# Efficient Detection of Pre-Proinsulin by Double Antibody Sandwich ELISA
## 1 Introduction  
Diabetes mellitus, commonly known as diabetes, is a metabolic disease characterized by high blood glucose levels due to defective insulin secretion or impaired insulin action [1]. Diabetes includes mainly Type I and Type II diabetes, and is one of the major diseases that threaten human health and life. Type I diabetes is characterized by a loss of the ability of pancreatic beta cells to secrete insulin, and patients rely on insulin therapy to maintain their lives. Type II diabetes, which accounts for approximately 90% of adult-onset diabetes, is caused by a combination of insulin secretion, insulin resistance, hepatic glucose output and lipid disorders [2]. Insulin can regulate glucose metabolism and control blood glucose balance and is the main drug for treating diabetes [3]. In addition, insulin therapy has shown significant advantages over anti-diabetic drugs in both type I and type II diabetes [4]. Insulin, originally produced as preproinsulin, is transformed into a proinsulin by proteolytic action and finally into the active polypeptide hormone, insulin [5]. Recently, developments in recombinant DNA technology and protein engineering have led to the increasing availability of human insulins and modified insulin analogues with improved pharmacokinetic properties [6]. Most of the insulins currently used in clinical trials are formed by recombinant DNA expression in E. coli or yeast to form inactive pre-proinsulin (PPI), subsequently enzymatically cleaved and purified to obtain high purity insulin. The efficient and stable expression of PPI is a prerequisite for obtaining high-yield insulin [7, 8]. PPI, although low in molecular weight (only 15 kD), remains immunogenic and its prolonged presence can produce antibodies and thereby reduce the pharmacologicaleffects of insulin [9]. However, undigested and unremoved PPI seriously affects the quality and safety of insulin [10; 11]. Therefore, the accurate detection of PPI expression and residues in insulin is very critical and urgent in the quality control of insulin biosynthesis.  
According to the European Pharmacopeia, PPI in insulin must be detected and controlled; however, no specific detection method has been proposed. Currently, the commonly used methods include radioimmunoassay and high-performance liquid chromatography (HPLC) [12; 13; 14]. Radioimmunoassay has been gradually eliminated due to inadequate prices, pollution to the environment and harm to human health [15]. High-performance liquid chromatography is often used to detect the PPI content in the purification process; however, its poor sensitivity leads to its inability to meet the requirements for detecting PPI residues in the final product [16].  
Enzyme-linked immunosorbent assays have high sensitivity and can be used to detect trace amounts of PPI. However, due to the large differences in the contents of PPI produced by different processes, using a universal ELISA kit for determination is currently impossible. In this study, a PPI molecule consists of the A-chain of insulin, B-chain of insulin, a guide peptide (SOD) and a -Lys-Arg- linker. After trypsin digestion, SOD and single-chain Lys-Arg-insulin (sIKR) were produced. As the enzyme digestion proceeded, the junction of the A-chain and arginine was cleaved, but only the remaining B-chain was linked to Arg-Lys-, and the free A-chain was correctly paired with the B-chain to form Arg-Lys-insulin (IKR). The IKR was turned into lys-insulin (IK) after the arginine residue was removed by carboxopeptidase B, and the lysine residue in IK was further removed to form insulin with the correct spatial conformation. To explore a highly targeted and sensitive method, we prepared six anti-PII monoclonal antibodies using the hybridoma technique and preliminarily established double-antibody sandwich ELISA to determine the PPI residue in recombinant human insulin.