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PubMed1980

Activation of the fourth component of complement (C4): assessment by rocket immunoelectrophoresis and correlation with the metabolism of C4.

The classical pathway of complement (C) is activated in several diseases, and this activation characteristically involves the activation of C4, the fourth component of C. Since activation of C4 ultimately produces the polypeptide fragment C4d, we have applied immunoelectrophoresis in gels containing antibodies that precipitate C4d and C4 (rocket immunoelectrophoresis) as a means of detecting and quantitating C4 activation. Plasma C4 produced a single precipitin line after rocket immunoelectrophoresis, whereas plasma containing C4d produced two precipitin lines corresponding to C4d and C4. The areas enclosed by the respective precipitin line(s) were quantitated by planimetry and approximated the amounts of C4d and C4 present in the specimen. The ratio of the areas of C4d/C4 as measured in this analysis correlated significantly with the in vivo metabolism of radiolabeled C4. Our method detected C4d in the plasmas of some patients with rheumatoid arthritis, hereditary angioedema, systemic lupus erythematosus, or chronic urticaria with hypocomplementemia. These studies indicated that determination of the C4d/C4 ratio is useful in the evaluation of in vivo C activation and also may be applicable to the study and management of diseases associated with C activation.

Angioedema↗
PubMed1978

Immune complex detection and complement activity in rheumatoid arthritis: a comparative study of a radioimmunoassay using monoclonal rheumatoid factor, gel diffusion techniques and C4 activity.

Paired sera and synovial fluids from forty-nine patients with rheumatoid arthritis and twenty-five with other forms of arthritis were tested for immune complexes by a radioimmunoassay using monoclonal rheumatoid factor and gel diffusion procedures with monoclonal rheumatoid factor and C1q. Synovial fluid hemolytic C4 and C4 adjusted for IgG concentration were determined in both groups of patients. Immune complexes were detected at similar high frequencies in the rheumatoid synovial fluids by precipitin formation with monoclonal rheumatoid factor (68%) and C1q (71%). In contrast, immune complexes in rheumatoid sera were detected in low frequency by precipitin reactions with monoclonal rheumatoid factor (10%) and C1q (0%). Using the monoclonal rheumatoid factor radioimmunoassay, thirty-one (63%) synovial fluids exceeded the mean non-RA binding activity by one standard deviation. Similarly, twenty-four (49%) rheumatoid sera exceeded the mean non-RA binding activity to one standard deviation. Synovial fluid C4 adjusted for IgG as well as IgG alone distinguished between the two groups of patients whereas the C4 values did not. The C4/IgG value showed a strong negative correlation with the monoclonal rheumatoid factor radioimmunoassay and C1q precipitin formation.

Antigen-Antibody Complex↗
PubMed1977

Detection of antibody to the antiphagocytic factor produced by group E streptococci.

Tests for opsonic activity, long chain formation, and precipitins were investigated for ability to detect antibodies to the antiphagocytic factor of group E streptococci. Rabbits, hyperimmunized to serum-grown group E streptococci developed antibodies to the antiphagocytic factor detectable after 2 weeks by the opsonic activity and long chain tests and at 4 to 6 weeks by a precipitin test in which a slide micro double diffusion method was used. Antiphagocytic factor antibodies were dected in swine serums 2 weeks after the swine were experimentally infected, and the antibodies persisted at the end of the experiment at 20 weeks. Demonstration of the antibodies in swine serum was made with the opsonic activity or long chains tests but not with the precipitin test.

Animals↗
PubMed1998

Pseudomonas aeruginosa and Burkholderia cepacia infection in cystic fibrosis patients treated in Toronto and Copenhagen.

