Establishment and evaluation of a general dissociation technique for antibodies in circulating immune complexes

Abstract

This study aimed to establish a general and efficient dissociation technique for detecting antibodies in circulating immune complexes (CICs) in serum and to evaluate its clinical application. CICs were efficiently separated from specimens using polyethylene glycol double-precipitation. The best conditions for anti-HBs dissociation from HBsAg-ICs were a pH of 1.80, incubation at 15 °C for 5-10 min, and detection within 10 min after neutralization. The mean dissociation rate, reproducibility, mean dissociation recovery rate and specificity of the new technique were 64.3%, < 5.97, 95.4 and 100%, respectively. They had a favourable linear relationship (r = 0.9932), and the stability of the reagents exceeded 24 months, except the CIC antibody dissociation reagent (> 12 months). Conditions for the dissociation of other CICs tested were similar, but there were differences in the rate of antibody dissociation. Different HBV-M patterns had significantly different levels and rates of antibody dissociation from HBsAg-IC (P < 0.05), and the detection rates of the corresponding antibodies in HCV, core-anti-HCV core antibody (HCV-ICs), HIV P24-anti-HIV P24 antibody (HIV-ICs), insulin-anti-insulin antibody (INS-ICs) and thyroid globulin-anti-thyroid globulin antibody CICs (TG-ICs) were 34.8, 66.7, 20 and 14.3%, respectively. These data suggest that our CIC antibody dissociation technique is a good general pretreatment technique for the detection of antibodies after the precipitation, separation and dissociation of multiple CICs.

Dissociation Phenomenon of Erythrocyte Agglutination and Its Application to Assay of Functional Activity of the Complement System in Clinical Laboratory

“…Firstly, the relative affinity of antibodies for antigens, wherein high‐affinity antibodies strongly bind to antigens that are difficult to dissociate, while low‐affinity antibodies weakly bind to antigens that are easily dissociated [ 33 , 34 ]. Secondly, environmental factors such as high or low pH and increased ionic strength can reduce or eliminate the electrostatic gravitational force between antigen and antibody, leading to immune complex dissociation [ 3 , 35 ]. Erythrocyte agglutination dissociation based on erythrocyte fragments refers to the dissociation of erythrocyte agglutination induced by the addition of homotypic erythrocyte fragments without alteration of the existing agglutination, antibodies, or reaction conditions.…”

Circulating immune complexes and mutations of HBsAg are associated with the undetectable HBsAg in anti-HBs and HBeAg positive occult hepatitis B virus infection

“…In this case, the positive result of anti-HBs indicates that the host has generated strong humoral immune responses to the virus, which reacts with the free HBsAg in plasma and forms CICs, resulting in undetectable HBsAg. Several methods for detecting CICs have been reported ( Stanojevic et al, 2000 ; Zhang et al, 2011 ; Matsumoto et al, 2017 ; Wang et al, 2019 ). Through preliminary experiments, we found that PEG-double precipitation combining HCl and trypsin digestion was better to detect HBsAg-CICs.…”

“…The 0.15 M borate buffer containing 0.132 M NaCl was added with 8 and 7% polyethylene glycol (PEG) 6000 to produce CIC separating solution A and CIC separating solution B, respectively ( Dai et al, 2018 ; Wang et al, 2019 ). The parameters for the quality control were a pH of 8.40–8.80 with the osmolarity at 500–530 osm.…”

Research Progress on the Mechanism of Persistent Low-Level HBsAg Expression in the Serum of Patients with Chronic HBV Infection

“…Because the body is in a low response state for a long time, the body's ability to clear CIC decreases. As a result, the concentration of free HBsAg in serum decreases or cannot even be detected [ 67 ]. At the same time, the use of immunosuppressants is also related to a low-level state of HBsAg.…”