A rapid latex agglutination test for detection of antibodies in tuberculosis and Hansen's disease.

Antigens of Mycobacterium w, a saprophytic fast growing organism having antigenic epitopes cross-reactive with Mycobacterium leprae and Mycobacterium tuberculosis, were coated on to latex beads (0.33 micron Zn size), and the reactivity tested with sera of tuberculosis and Hansen's disease (HD) patients. Seventy nine percent of lepromatous leprosy (LL) and eighty five percent of pulmonary tuberculosis (TB) patients sera showed an agglutination reaction easily read by naked eye. Specificity of the test was further checked by testing sera of non-mycobacterial infection cases and all of them were found negative. Among apparently healthy controls, 4.3% were found positive from non-endemic and 8.8% from endemic area. The sensitivity of the assay is further enhanced from 78.7% to 90.4% and 85.7% to 91.6% in both LL HD and pulmonary tuberculosis respectively, by using immune complexes extracted from the patients sera. Potential of these antigen coated beads to detect the two major human mycobacterial disease, LL HD and pulmonary TB was also put evidence in a double blind study on coded sera samples obtained from various hospitals in India. The antigen coated beads are stable for upto 6 months at 4 degrees C. The latex slide agglutination test reported here, is simple, rapid, easy to perform and can be used even in rural areas of developing countries.     

A study of Mycobacterium tuberculosis antigen and antibody in cerebrospinal fluid and blood in tuberculous meningitis

Radioimmunoassay (RIA) techniques have been evaluated to detect specific tubercular antigen (TB Ag) and antitubercular antibody (TB Ab) in CSF and serum of patients with tuberculous meningitis (TBM). A solid-phase RIA using H37RV sonicate antigen of Mycobacterium tuberculosis, anti-BCG antibody, and staphylococcal protein A was standardized. TB Ag and TB Ab levels were noted to be significantly elevated in cerebrospinal fluid (CSF) as well in circulating immune complexes (CIC) isolated from serum samples of TBM patients as compared to control group (P less than 0.01). Detectability of disease by demonstrating elevated TB Ag and/or TB Ab levels in either CSF or CIC or both was 95%. There was no correlation between individual levels of TB Ag and TB Ab in CSF and in circulation. A follow-up study in patient over a period of 4-12 weeks revealed that TB antigen and/or TB Ab persisted in the majority of the cases for several weeks despite chemotherapy.     

Evaluation of a monoclonal antibody (TB72) based serological test for tuberculosis.

A serological test for tuberculosis (TB), based on competitive inhibition by human sera of antigen binding by a murine monoclonal antibody (TB72) has been performed using solid phase radioimmunoassay. The test was evaluated on sera from patients with pulmonary or extra-pulmonary active TB as well as from control hospitalized patients--half of whom had various chest complaints. Positive values were found in 74% of all or 55% of untreated active pulmonary TB. Patients with extrapulmonary TB segregated, whereby antibody titres were increased in cases of pleural, peritoneal, pericardial or bone infection but only marginal or negative values were found in the majority of patients with lymphatic and genitourinary infection. None of the subjects with suspected but excluded tuberculosis or sarcoidosis had demonstrable TB72 antibodies. Generally there was a positive correlation between the levels of TB72 like and 'total' Mycobacterium tuberculosis sonicate binding antibodies. In particular, the titre of sonicate binding antibodies did not exceed background levels in patients with active pulmonary tuberculosis who were negative in the TB72 competition test. None of these false negative patients received drug therapy whereas most patients with the highest antibody levels were under chemotherapy for 1-8 months prior to serum testing. The serodiagnostic value of the TB72 based competition test has been discussed.     

