Antigens In Circulating Immune Complexes Research Articles

Immune complexome analysis of antigens in circulating immune complexes from patients with acute cellular rejection after living donor liver transplantation

Liver transplantation is a life-saving procedure for many end-stage liver diseases; however, rejection after transplantation is still occurs in some recipients. The most common form of rejection is T cell-related acute cellular rejection (ACR). To understand the mechanism of rejection, it is necessary to identify immune targets. Since the development of B cell immunity depends upon concordant T cell immunity, we hypothesized that rejection-specific antigens in circulating immune complexes (CICs) may be present in the sera of recipients experiencing rejection, and as such, may be useful as diagnostic biomarkers for ACR. The purpose of this study was to investigate rejection-specific antigens in CICs (CIC-antigens) in serum of ACR patients. We applied immune complexome analysis, in which CICs are separated from whole serum and then subjected to direct tryptic digestion and identification of CIC-antigens by nano-liquid chromatography-tandem mass spectrometry, to sera of 32 living donor liver transplant recipients (10 recipients experienced ACR and the others did not experience). CIC-antigens were compared between rejection and non-rejection groups to elucidate those that were only detected in the rejection group. We identified 11 CIC-antigens that were only detected in patients who experienced rejection, 4 of which (thrombospondin-1, apolipoprotein E, apolipoprotein C-III, and complement factor H) were only detected during ACR.

Immune Complexome Analysis of Serum and Its Application in Screening for Immune Complex Antigens in Rheumatoid Arthritis

Analysis of circulating immune complexes (CICs) produced during an immune response may be useful in elucidating some aspects of this process. Identification of antigens incorporated into CICs provides information that may be helpful in developing diagnostic and treatment strategies for autoimmune diseases, infection, cancer, and transplantation therapy, and such information might be more relevant than information on free antigens. Because CICs may contain many antigens, comprehensive identification and profiling of such antigens is more effective than immunoblotting detection. We developed a novel proteomic strategy (immune complexome analysis) in which immune complexes (ICs) are separated from serum, digested directly with trypsin, and then subjected to nano-liquid chromatography-tandem mass spectrometry for identifying and profiling antigens in CICs. We applied this strategy to the analysis of CICs in 21 rheumatoid arthritis (RA) patients. Serum samples from 13 healthy donors and 8 osteoarthritis patients were used as controls. CICs containing thrombospondin-1 (TSP-1) and platelet factor 4 (PF4) were found in the serum of 81% and 52% of RA patients, respectively, and in none of the controls. The ICs in the serum of a majority of the RA patients contained TSP-1 or PF4, and these ICs may have potential as alternative biomarkers. Our technique for immune complexome analysis uses routine clinical samples, simple protocols, and widely available equipment. This method may be generally applicable to the study of the relationship between CICs and certain diseases associated with the immune response in animals and humans.

Characterization of entamoeba histolytica antigens in circulating immune complexes in sera of patients with amoebiasis.

Isolated circulating immune complexes (CICs) from sera of patients with amoebiasis were characterized to determine Entamoeba histolytica antigens that participate in the disease process. In total, 116 serum samples were collected before starting anti-amoebic therapy, and their CICs were isolated by differential polyethylene glycol precipitation. The presence of amoeba-specific antigens in CICs was detected by antigen capture enzyme-linked immunosorbent assay (ELISA) and by immunoblot assay. Antigen capture ELISA showed significantly higher optical density (p < 0.001) in all patients with amoebiasis than in the normal healthy controls and patients of non-amoebic hepatic disorder. Immunoblot assay detected amoeba-specific CICs in all 18 patients (100%) with confirmed amoebic liver abscess, 28 (80%) of 35 patients with clinically-suspected amoebic liver abscess, and 18 (78.26%) of 23 patients with amoebic colitis. No patients with non-amoebic hepatic disorders and healthy control subjects had any detectable level of amoebic antigens in CICs. Immunoblot assay revealed E. histolytica antigens of relative molecular masses of 35, 56, 70, and 90 kDa present in CICs of 64 of 76 patients with amoebiasis. The 35-kDa polypeptide was observed in 52 patients (81.25%). The results of the study suggest that the 35-kDa polypeptide antigen can be a diagnostic marker in active amoebiasis.

