Extensive endoscopic image-guided sinus surgery decreases BPI-ANCA in patients with cystic fibrosis.

Antineutrophil cytoplasm autoantibodies (ANCA) directed against bactericidal/permeability-increasing protein (BPI) are common in patients with cystic fibrosis (CF), and serum levels are correlated with lung colonization by Pseudomonas aeruginosa and the severity of lung damage. The production of BPI-ANCA may be due to the costimulation of BPI when mounting an immune response against P. aeruginosa. The effect of surgery aiming to eradicate bacteria and infected tissue on BPI-ANCA levels is sparsely described. A cohort of patients with CF were included: 53 patients having extensive image-guided sinus surgery (EIGSS) with topical postoperative antibiotic treatment, 131 non-operated controls and 36 who had double lung transplantation (LTX). In all 219 patients, serum samples before and after surgery or at similar intervals were analysed for IgG and IgA BPI-ANCA. The EIGSS group showed a highly significant decrease in both IgA and IgG BPI-ANCA levels compared with their own preoperative values and control group values (P < 0.001-0.02). The LTX patients also showed a highly significant decrease in both IgA and IgG BPI-ANCA levels (P < 0.001). EIGSS and LTX decrease IgA and IgG BPI-ANCA levels in patients with CF, indicating that extensive removal of infected tissue influences the pathogenic process of autoantibody production. The results shown herein are in favour of applying EIGSS in selected patients with CF and for using BPI-ANCA as a surrogate marker for guiding further therapeutic interventions.

Circulating antibodies to mouse laminin in Chagas disease, American cutaneous leishmaniasis, and normal individuals recognize terminal galactosyl(alpha 1-3)-galactose epitopes.

Sera from patients with American cutaneous leishmaniasis and Chagas disease and from monkeys infected with either Trypanosoma cruzi or Trypanosoma rhodesiense show, in RIAs, strong binding to mouse laminin. A distinct although weaker binding activity is also detected in normal human sera. The antibodies recognize a common carbohydrate epitope present on mouse laminin, which was assigned to a terminal galactosyl(alpha 1-3)-galactose group. Distinct crossreactions were observed with some other basement membrane proteins, rabbit glycosphingolipids, defucosylated human B blood group substance and components produced by some human tumor cells. Only little activity was, however, found on laminin obtained from human placenta. The data indicate that the antibodies arising in infectious diseases are stimulated by similar carbohydrate epitopes present on the surface of parasites. Tissue-specific occurrence of such epitopes may exist and explain the involvement of distinct tissues in autoimmune disorders.

Depressed activation of the lectin pathway of complement in hereditary angioedema.

The possibility of simultaneous measurement of the classical pathway (CP), mannan-binding lectin (MBL)--lectin pathway (LP) and alternative pathway (AP) of complement activation by the recently developed Wielisa method allowed us to investigate the in vivo significance of the C1-inhibitor (C1INH) in three complement activation pathways. Functional activity of the CP, LP and AP were measured in the sera of 68 adult patients with hereditary angioedema (HAE) and 64 healthy controls. In addition, the level of C1q, MBL, MBL-associated serine protease-2 (MASP-2), C4-, C3- and C1INH was measured by standard laboratory methods. MBL-2 genotypes were determined by polymerase chain reaction. Besides the complement alterations (low CP and C1INH activity, low C4-, C1INH concentrations), which characterize HAE, the level of MASP-2 was also lower (P = 0.0001) in patients compared with controls. Depressed LP activity was found in patients compared with controls (P = 0.0008) in homozygous carriers of the normal MBL genotype (A/A), but not in carriers of variant genotypes (A/O, O/O). Activity of CP correlated with LP in patients (Spearman's r = 0.64; P < 0.0001), but no significant correlation was found in the control group and no correlation with AP was observed. In contrast, the activity of CP and AP correlated (Spearman's r = 0.47; P < 0.0001) in healthy controls, but there was no significant correlation in the HAE patients. We conclude that the activation of LP might also occur in subjects with C1INH deficiency, which is reflected by the low MASP-2 and C4 levels.

Calibration of low-level beta-gamma coincidence detector systems for xenon isotope detection.