Glycolipids of Mycobacterium tuberculosis strain H37Rv are potential serological markers for diagnosis of active tuberculosis

A simple and cost-effective diagnostic tool (TB Screen Test) for the screening of patients with pulmonary and extrapulmonary tuberculosis and for differentiation of those individuals from individuals without tuberculosis, other common infections, and healthy controls has been developed. The serological responses of purified mycobacterial glycolipid antigens were examined by a liposome agglutination assay. The assay was able to detect very low antiglycolipid antibody concentrations in the infected individuals. The sera from the tuberculosis patient group had significantly higher concentrations of antiglycolipid antibody than the sera from uninfected control subjects, with 94% sensitivity and 98.3% specificity. Glycolipids of Mycobacterium tuberculosis H37Rv antigens were isolated, purified, and characterized. After interchelation with liposome particles, these purified antigens specifically bound to the antiglycolipid antibodies present in the sera of patients with tuberculosis, resulting in the formation of a blue agglutination. This protocol clearly differentiates healthy controls and M. bovis BCG-vaccinated subjects from those with active tuberculosis. The resultant diagnostic tool, the TB Screen Test, is more economical and rapid (4 min) than other currently available products and can be used for the mass screening of a heavily afflicted population.     

Glycolipids of Mycobacterium tuberculosis Strain H37Rv Are Potential Serological Markers for Diagnosis of Active Tuberculosis

A simple and cost-effective diagnostic tool (TB Screen Test) for the screening of patients with pulmonary and extrapulmonary tuberculosis and for differentiation of those individuals from individuals without tuberculosis, other common infections, and healthy controls has been developed. The serological responses of purified mycobacterial glycolipid antigens were examined by a liposome agglutination assay. The assay was able to detect very low antiglycolipid antibody concentrations in the infected individuals. The sera from the tuberculosis patient group had significantly higher concentrations of antiglycolipid antibody than the sera from uninfected control subjects, with 94% sensitivity and 98.3% specificity. Glycolipids of Mycobacterium tuberculosis H37Rv antigens were isolated, purified, and characterized. After interchelation with liposome particles, these purified antigens specifically bound to the antiglycolipid antibodies present in the sera of patients with tuberculosis, resulting in the formation of a blue agglutination. This protocol clearly differentiates healthy controls and M. bovis BCG-vaccinated subjects from those with active tuberculosis. The resultant diagnostic tool, the TB Screen Test, is more economical and rapid (4 min) than other currently available products and can be used for the mass screening of a heavily afflicted population.     

流行性出血热循环免疫复合物各组分的动态观察及意义

应用ＰＥＧ沉淀法对２７例１１４份流行性出血热病人血清中循环免疫复合物进行了检测，不同病期循环免疫复合物检出率分别为：发热期８８．８％，少尿期８２．５％，多尿期４７．８％，恢复期２１．４％。对流行性出血热循环免疫复合物组分的动态观察发现，在不同病期循环免疫复合物中绝大多数可检出流行性出血热病毒抗原及免疫球蛋白与补体Ｃ３，表明在流行性出血热患者体内检出的循环免疫复合物是特异性病毒抗原刺激机体免疫应答的     

Detection of circulating immune complexes with polyethylene glycol precipitation F(ab')2 anti-C3 ELISA

A polyethylene glycol (PEG) precipitation F(ab')2 anti-C3 ELISA for the detection of complement-fixing IgG circulating immune complexes (CIC) is described. For this assay, test sera were treated with 3.5% PEG and then measured with F(ab')2 anti-C3 ELISA. The lower detection limit was 4 micrograms/ml of heat aggregated human IgG (HAHG). Intra-assay coefficient of variation (CV) was 4.9-8.3%. High levels of CIC are found in the sera of patients with systemic lupus erythematosus (SLE), hepatitis B and stomach cancer.     

Time course of anti-SL-IV immunoglobulin G antibodies in patients with tuberculosis and tuberculosis-associated AIDS.

