A novel general and efficient technique for dissociating antigen in circulating immune complexes

Abstract

Circulating immune complexes (CICs) are produced during the immune response. It is more clinically important to establish a general and efficient CICs dissociation technique for the detection of antigens for CICs other than the detection of free antigens in the serum. Polyethylene glycol (PEG) two-precipitation separation and glycine-HCl as a buffer system were employed to develop a general and efficient buffer dissociation technique to separate CICs from serum and dissociate antigens from CICs. The measurement value of new PEG two-precipitation separation technique was higher than traditional PEG precipitation separation technique. There were slight differences in the dissociation conditions of HCV Core-IC, HIV P24-IC, Ins-IC and TG-IC as compared to HBsAg-IC. The detection of antigens in HBsAg-IC, HCV Core-IC, HIV P24-IC, Ins-IC and TG-IC with this technique was superior to that with HCl Dissociation, Trypsin Digestion or Immune Complex Transfer technique. PEG two-precipitation dissociation technique may reduce macromolecular protein and the adhesion of free antigens during the co-precipitation, which increases the efficiency of separation and precipitation of CICs. This technique also avoids the damage of reagents to antigens, assuring the repeatability, reliability and validity. Thus, this technique is application in samples negative or positive for free antigens.

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

“…CIC separating agent A, CIC separating agent B, and CIC dissolving agent were prepared as previously reported with slight modifications [19]. Briefly, to prepare the CIC separating agent, 0.15 M borate buffer containing 0.132 M NaCl and 0.02% ProClin 300 biological preservative was mixed with 8% and 7% polyethylene glycol (PEG) 6000 to yield CIC separating agent A and CIC separating agent B, respectively.…”

“…Four free HBsAg-positive samples (samples 1–4: 2.68–182.92 IU/ml), 3 free anti-HBs-positive samples (samples 5–7: 23.89–897.32 mIU/ml, of which sample 6 and 7 were from acute HBV-infected patients in the recovery phase), 1 sample (sample 8) negative for both free HBsAg and free anti-HBs, and the prepared HBsAg-IC positive control I and positive control II were independently deposited and separated using the PEG double-precipitation separation method [19] and the traditional PEG precipitation separation method [22]. The CIC antibody dissociation technique developed by our group was then used to dissociate anti-HBs from HBsAg-ICs and the corresponding blank control.…”

“…The dissociation of anti-HBs from HBsAg-ICs was performed according to a previous method [19], with slight modifications. Briefly, the HBsAg-IC positive control II was precipitated by the double-precipitation separation method under conditions including 0.06 M glycine-HCl buffer at different pH values (1.0, 1.4, 1.8 and 2.2), temperatures (4 °C, 15 °C, 25 °C and 37 °C), dissociation times (5 min, 8 min, 10 min and 20 min), shaking frequencies (0,20,60, and 100 cycles /min) and residence times after dissociation (0 min, 5 min, 10 min, and 15 min).…”

“…The currently available methods for detecting CICs include the following: (1) the detection of total CICs via antigen-nonspecific methods (such as physical techniques, complement techniques, antiglobulin techniques, and cellular techniques) [12] and antigen-specific methods (such as in two-component-determined CICs) [13]; (2) antigen-specific methods for detecting antigens in CICs (such as the HCl dissociation technique [14], the surfactant dissociation technique [15], the trypsin digestion technique [16], the immune complex transfer technique [17], immune complexome analysis [18] and the CIC antigen dissociation technique [19]); and (3) antibody-specific methods for detecting antibodies in CICs (such as the dissociation enzyme-linked immunosorbent assay (ELISA) for anti-Leishmania IgG in ICs [20]). The above methods, except for the CIC antigen dissociation technique [19], have poor specificity [12, 21] and poor sensitivity; therefore, these approaches may not meet the requirements for clinical diagnosis and scientific studies [12, 13, 15, 21] and may have limited applications [13–18, 20]. In particular, little is known about antibody-specific methods for detecting antibodies in CICs.…”

“…In particular, little is known about antibody-specific methods for detecting antibodies in CICs. Based on our previous findings [19] and previously reported studies [20], we developed a general and efficient dissociation technique to detect antibodies in CICs (also known as the antibody-specific method for detecting CICs). This technique can be used for dissociation of specific antibodies from CICs in serum samples.…”