detected, easily leading to cross reactions; in addition, the high concentration of insulin in the detection samples tends to interfere with the detection results. In this study, the purified insulin precursor was collected in the production process of recombinant human insulin in a Hefei Tianmai biology company as an antigen to prepare monoclonal antibodies. In order to establish a double antibody sandwich ELISA for the detection of insulin precursors, the monoclonal antibodies obtained after purification were screened against each other to obtain the best antibody pairing, and subsequently, the specificity of the paired antibodies was assayed to screen out the antibody combinations with good specificity. In addition, the accuracy and sensitivity of the method were verified.