Immunoglobulin G (IgG) and IgM antibodies against the SL-IV antigen of Mycobacterium tuberculosis in the sera of patients with tuberculosis with negative serology for human immunodeficiency virus (HIV) infection (TB group; n = 97), patients with tuberculosis with positive serology for HIV infection (TB-HIV group; n = 59), and healthy controls (n = 289) were determined by enzyme-linked immunosorbent assay. All sera were obtained at the onset of tuberculosis, i.e., when clinical symptoms appeared. Clinical specimens were collected and cultured for the isolation of M. tuberculosis, and treatment with antituberculous drugs was started. Sera were also obtained from patients in the TB group at fixed intervals during treatment; sera were available from 13 patients in the TB-HIV group before the onset of tuberculosis. The best specificity and positive predictive values were obtained with the IgG assays. In the IgG assays at specificities above 96.0%, the sensitivities of the tests were 45.3 and 72.8% for the TB and TB-HIV groups, respectively, and the sensitivity was 51.9% when data from both groups were combined for analysis. For the TB group, results of this study indicated that the levels of IgG antibodies remain high during treatment. Thus, repetitive serological assays may not be useful for treatment follow-up. In the TB-HIV group, 12 of 13 patients had IgG-specific antibodies against the SL-IV antigen between 1 and 30 months before the onset of tuberculosis, so we suggest that the IgG antibody assay against SL-IV may be helpful for identifying tuberculosis in patients infected with HIV.     

Detection of mycobacterial antigen in circulating immune complexes in patients with childhood tuberculosis

The presence of both components (antigen and antibody) in circulating immune complexes (CIC) were detected in tuberculosis in children. Fifty two patients with pulmonary and extrapulmonary tuberculosis showed the presence of either components or both. CIC--antigen was present in 92.3% (48/52) and CIC antibody in 88.96% (46,52). Out of these 52 patients, 20 were proved cases, CIC antigen (ag) and CIC--antibody (ab) were present in 100% (20/20). In the control group both CIC-ag and CIC-ab and CIC = ab can be taken as an additional marker in diagnosis of tuberculosis.     

Circulating immune complexes among diabetic children

Insulin dependent diabetes mellitus (IDDM) is an autoimmune disease associated with the presence of different types of autoantibodies. The presence of these antibodies and the corresponding antigens in the circulation leads to the formation of circulating immune complexes (CIC). CIC are known to persist in the blood for long periods of time. Such CIC following deposition in the small blood vessels have the potential to lead to microangiopathy with debilitating clinical consequences. The aim of our pilot study was to investigate whether a correlation exists between CIC and the development of microvascular complications in diabetic children. Isolation of a new glycoprotein complement inhibition factor (CIF) from the parasitic plant Cuscuta europea seed, which appears to bind specifically to complement component C3 has provided an unique tool for the measurement of immune complexes by means of ELISA-type techniques (CIF-ELISA). We studied the levels of CIC (IgG, IgM and IgA) in 58 diabetic children (mean age 12.28 +/- 4.04 years, diabetes duration 5.3 +/- 3.7 years), 29 of them had vascular complications (group 1) and the other 29 were without vascular complications (group 2). As controls, we studied sera samples from 21 healthy children (mean age 13.54 +/- 4.03 years). Sera from the diabetic patients showed statistically significant higher levels of CIC IgG (p = 0.03) than sera from the control group. In sera from group 1 values of CIC IgG showed statistically significant higher levels than controls (0.720 +/- 0.31 vs. 0.46 +/- 0.045; p = 0.011) Sera from 59% of the patients were positive for CIC IgG, 36% for CIC IgM and 9% for CIC IgA. Among 26 patients with microalbuminuria, sera from 17/26 (65%) were positive for CIC IgG, 8/26 (31%) for CIC IgM and 2/26 (8%) for CIC IgA. CIC IgG correlated with HbAlc (r = 0.51; p = 0.005) and microalbuminuria (r = 0.42, p = 0.033). CIC IgA correlated with age (r = 0.44, p = 0.03). CIC IgM correlated with the duration of diabetes (r = 0.63, p = 0.02). These findings suggest that elevated levels of CIC IgG are associated with the development of early diabetic nephropathy.     