Differences in the course of pulmonary disease in cystic fibrosis (CF) may be altered by different treatment strategies in different CF centers. The Copenhagen clinic uses scheduled, regular and very aggressive treatment of lung infection. The Toronto clinic treats pulmonary infection with oral, inhaled, or intravenous antibiotics, and has emphasized aggressive nutritional therapy. This study compared the clinical status of CF patients treated in the two centers (Toronto, Canada, n=302, and Copenhagen, Denmark, n=214) using a cross-sectional design in terms of Pseudomonas aeruginosa (PA) and Burkholderia cepacia (BC) lung infections, pulmonary function, and levels of PA and BC precipitating antibodies (precipitins). Median ages were similar, but the age distribution was significantly different, with a higher proportion of patients under 10 and > or = 25 years in Toronto, and higher proportion of patients 11-24 years of age in Copenhagen. A higher number of female patients was observed in Copenhagen than in Toronto. Seventy-nine percent of Copenhagen patients, and 52% of Toronto patients were deltaF508 homozygous. Of all the patients, 20.1% of Copenhagen patients and 38% of Toronto patients were deltaF508 heterozygous. Ten percent of Toronto patients had two uncommon mutations. Pulmonary function and nutritional status in both groups were similar despite varying treatment strategies. The prevalence of PA was lower in Danish children and higher in Danish adults than in Canada. These differences are probably due to cohort isolation, which was introduced in Copenhagen in 1981. The prevalence of BC was higher in Toronto than in Copenhagen patients at all ages. In both centers, the number of PA and BC precipitins increased with age in patients chronically infected with PA and BC, respectively, and the number of both PA and BC precipitins rose with declining lung function. This study suggests that the clinic populations had similar pulmonary and nutritional statuses despite differing clinic antibiotic treatment strategies. Microbial colonization seemed to differ, at least in part, because of differences in cohort isolation strategies. Early, aggressive anti-pseudomonal chemotherapy may have reduced pseudomonal colonization among younger patients in Copenhagen. Future studies will be required to assess the impact of this on this cohort's outcome.

Adolescent↗
PubMed1992

Immunological status and respiratory findings in furriers.

We studied 42 women occupationally exposed in the fur manufacturing industry (mean age: 34 years, mean duration of exposure: 11 years). The highest prevalence of positive immediate skin reactions to antigen of animal hair was found for marten (10%), followed by fox and lamb (7%), mink (5%), and Chinese lamb, domestic fox, and Chinese calf (2%). Precipitating antibodies were demonstrated for lamb (17%), astrakhan (14%), mink, domestic fox and skunk (12%), Chinese lamb (10%), and Chinese calf (7%). Increased total IgE was found in 9.5% of subjects. Chronic symptoms were consistently more prevalent among workers with positive skin tests and positive precipitins than among workers with negative tests. A high prevalence of acute symptoms during the work shift was found among furriers. The prevalence of these symptoms was higher among workers with positive precipitating antibodies than among those with negative studies but not for workers with positive skin tests. Mean acute reductions in ventilatory capacity over the work shift were recorded for most ventilatory parameters. In general, greater drops in respiratory parameters occurred in individuals with positive precipitins (e.g., FEV1: -6.5% vs. -2.8%; positive vs. negative precipitins) but not in those with positive skin tests. Our study suggests that workers in the fur manufacturing industry develop acute and chronic respiratory problems often associated with specific indicators of atopy.

Acute Disease↗
PubMed1988

Structural and immunochemical aspects of Brucella abortus endotoxins.