The beta-gamma coincidence detector systems used for the measurement of the CTBT-relevant xenon isotopes (Xe-131m, Xe-133m, Xe-133 and Xe-135) in the International Monitoring System network and in the On-Site Inspection are reviewed. These detectors typically consist of a well-type or bore-through NaI crystal into which a measurement cell, serving also as a sample container, is inserted. This work describes the current calibration procedure for energy, resolution and efficiency, implementation challenges, availability and uncertainties of the specific nuclear decay data and the path forward to full calibration validation using GEANT4.

Development of a mobile radioxenon processing system for on-site inspections and the deployment in IFE14.

A mobile radioxenon gas processing system (XESPM-III) was developed for on-site inspections-targeting deployment in the Integrated Field Exercise in Jordan 2014 (IFE14)-in order to monitor radioxenon isotopes (131m,133,133m,135Xe) from the subsoil and atmosphere. XESPM-III is composed of primarily three units, the sampling unit, the purification unit and finally the quantification unit. The function of the sampling unit is to pre-enrich xenon by removal of impurities in the gas sample, while the purification unit further purifies, separates impurities and prepares a small-volume sample with relatively high concentration of xenon gas-both stable and radioactive xenon (if present). The quantification unit quantifies the stable xenon which provides information of the gas recovery (yield) of the gas sampling and purification process. In one cycle (7.5 h) XESPM-III can process either two 4 m3 volume samples or two pairs 2 m3 samples each; 24 h maximum throughput is thus twelve 2 m3 samples or six 4 m3 samples; final purified gas sample volume is approx. 7 cm3 (Xe + N2 used as carrier gas); gas recovery (yield) is >70%; radon removal coefficient is 10-6; cross contamination between subsequent samples is <1%; Its flexible design, that does not include a spectrometry system, allows it to be used with various spectrometric systems (HPGe, beta-gamma coincidence) for the final measurement of the radioactive xenon concentrations in the sample. During the field deployment of the XESPM-III in IFE14 it was able to measure 133Xe in the range of 0.18-0.54 Bq/m3 in spiked subsoil gas.

Human glomerular basement membrane. Heterogeneity of antigenic determinants.

Human glomerular basement membrane was solubilized by digestion with proteolytic enzymes and immunoreactive components were quantitated and characterized by using rabbit antibodies raised against the particulate membrane. A number of antigens were demonstrated but they did not separate on gel filtration. However, two antigenic components in a collagenase digest of the membrane could be separated and isolated by Sepharose 6B chromatography. Chemical characterization suggests that both fragments are noncollagenous glycopeptides (molecular weights approx. 1,000,000 and 60,000--200,000, respectively).

Human glomerular basement membrane. Antigenic determinants in human urine.

Antisera against particulate human glomerular basement membrane prepared from cadaver kidneys were raised in rabbits. It was shown that both normal individuals and patients with glomerular and tubular diseases excrete in their urine several antigens reactive with these antibodies. One antigen crossreacted immunologically with an antigen from human glomerular basement membrane while several others did not. One of the urinary antigens and the antigen crossreacting with the basement membrane were separated from the others by ion exchange chromatography and gel filtration, respectively. The pattern of anttigen excretion differed depending on the underlying renal disease but the multitude of different antigens detected complicates the interpretation of the patterns of excretion in different diseases.

Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA.

Primary defence against invading microorganisms depends on a functional innate immune system and the complement system plays a major role in such immunity. Deficiencies in one of the components of the complement system can cause severe and recurrent infections, systemic diseases, such as systemic lupus erythematosus (SLE) and renal disease. Screening for complement deficiencies in the classical or alternative complement pathways has mainly been performed by haemolytic assays. Here, we describe a simple ELISA-based format for the evaluation of three pathways of complement activation. The assays are based on specific coatings for each pathway in combination with specific buffer systems. We have standardized these assays and defined cut off values to detect complement deficiencies at the different levels of the complement system. The results demonstrate the value of these ELISA-based procedures for the functional assessment of complement deficiencies in clinical practice. The assay is now available commercially in kit form.

Alport syndrome in southern Sweden.