Assays for total and antigen-specific polymeric IgA in serum based on binding to secretory component

Binding assays with secretory component (SC) were used to detect polymeric IgA antibody to E. coli lipopolysaccharide and to estimate total polymeric IgA in sera from 14 patients with alcoholic liver disease and eight normal controls. Radioiodinated human SC was shown to bind to polymeric IgA and IgM but not to monomeric IgA, secretory IgA or IgG. Serum aliquots (0.5 ml) were totally depleted of IgM using 2 ml anti-IgM affinity columns and the effluent sera were titrated in microtitre plates coated with lipopolysaccharide, the binding of polymeric IgA being detected by adding 10 ng radiolabelled SC. Total polymeric IgA was measured via its capacity to inhibit the binding of 5 ng labelled SC to IgM coated wells, quantitation being achieved by comparison with the inhibition produced by purified polymeric IgA. Total lipopolysaccharide-specific IgA antibody was detected by ELISA in sera from both patients and controls, 1185 +/- 793 and 56 +/- 19 U/100 microliters (mean +/- SD), respectively; but polymeric IgA antibody was detected only in patients' sera (131 +/- 214 U/100 microliters). The concentration of total polymeric IgA was higher in patients' sera than in control sera (488 +/- 333 and less than 120 micrograms/ml respectively).     

Detection of circulating immune complexes in patients with glomerulonephritis.

143 patients were evaluated clinically on the basis of the renal biopsy. Three methods for detecting circulating immune complexes (CIC) were employed: complement consumption test, inhibition of erythrocyte antibody IgG-EA rosette forming test and optical density of 4% PEG precipitated sera. CIC were present in the sera of all patients with acute poststreptococcal glomerulonephritis (2 weeks after streptococcal infection of the throat). In a group of patients with chronic glomerulonephritis the highest values in positive results were observed in lupus nephritis, chronic proliferative glomerulonephritis and chronic submicroscopic glomerulonephritis. These results were compared with levels of total hemolytic complement, C3, C4 components and serum immunoglobulins (IgA, IgG, IgM).     

An enzyme-linked immunosorbent assay for immune complex of HTLV-I

A sandwich enzyme-linked immunosorbent assay (ELISA) for immune complexes of human T cell leukemia virus type I (HTLV-I) was developed using monoclonal antibody (MoAb) 3G1 which recognizes a different epitope on HTLV-I to that with which natural human anti-HTLV-I antibody binds. The assay was capable of titrating artificial immune complexes not only at antigen-antibody equivalence but also at antibody excess. Although the antigen-antibody ratios could not be determined in the individual sera from patients with overt ATL, the level of immune complexes in three out of four sera was estimated to be 250 +/- 36 ng/ml. Immune complexes of HTLV-I could not be identified in sera obtained from one patient with overt ATL, three healthy HTLV-I carriers and three normal human controls.     

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.     

Characterization of immune complexes in acute lymphoblastic leukaemia.

Sera from 120 children and young adults with acute leukaemia (59), various other tumours (53) and histiocytosis X (eight) were studied for the presence and characteristics of circulating immune complexes (CIC). Serial and parallel testing was performed using: C1q binding (solid phase), Raji cell radioimmunoassay and anti-C3 (solid phase). CIC were detected in 36 of 56 (64%) patients with acute lymphoblastic leukaemia (ALL) and in 62% of other tumour subjects. In the ALL sera, the mean positive C1q binding was 5.4 s.d., Raji cell 4.2 s.d. and anti-C3 4.4 s.d. In 12 ALL sera CIC were characterized for molecular size by sucrose gradient centrifugation. Most samples showed high molecular weight (19S) complexes but intermediate (11-14S) and smaller (8-9S) complexes were also detected. There was no apparent relationship between the presence, amount or physical size of the detectable CICs and clinical course of the patients studied; 12 patients with ALL in long term remission showed presence of CIC at some time during their course. Immune complexes precipitated from leukaemic sera were also examined for the presence of common ALL antigen (cALL) and Ia(DR) antigens utilizing rabbit antisera and mouse monoclonal antibodies. Experiments with isolated immune complexes from ALL sera provided no positive evidence for the presence of cALL antigen or Ia antigen within immune complex materials from ALL patients.     