Detection of microfilarial antigen in circulating immune complex from sera of Wuchereria bancrofti-infected individuals.

Polyethylene glycol-precipitated circulating immune complexes (CICs) from the sera of patients with Bancroftian filariasis were examined for parasite antigen content by an ELISA. Of the 227 patients, 214 were asymptomatic microfilariae carriers with microfilaremias ranging between six and 14,000/ml blood, and 13 were symptomatic patients with chronic filariasis without microfilaremia. In addition, the sera of 10 of the patients treated with diethyl carbamazine were also examined. These assays were devised using mouse monoclonal antibody raised against microfilariae of Wuchereria bancrofti. Using an acid-phase covalent-bound plate for the ELISA makes it possible to assay the antigen contents simply. Microfilarial antigens were detected from 92 (43.0%) of microfilaria (mf)-positive individuals. Furthermore, the level of antigen in CIC was correlated with mf counts from night blood Nuclepore filtration results. Antigen was not detected in CIC from patients with a variety of nonfilarial helminth infections. These findings indicate that microfilariae play an important role of CIC formation in lymphatic filariasis.

Analysis of circulating immune complexes (CIC) in tuberculosis: Levels of specific antibody and antigens in CIC and relationship with serum antibody

Analysis of circulating immune complexes (CIC) in tuberculosis: Levels of specific antibody and antigens in CIC and relationship with serum antibody

Analysis of circulating immune complexes (CIC) in tuberculosis: Levels of specific antibody and antigens in CIC and relationship with serum antibody

Eighty sera from tuberculosis (TB) patients, 16 Indian and 10 American control sera were analyzed by ELISA for relative titres of antibody against mycobacterial antigens. Levels of specific antibody and mycobacterial Ag in circulating immune complexes (CIC) isolated from these sera were also studied. All these parameters were found to be elevated in TB sera as compared to control sera. Maximum increase was however noted in CIC specific antibody titres. A good correlation was observed between serum and CIC levels of specific antibody (r = 0.72) and between specific antigen (Ag) and antibody (Ab) levels within CIC (r = 0.64). In a few of the TB sera examined, CIC specific Ab contributed less than 1% to the Ab titres in sera. In order to examine the differences between different subgroups within TB patients, a statistical analysis of variance was performed. Sex of the patients had no effect on any parameter. Sputum-positive patients had significantly higher levels of CIC Ag and Ab than the sputum-negative patients, although no significant difference occurred in respect to serum Ab. All three parameters were significantly higher in patients on chemotherapy as compared to fresh untreated cases. The relevance of these observations to the development of a CIC-based immunodiagnostic assay for TB is discussed.

Uses and limitations in the demonstration of specific circulating immune complexes in patients with amoebiasis

A micro-enzyme linked immunosorbent assay (micro-ELISA) has been evaluated as a diagnostic test to detect amoebic antigen in polyethylene glycol (PEG) precipitated circulating immune complexes (CIC) in sera from patients with amoebiasis. The immune complexes were captured on rabbit anti-amoebic IgG-coated wells of microtitration plates and the complexed antigen was detected by enzyme linked antihuman immunoglobulins. A titre of greater than 160 for the immune complexes was considered to be of clinical significance. The immunoassay detected amoebic, antigen-specific CIC in 35 (94.5%) of 37 patients with confirmed amoebic liver abscess. Twenty (55.5%) of 36 clinically suspected cases of amoebic liver abscess had amoebic antigen-specific CIC and responded favourably to anti-amoebic chemotherapy. Only two (20%) of 10 cases of non-dysenteric symptomatic intestinal amoebic infection had amoebic antigen-specific CIC. One (10%) of 10 patients with non-amoebic intestinal disorders also had amoebic antigen in CIC. However, none of 15 cases of non-amoebic hepatic disorders that included hydatid disease, metastatic adenocarcinoma, hepatocellular carcinoma, cholecystitis and choledocal cyst, 13 cases of rheumatoid arthritis and 25 apparently healthy subjects had amoebic antigen in CIC. The levels of the amoebic antigen-specific CIC did not correlate (p greater than 0.05) with either the number of abscess(es) or lobe(s) of the liver involved. However, the levels of antigen-specific CIC were higher (p less than 0.01) in patients with a liver size of more than 5 cm below the right costal margin. Antigen-specific CIC levels tended to decline or disappear during 3-6 months following completion of therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydatid disease: analysis of parasite antigens in circulating immune complexes and in preformed hydatid antigen-antibody complexes.