AIM: The aim of the present investigation is to study the epidemiology of Alport syndrome in southern Sweden, to search for mutations in the COL4A5 gene and to estimate the mutation frequency. PATIENTS AND METHODS: Patients with suspected Alport syndrome were identified in an area with a population of 1.45 million. Clinical criteria were used to establish the diagnosis and samples for mutation analysis were collected. Mutation analyses were performed with Single-Stranded Conformation Polymorphism analysis (SSCP) of PCR-amplified genomic DNA. RESULTS: Altogether 25 families with hereditary nephritis were identified. Alport syndrome with X-linked transmission was evident in 14 families, with juvenile (< 31 years) progression to end-stage renal failure (ESRF) in ten, and adult (> or = 31 years) in four families. CONCLUSION: The frequency of males with X-linked disease was calculated to one in 17,000 male births (95% confidence interval (CI) 1/10,500-1/28,600), and the prevalence to one in 40,000. A total of seven females with ESRF were identified, with a median age at ESRF of 45 years. The male to female ratio of cases with ESRF was 4.9 to 1. The risk of developing ESRF among females was from the expected incidence roughly estimated to 12%. Patients with X-linked disease constituted 1.8% of patients with ESRF in the examined area. A mutation was identified positive in 10 of 14 families with X-linked disease, but never in families not fulfilling the clinical criteria for Alport syndrome. In families with juvenile phenotype and positive mutation analysis, the mutation frequency was calculated to between 1/78,000 and 1/198,000 (95% CI 1/42,000-1/177,000) if the effective fertility was estimated to be between 0 and 0.2.

A rapid and semi-quantitative method for the detection of autoantibodies by multiple spot immunoassay.

A method for rapid simultaneous determination of multiple autoantibodies in sera has been developed. 42 different antigens were coated onto a nitrocellulose membrane in a miniblotter apparatus with 42 lanes. The membrane was also coated with different concentrations of human IgG to create a standard curve. The blotting apparatus after coating, blocking and washing was turned 90 degrees so that all lanes crossed the antigen coated lanes. Thereafter, 42 sera were incubated with the antigens. After this stage the membrane was treated as in the usual dot-blot procedure. After incubation with alkaline phosphatase labelled anti-human IgG, staining and drying of the membrane, the staining intensity of individual lanes was scanned into a data file and analyzed by computer. By this method it was possible in 4 h to examine 42 sera for autoantibodies against 42 antigens, i.e., more than 1600 tests. Furthermore, the amount of antibody could be semi-quantified, using the IgG standards. The method should be useful for rapid screening of autoantibodies in a routine laboratory.

A rapid assay for circulating anti-glomerular basement membrane antibodies in Goodpasture syndrome.

A rapid ELISA for the detection of circulating anti-glomerular basement membrane antibodies in Goodpasture syndrome is described. The specificity of the test was shown to be highly dependent on the antigens used. Using the purified Goodpasture antigen it was possible to shorten the incubation times to 10 min in a routine assay using alkaline phosphatase-labeled second antibodies and the total assay was complete in 30 min. 200 reference sera, 500 sera from patients with various types of glomerulonephritis and 32 sera from patients with Goodpasture syndrome were analyzed by this rapid assay. The assay was able to discriminate between Goodpasture syndrome and other forms of glomerulonephritis. Using enzyme amplification it was possible to further shorten the incubation times to 1 min and the total time of the assay to 6 min.

Measurements of proteinase 3 and its complexes with alpha 1-proteinase inhibitor and anti-neutrophil cytoplasm antibodies (ANCA) in plasma.

Wegener's granulomatosis (WG) is a systemic vasculitis which is diagnosed on clinicopathological findings. The diagnosis may be aided by the presence of anti-neutrophil cytoplasm antibodies (ANCA). In WG, ANCA are primarily directed to proteinase 3 (PR3), a serine protease of the azurophilic granules of the neutrophilic granulocyte. The main plasma inhibitor of PR3 is alpha 1-proteinase inhibitor (PI). To study if free PR3 or complexes between the enzyme and PI or PR3 and ANCA could be found in the plasma from patients with WG we have developed three ELISA systems for the detection of these complexes and free PR3. In all three assays monoclonal antibodies against PR3 were used as capture antibodies. After incubation with plasma, free PR3 was detected by affinity purified rabbit anti-PR3 followed by alkaline phosphatase-labelled swine anti-rabbit IgG. Serial dilutions of purified PR3 was used as standard. The detection limit was 3 ng/ml. PR3 complexed with PI was measured by rabbit anti-PI antibodies and alkaline phosphatase-labelled swine anti-rabbit IgG. Pre-formed in vitro complexes of PR3/PI in serial dilutions were used as standard. The detection limit of this assay was 1 ng/ml. PR3/IgG-ANCA complexes were detected by alkaline phosphatase labelled goat anti-human IgG. A positive plasma sample in serial dilutions was used as standard. Plasma samples from nine patients with WG, eight patients with fever of infectious origin without evidence of vasculitis and ten healthy donors were examined by these methods. Free PR3 could not be found in any of the plasma samples. PR3/PI complexes were detected in healthy donors at levels between 41-85 ng/ml. All WG patients, both active and inactive, had PR3/PI concentrations above this level, and so had all patients with fever. PR3/IgG-ANCA was found in three of the patients with WG, two being ANCA negative with inactive disease and one was ANCA positive with active disease. Thus, the developed methods can be useful for future studies of the clinical relevance of these complexes in patients with WG and possibly other vasculitides.