Evidence for a disease specific antigen in circulating immune complexes in ankylosing spondylitis.

The presence of circulating immune complexes (CIC) has been documented in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and ankylosing spondylitis (AS) by various investigators. It has been suggested that these may be used as probes to identify antigens playing a role in these pathological processes. Using a solid-phase cross-reaction assay to establish if these complexes reacted with each other in specific disease groups, it was found that polyethylene glycol (PEG) precipitates of six AS patients cross reacted in 29 of 36 tests, but reacted with SLE and RA PEG precipitates in only two of 24 tests in each case. SLE PEG precipitates cross-reacted in four of 14 tests and reacted with none of the six AS and four RA precipitates. Similarly, RA PEG precipitates did not cross-react (none of 16 tests), nor did they react with AS (none of 24 or SLE precipitates (none of 16). Similar results were observed when IgG, obtained after acid dissociation on sucrose density gradients of CIC isolated by PEG precipitation and staphylococcal protein A chromatography, was used as the solid phase component. F (ab')2 fragments with similar antibody specificity were obtained by pepsin digestion of isolated CIC from three of six AS patients. These bound radiolabelled AS PEG precipitates (2.02-2.40%) significantly more than SLE (0.22-0.28%) or RA (0.29-0.35%) precipitates. These studies demonstrate the feasibility of obtaining F (ab')2 fragments with antibody activity from isolated CIC and the presence of a disease specific antibody specificity in AS CIC. The nature of the antigen involved remains to be elucidated. Such a cross-reactive antibody specificity was not found in RA nor SLE CIC.     

Pulmonary tuberculosis and serum IgE

Several recent studies indicate that mycobacterium or viral infection may reduce IgE levels or suppress atopy or both. The present study was undertaken to investigate whether Mycobacterium tuberculosis infection and its successful treatment down-regulate serum total IgE levels, a marker of a Th2 response, due to enhancement of a Th1 response in adult patients with tuberculosis (TB). We prospectively studied the changes in serum total IgE and DTH response to tuberculin, a marker of a Th1 response in 10 healthy controls, 20 patients with pulmonary TB, and 19 asthma patients without TB. Measurement of serum total IgE and tuberculin skin tests were performed before initiation of treatment and after successful completion of 6 months treatment in TB patients, and at the corresponding intervals in controls and asthmatics. The initial serum total IgE concentrations were significantly higher in TB patients than in healthy controls (282 +/- 26 U/ml (mean +/- s.e.m.) in TB patients versus 126 +/- 56 U/ml in controls; P = 0.03). However, serum total IgE concentrations significantly decreased (282 +/- 26 U/ml before versus 151 +/- 12 U/ml after treatment; P = 0.03) and tuberculin indurations significantly increased (23.6 +/- 1.8 mm before versus 29.6 +/- 2.1 mm after treatment; P = 0.04) in TB patients. In contrast, initial serum IgE concentrations and tuberculin indurations did not differ significantly from post-observation data in both healthy controls and asthmatics (P>0.30). The present study confirmed that immune responses to M. tuberculosis down-regulate a Th2 immune response, and might contribute to the decreased prevalence of allergic disorders.     