Fifty-three sera from 29 patients with hydatid disease, all but one positive for specific anti-parasite antibodies and all negative for specific circulating antigens, were studied for the presence of circulating immune complexes (CIC) by conglutinin binding-assay (KgBA). Fourteen serum samples (26%) from eight patients (27%) were positive. These positive sera were pooled for each patient and the eight samples were PEG-precipitated and analysed for the presence of specific Echinococcus granulosus antigens in the CIC using a human anti-human-hydatid cyst fluid antiserum capable of recognizing the major antigenic systems of the parasite namely, antigens 4 and 5. The assays utilized for detecting antigen in CIC were: (a) blotting on nitrocellulose paper after sodium dodecil sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and specific immunological detection; (b) ultracentrifugation in acid buffer and subsequent detection of antigens by a sandwich-radioimmuno assay (RIA); (c) protein separation by isoelectric focusing (IEF) and specific immunological recognition. In addition, all positive sera were analysed for the presence of antigen in the CIC by a modified KgBA and by polyethylenglicol (PEG)-precipitation in acid buffer followed by immunological recognition of antigen. All tests gave negative results with the patients' samples, but were positive with preformed in vitro complexes between parasite antigens and corresponding antibodies. Failure to detect antigen in the CIC could be due to: 1) insufficient sensitivity of the assays used to detect hydatid antigens in CIC; 2) rapid clearance of antigen or CIC from the circulation; 3) presence of parasite antigen not recognized by the antiserum employed; 4) production of CIC as a result of polyclonal B-cell activation. This last hypothesis is supported by the demonstration of IgM-rheumatoid factor (RF) and anti-F(ab')2 antibodies respectively in 11 (44%) and 13 (52%) out of 25 patients.

Specific circulating immune complexes in amoebic liver abscess.

A micro enzyme-linked immunosorbent assay was used to detect the presence of amoebic antigen in polyethylene-glycol-precipitated circulating immune complexes (CIC). A cutoff dilution of greater than 1:160 for the precipitates was taken to be of clinical significance. Among the patients with amoebic liver abscess, 93% (14 of 15 confirmed cases) had indications of amoebic antigen in CIC. In 57% of the suspected cases of amoebic liver abscess, amoebic antigen could be detected in CIC. The specificity of the technique for the demonstration of specific amoebic antigen is indicated by the undetectable levels of amoebic antigen in CIC in serum samples from nine cases of nonamoebic hepatic diseases and 10 apparently healthy subjects. It is thus believed that the specific diagnosis of an individual case of amoebic liver abscess can be made by demonstrating specific amoebic antigen in CIC.

Significance of circulating HBs antigen-antibody immune complexes in patients with HBs antigen-positive liver disease.

The nature of circulating immune complexes (CIC) which appear in patients with type B hepatitis was investigated using a method of Raji cell fluorescent immunoassay. CIC were found in seventeen of thirty-five cases (48.6%) with HBs antigen (HBsAg)-positive liver diseases (4/8 cases with acute hepatitis, 9/18 cases with chronic hepatitis, and 4/9 cases with liver cirrhosis), whereas no CIC were detectable in sera of ten asymptomatic, healthy carriers with HB virus. Among the seventeen cases with CIC-positive liver diseases, HBs antigen-antibody immune complexes (HBsIC) were demonstrated in eleven (65%). A high incidence (54%) of proteinuria was observed in patients with CIC-positive liver disease compared to those without them (10%). Moreover, 83% of patients with HBsIC were associated with proteinuria. A case of fulminant type B hepatitis showed high titers of both CIC and HBsIC during the acute phase of the disease; in the recovery stage, the titers decreased to within normal ranges. These results demonstrate that HBsAg is a possible antigen in CIC during type B hepatitis. Determination of serum HBsIC is significant for the clinical evaluation of HB virus-related liver diseases.