An ELISA for the detection of anti-neutrophil cytoplasm antibodies (ANCA).

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of circulating anti-neutrophil cytoplasm antibodies (ANCA), which are defined by a diffuse, granular staining of the cytoplasm of alcohol-fixed human neutrophils by indirect immunofluorescence (IIF). Detection of antineutrophil cytoplasm antibodies has a high sensitivity and specificity for active Wegener's granulomatosis (WG) and reflects the effect of treatment. In the present enzyme-linked assay, immunoplates were coated with the cytoplasmic alpha fraction of neutrophils obtained from apparently healthy human donors by nitrogen bomb cavitation and subsequent Percoll gradient centrifugation. Alkaline phosphatase-labelled anti-human IgG was used as a secondary antibody. Diluted sera from 70 patients with WG and 16 patients with other diseases with anti-myeloperoxidase antibodies (anti-MPO) were examined. It is concluded that the ELISA accurately detects IIF ANCA positive patients with WG, is helpful in detecting WG patients in remission, is not influenced by the presence of anti-MPO and may help in detecting ANCA in cases with granulocyte-specific anti-nuclear antibodies since this IIF pattern obscures the IIF ANCA patterns. The ELISA with titration can be carried out in 3.5 h whereas a rapid test just to detect ANCA can be performed in 30 min.

Capture-ELISA based on recombinant PR3 is sensitive for PR3-ANCA testing and allows detection of PR3 and PR3-ANCA/PR3 immunecomplexes.

Proteinase 3 (PR3), a constituent of azurophil granules of neutrophils (polymorphonuclear cells, PMNs), is the target antigen for most anti-neutrophil cytoplasmic antibodies (c-ANCA) in Wegener's granulomatosis (WG). We have recently developed an expression system for recombinant PR3 (rPR3) that is recognized by c-ANCA. Here, we report on the development and characterization of two monoclonal antibodies (moABs) and a rabbit polyclonal antiserum generated against this rPR3. Epitope competition analysis indicates that the moABs MCPR3-1 and MCPR3-2 recognize overlapping epitopes on the PR3 molecule that are distinct from the ones recognized by moABs 4A5 and 6A6 developed by others. Since MCPR3-2 does not appear to compete for epitopes recognized by a sizable proportion of PR3-ANCA, we used it to develop a sensitive capture enzyme linked immunosorbent assay (ELISA) for clinical PR3-ANCA testing. Both purified PMN PR3 and crude human mast cell line (HMC-1)/PR3-S176A cell lysates were used as sources of PR3 target antigen in this assay with equal analytical sensitivity and specificity. Of 109 patients with ANCA-associated disease, 91 (83.5%) and 90 (82.6%) were PR3-ANCA positive by capture ELISA when PMN-PR3 and HMC-1/PR3-S176A cell lysates were used as antigen, respectively. When HMC-1/PR3 and HMC-1/PR3-S176A cells were used as indirect immunofluorescence (IIF) substrate, 88 (80.7%) and 92 (84.4%) were PR3-ANCA positive, respectively. These differences were not statistically significant. Only 1 of 151 controls without defined ANCA-associated disease tested positive by capture ELISA with either target antigen (both negative by PR3-ANCA specific IIF). The capture ELISA can also be used to detect of PR3-ANCA immunecomplexes and, in combination with the rabbit antiserum, for the quantitative measurement of PR3 in biological fluids.