Immunoassay of hepatitis b surface antigen by particle counting after pepsin digestion

Particle counting immunoassay is based on latex agglutination, the reaction being measured by instrument counting of the particles remaining unagglutinated. Most interference which generally affects latex agglutination can be avoided by pepsin digestion of the sample, provided the antigen (Ag) of interest resists pepsin, which is the case of the hepatitis B surface antigen (HBsAg). Pepsin treatment has the additional advantage of inactivating antibodies and so releasing the Ag from immune complexes. We have set up an assay of HBsAg, proceeding in a prototype of Impact Instrument (Acade Diagnostic Systems, Belgium) at a rate of 60 samples· h⁻¹ and a total running time of 2 or 4 h. This assay was compared with Abbott radioimmunoassay (RIA) in 706 consecutive patients (A) and 31 selected sera for which values close to the cut-off had been obtained by RIA (B). In A, 38 sera were found positive and 668 negative by both methods. In B, RIA after neutralization classified the samples as positive (n = 14), negative (n = 14), or dubious (n = 3). Complete agreement between latex and RIA was achieved for nine positive, 12 negative, and two dubious samples. Of five RIA-positive samples, two were classified as latex-negative and three as dubious in the latex assay. One sample dubious in RIA was found latex-positive and two RIA-negative samples were found, respectively, latex-positive and dubious; when retested after pepsin digestion, the first of them became RIA-positive. That pepsin is useful to release antigen from immune complexes was also shown by addition of rabbit anti-HBs antibodies to five positive serum samples and by the recovery of the antigen both in RIA and latex agglutination after pepsin digestion.     

Detection of in vitro interferon-gamma and serum tumour necrosis factor-alpha in multidrug-resistant tuberculosis patients

Multidrug-resistant tuberculosis (MDR-TB) is known as having a poor prognosis with a weak response to therapy and very high death rates. The aim of this work was to assess the immune response to the RD1-encoded antigen ESAT-6 of Mycobacterium tuberculosis in MDR-TB patients and compare to non-resistant (NR) TB patients and healthy controls (HC). Evaluation of interferon (IFN)-gamma production showed that, although 55% of the MDR patients were responsive to ESAT-6, they produced lower IFN-gamma levels (553 +/- 11 pg/ml) when compared to NR-TB (1179 +/- 163 pg/ml; P < 0.05) but not to controls (412 +/- 65.7 pg/ml). Differences in the response to ESAT-6 and to its overlapping peptides mixture were also significant between MDR versus treated pulmonary NR-TB. Furthermore, a very low rate of response to PPD (23.5%) and to Ag85B (33.3%) was noted in MDR-TB patients as compared to the other groups. To determine the inflammatory response in patients' groups, detection of tumour necrosis factor (TNF)-alpha was assessed in their sera before and during chemotherapy. Mean TNF-alpha levels in MDR-TB (43.8 +/- 9 pg/ml) paralleled those found in treated pulmonary, and it was significantly different (P < 0.05) from the values found in untreated NR and HC. Interestingly, secretion of IFN-gamma and TNF-alpha were predominant in MDR patients who presented with bilateral pulmonary lesions and lung cavitation. The present data indicate that the overall immune response to mycobacterial antigens is decreased in resistant TB and the major role inflammatory cytokines may play in perpetuating pulmonary tissue damage.     

Isolation and characterization of circulating immune complexes from patients with pneumococcal pneumonia.

Circulating immune complexes (CIC) were isolated from serum samples from patients with bacteremic and nonbacteremic pneumococcal pneumonia. Overall, 63% (26 of 41) of patients with pneumococcal pneumonia had elevated levels of immunoglobulin G (IgG)-containing CIC. IgM-containing CIC were identified in samples from only three patients. Serum samples from nonbacteremic patients contained significantly higher levels of IgG-containing CIC (96.6 +/- 111.7 micrograms/ml) than did samples from bacteremic patients (31.7 +/- 26.9 micrograms/ml) during week 1 in hospital (P less than 0.05). Immune complexes levels did not correlate with IgG concentrations in serum or anticapsular antibody levels. Immune complexes from nonbacteremic patients had sedimentation coefficients of greater than 19s by density gradient ultracentrifugation. In contrast, CIC from bacteremic patients had smaller coefficients, of between 9s and 14s. Pneumococcal capsular antigens were identified in concentrated dissociated CIC from both patient groups by counterimmunoelectrophoresis.