Diagnostic Values Of Serum Hybrid Kappa\lambda Antibody For Autoimmune Pancreatitis And The Study Of Its Producing Mechanism

Autoimmune pancreatitis (AIP) is pancreatic manifestation of an IgG4-related systemic disease characterized by elevated serum IgG4 levels and extrapancreatic lesions, which accounts for 2% of chronic pancreatitis cases, usually presenting with obstructive jaundice due to enlargement of the pancreatic head or thickening of the lower bile duct wall. The only generally accepted serological marker currently used for the diagnosis of AIP is IgG4. The etiopathogenesis of AIP are largely unknown and the significance of IgG4 in the disease development still remains unclear.The classic antibody paradigm is that a single mature plasma cell produces symmetrical antibodies composed of one type of immunoglobulin heavy chain and one type of immunoglobulin light chain, either kappa (κ) or lambda (λ). Within the last 10 years, it has been shown that human IgG4 is a dynamic antibody, which is involved in a continuous process of half-molecule exchange. This process, also referred to as "Fab-arm exchange", can result in asymmetric antibodies with two different antigen-combining sites. Recently, one report showed that hybrid κ/λ, antibodies compose a substantial portion of IgG4 in normal human serum. These molecules are formed by two IgG4 heavy chains plus one κ and one λ light chain. Because AIP is characterized by an elevated serum IgG4 concentration, we attempted to investigate the diagnostic utility of hybrid κ/λ antibody in the diagnosis of AIP and its differentiation from pancreatic cancer.We established an enzyme-linked immunosorbent assay (ELISA) system to measure the levels of hybrid κ\λ antibodies in human sera. First, a double-antibody sandwich ELISA for hybrid κ\λ, antibody detection was developed, in which the plates were coated with anti-human lambda light chain and the peroxidase labeled anti-human kappa light chain was used as detection antibody by a three-step development optimization and validation strategy. Sera were obtained from 338 patients, including 61 with AIP,74 with pancreatic cancer,50 with acute pancreatitis,40 with ordinary chronic pancreatitis,15 with miscellaneous pancreatic diseases, and 98 with normal pancreas. Our study showed the median and range of hybrid κ\λ antibody in patients with AIP, acute pancreatitis, ordinary chronic pancreatitis, pancreatic cancer, miscellaneous pancreatic diseases and normal pancreas was 6.75AU/mL (range,1.53-55.5 AU/mL),2.06 AU/mL (range, 0.26-4.97 AU/mL),2.06 AU/mL (range,0.86-4.94 AU/mL),2.04 AU/mL (range, 0.93-6.1 AU/mL),1.61 AU/mL (range,0.94-4.87 AU/mL) and 2.05 AU/mL (range, 0.42-7.2 AU/mL), respectively. Levels of hybrid κ\λ antibody in AIP group were significantly higher than in the non-AIP groups (P<0.001). However, there was no significant difference between the non-AIP groups (p=0.66). In order to evaluate the utility of hybrid κ\λ antibody in the diagnosis of AIP, cut-off values were established to determine the sensitivity and specificity. The optimal cutoff value hybrid κ\λ antibody was 4.04 AU/ml from the ROC curve. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the diagnosis of AIP were 80.3%, 91%,66.2% and 95.5% respectively. The area under the curve was 0.93 (p<0.001). Furthermore, the combined measurement of serum hybrid κ\λ antibody and IgG4 tended to increase the sensitivity (90.2% vs.78.7%) without sacrificing the specificity (85.9% vs. 87.7%), compared to measurement of IgG4 alone.Next, we carried out a series of studies regarding the producing mechanism of hybrid κ\λ antibody in patients with AIP. Size exclusion chromatography (SEC), affinity purification and non-reducing SDS page polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that most of the hybrid κ\λ antibodies were monomeric. Levels of hybrid κ\λ antibody were significantly correlated with the concentrations of serum IgG4 (r =0.772, p<0.001). The predominant immunoglobulin type of hybrid κ\λ antibody was IgG4 which determined by a new magnetic separation technique. Furthermore, hybrid κ\λ antibody could be generated in vitro and the denaturing condition (GSH) was a necessary requisite for the reaction.Taken together, our study demonstrated hybrid κ\λ antibody is a new serological marker to diagnose autoimmune pancreatitis and differentiate it from pancreatic cancer. High levels of hybrid κ\λ, antibody in patients with AIP had a close relation with serum IgG4 and probably derived from Fab-arm exchange between different IgG4 molecules. Most of them existed as antibody monomers, which can help in a broader understanding of the role of IgG4 in AIP and other IgG4 related diseases (IgG4-RD).