Development and standardization of solid phase assays for the detection of anti-neutrophil cytoplasmic antibodies (ANCA). A report on the second phase of an international cooperative study on the standardization of ANCA assays.

Anti-neutrophil cytoplasmic antibodies (ANCA) are diagnostic markers for systemic vasculitis. They are classically detected by an indirect immunofluorescence test using normal donor neutrophils as substrate. This assay lacks antigenic specificity and is not quantitative. The 'EC/BCR Project for ANCA Assay Standardization' is an international collaboration study with the aim to develop and standardize solid phase assays for ANCA detection. In this part of the study the isolation and characterization of proteinase-3 and myeloperoxidase, the two main target molecules for ANCA, and the development and standardization of ELISAs with these antigens are described. Six laboratories successfully isolated purified proteinase-3 preparations that could be used. Three of these preparations, together with one myeloperoxidase preparation, were subsequently used for ANCA testing by ELISA. The ELISA technique was standardized in two rounds of testing in the 14 participating laboratories. The coefficient of variation of these new assays decreased from values of approx. 50% in the first round to approx. 20% in the second round. We conclude that purified proteinase-3 and myeloperoxidase can be used in standardized ELISAs for ANCA detection. Whether such procedures offer advantages over the IIF test will be determined in a prospective clinical study.

Systemic vasculitis in a kidney transplant population.

BACKGROUND: Systemic vasculitis as original disease might adversely influence the result of kidney transplantation. METHODS: The clinical course after 32 transplantations to 26 patients with microscopic polyangiitis, Wegener's granulomatosis, Henoch-Schonlein purpura, thrombotic thrombocytopenic purpura/hemolytic uremic syndrome, or Goodpasture's disease was evaluated. The median follow-up time was 82 months (range, 4-132 months). Frozen sera from 25 transplantations were analyzed for Goodpasture antibodies, myeloperoxidase antineutrophil cytoplasmic antibodies (ANCA), and proteinase 3 ANCA. RESULTS: Survival of patients and grafts did not differ between patients and matched controls. Recurrent vasculitis occurred with seven grafts (four patients with microscopic polyangiitis or Wegener's granulomatosis, two patients with Henoch-Schonlein purpura, and one patient thrombotic thrombocytopenic purpura). New-onset hematuria was the initial renal symptom in five patients. Treatment with corticosteroids, cyclophosphamide, and/or plasma exchange was most often effective, but two grafts were lost. Proteinase 3 ANCA titers were increased to 12-738 U/ml before seven transplants. The patient with the lowest titer lost his graft due to recurrence, two other patients had reversible recurrence after 1 year and 5 years, two patients lost their grafts due to unknown/unrelated causes, and two patients' grafts remain without recurrence. Myeloperoxidase ANCA were increased to 22-39 U/ml before two transplants, which have been uneventful for 4 years. CONCLUSIONS: An awareness of the small but perpetual risk of recurrence facilitates early treatment that may save the transplant. Testing for hematuria and early transplant biopsies, and possibly monitoring of ANCA titers, are essential, but pretransplant ANCA titers have no predictive value in asymptomatic patients. Results of kidney transplantation in patients with vasculitis are as good as in other patients.

The prevalence and clinical significance of alpha 1-antitrypsin deficiency (PiZ) and ANCA specificities (proteinase 3, BPI) in patients with ulcerative colitis.

Our aim was to determine the prevalence of the PiZ allele for alpha 1-antitrypsin (AAT) deficiency and some relevant antineutrophil cytoplasmic antibody (ANCA) specificities in patients with ulcerative colitis (UC), and explore a possible association between these markers. In addition, we studied the relation to disease extension and activity. Sera from 141 patients with UC (72 women) were analyzed while 50 blood donors and 54 patients with acute myocardial infarction served as controls. Serum samples were screened for PiZ with ELISA and phenotyped by isoelectric focusing. BPI-ANCA and PR3-ANCA were detected by ELISA. Results were that 8.5% (12/141) of the patients with UC were PiZ carriers, higher than expected in the general Swedish population (4.7%) (p = 0.03). There was a significant difference between PiZ-carriers and non-PiZ-carriers in the extension and severity of colitis (odds ratio = 4.1, confidence interval = 1.1, 14.9; p = 0.028, and odds ratio = 9.0, confidence interval = 1.1, 73.3; p = 0.015; respectively). BPI-ANCA and PR3-ANCA were detected in 20.5% (29/141) and 12% (17/141) (p < 0.05 compared with controls for all parameters). Occurrence of BPI-ANCA and PR3-ANCA was not related to extension or severity of colitis (p > 0.05 for both variables). We observed no association between PiZ-carrier status and occurrence of BPI-ANCA or PR3-ANCA. The increased frequency of heterozygosity for the PiZ variant of AAT deficiency among patients with UC might imply a role played by protease inhibitors for regulation of inflammation and immunologic response in UC.

International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA)

Antineutrophil cytoplasmic antibody (ANCA) tests are used to diagnose and monitor inflammatory activity in the primary systemic small vessel vasculitides. ANCA is best demonstrated in these diseases by using a combination of indirect immunofluorescence (IIF) of normal peripheral blood neutrophils and enzyme-linked immunosorbent assays (ELISAs) that detect ANCA specific for proteinase 3 (PR3) or myeloperoxidase (MPO). For ANCA testing in "new" patients, IIF must be performed on all serum samples. Serum samples containing ANCA, any other cytoplasmic fluorescence, or an antinuclear antibody (ANA) that results in homogeneous or peripheral nuclear fluorescence then should be tested in ELISAs for PR3-ANCA and MPO-ANCA. Optimally, ELISAs for PR3-ANCA and MPO-ANCA should be performed on all serum samples. Inclusion of the most recent positive sample in the IIF or ELISA may help demonstrate a change in antibody level. Reports should use recommended terms. Any report of positive neutrophil fluorescence issued before the ELISA results are available should indicate that positive fluorescence alone is not specific for the diagnosis of Wegener granulomatosis or microscopic polyangiitis and that decisions about treatment should not be based solely on the ANCA results.

Immunoreactivity of the JK-132 monoclonal antibody directed against basement membrane collagen in normal and diabetic glomeruli.

The possible involvement of basement membrane-associated collagen (recognized by the monoclonal antibody JK-132) in the evolution of diabetic nephropathy was studied in kidney specimens from seven patients with noninsulin-dependent diabetes mellitus, and its distribution was compared with those of antibodies against alpha 1 to alpha 4 chains of type IV collagen. JK-132, a monoclonal antibody against basement membrane-associated collagen, reacted immunohistochemically exclusively with the mesangial matrix of the glomerular capillary. In contrast, antibodies to the alpha 1 and alpha 2 chains (IV) reacted strongly with mesangial matrix, and less strongly with the glomerular basement membrane (GBM). Antibodies to the alpha 3 and alpha 4 chains (IV) reacted mainly with GBM. In diabetes, JK-132 reacted most extensively with the expanded mesangial matrix, its staining intensity increasing with progression of the diabetic glomerulosclerosis. Antibodies to the alpha 1 and alpha 2 chains (IV) reacted prominently with the expanded mesangial matrix but less strongly with the GBM. Antibodies to the alpha 3 and alpha 4 chains reacted intensely with the thickened GBM. These results suggest that basement membrane-associated collagen differs from alpha 1 to alpha 4 chains of type IV collagen and that basement membrane-associated collagen is a good marker of mesangial expansion in diabetic nephropathy.

The involvement of type IV collagen in Goodpasture's syndrome.

Goodpasture's syndrome, involving lung and kidney, is considered to be caused by autoantibodies to basement membranes. This paper has described the isolation and identification of the antigen, which is isolated from collagenase digests of glomerular basement membrane, as a monomer protein of 26,000 daltons and two dimers of about 50,000 daltons. Further analyses indicated that the antigenic protein is derived from the globular domain of type IV collagen corresponding to the NCl peptide. All 22 patients with Goodpasture's syndrome studied had circulating antibodies to this antigen, a few had additional antibodies to laminin, and only one also had antibodies to the 7S collagen domain. No other patient with glomerulonephritis had circulating antibodies to the antigen. The isolated protein can therefore be used in an assay specific for Goodpasture's syndrome. Interestingly the protein antigen could be identified in glomerular, lung, and placenta basement membranes, although the components reacting with the antibodies represented different proportions of the preparations.