Smooth lipopolysaccharide (sLPS) of Brucella abortus, which is the most immunodominant component among the antigens of B. abortus isolated, has been used for diagnosis for decades. High yields of sLPS can be prepared by a modification of the procedures of Moreno et al. (J. Bacteriol. 138:361-369, 1979). Washed B. abortus cells can be disrupted by 21 freeze-quick thaw cycles and ultrasonication to separate non-membrane-bound material; then phenol extraction is performed 3 times and the phenol fraction is washed with H2O intensively. The membrane-bound sLPS can be fractionated into 3 to 5 groups according to the extent of dialysis and centrifugation. These membrane bound sLPS fractions show marked individual differences in their precipitin profile and chemical composition. Their protein content varies from 16% to 42% as determined by dye binding test and 17 to 60% by Lowry phenol method using bovine serum albumin as the standard, which indicates that these proteins associated with LPS may play important roles in the immunochemical interactions, solubility, and the heterogeneity of B. abortus lipopolysaccharides. Compared to previously published methods, a higher yield of sLPS, ranging from 3.6% to 7.7% of dried bacteria, is obtained. Group f5A, which has a standard bell shaped curve in the precipitin assay, is one of the major fractions in all three strains (1119.3, 19, 2308). The protein free sLPS (less than 1% of Lowry reactive component) can be prepared by pronase digestion. The immunochemical reactivity remains about the same before and after this treatment. The O-chains of the major fraction (f5A) of B. abortus (Strains 2308 and 19) membrane bound smooth lipopolysaccharide (sLPS) are obtained by hydrolysis of f5A native sLPS in 1% acetic acid at 100 degrees C for 2 hours. After hydrolysis, the O-chains are separated from the lipid A protein complex by centrifugation, and from small fragments by ultrafiltration of a molecular weight cut-off (MWCO) of 1.0 x 10(3). These carbohydrate haptens can be identified by precipitin-inhibition assay and further fractionated by both membrane filtration and dialysis. The size distributions of carbohydrate haptens of the endotoxins (f5A) ranged from several oligosaccharides up to 1.0 x 10(4) MWCO. Three major fractions of MWCO 8.0-10.0 x 10(3), 3.5-5.0 x 10(3), and less than 1.0 x 10(3) for both strains 2308 and 19 contain more than 85% of the total immunreactive materials.(ABSTRACT TRUNCATED AT 400 WORDS)

Brucella abortus↗
PubMed1987

Strain specific antibodies revealed by immunoabsorption studies with avian infectious bronchitis virus.

Rabbit antisera prepared against the Massachusetts 41 (M41) strain of avian infectious bronchitis virus (IBV) and absorbed with chick embryo immunoabsorbent produced multiple precipitin lines in immunodouble-diffusion (IDD) tests with homologous or heterologous strains of virus. These precipitin lines were all removed by absorption with concentrated M41 virus preparations, but repeated absorption with concentrated, purified preparations of IBV strains: T, Holte, Connecticut, Beaudette or H120 failed to remove all precipitin lines produced to M41 virus, although all those to the heterologous viruses were removed. The remaining line(s) produced with M41 virus by sera absorbed with different heterologous viruses showed identity in IDD tests and was associated with the surface projections of the virus.

Animals↗
PubMed1986

Identification of myeloperoxidase in human colostrum.

The properties of a peroxidase in human colostrum were studied using antiserum against human myeloperoxidase. The peroxidase in human colostrum gave a single precipitin line against the antiserum on double immunodiffusion, and this precipitin line fused completely with the precipitin line formed between myeloperoxidase and the antiserum. The peroxidase activity in human colostrum was precipitated completely with anti-myeloperoxidase IgG, like myeloperoxidase activity. The peroxidase of colostral whey was purified to homogeneity. The purified enzyme consisted of two subunits of Mr 59,000 and 15,000, corresponding in size to the two subunits of myeloperoxidase. Immunostaining of a protein blot from a sodium dodecyl sulfate-polyacrylamide electrophoresis gel also showed that the peroxidase in the whey extract consisted of the same two subunits as myeloperoxidase. These results indicate that the peroxidase of human colostrum is identical with myeloperoxidase.

Chemical Precipitation↗
PubMed1975

A critical evaluation of the proposal that serum apolipoproteins are the major constituents of the human erythrocyte membrane.

1. The EDTA and Triton X-100 extracts of human erythrocyte ghosts gave no precipitin lines in double diffusion analyses with antibodies to either lipoprotein A, lipoprotein B, lipoprotein C, lipoprotein D, Lp(a) lipoprotein or arginine-rich apolipoprotein of normal human serum (for nomenclature for serum lipoprotein families and apolipoptoteins, see Alaupovic, P., Kostner, G., Lee, D. M., McConathy, W.J. and Magnani, HN. (1972) Expo. Annu. Biochem. Med. 31, 145-160 and Alaupovic, P., Lee, D.M. and McConathy, W.J., (1972) Biochim. Biophys. Acta 260, 689-707.) These membrane preparations also reacted negatively with commercially available antisera to alpha- and beta-lipoproteins. 2. The normal serum very low density, low density and high density lipoproteins formed no precipitin lines with antibodies to either intact or EDTA-extracted ghosts. 3. The serum apolipoproteins and their constitutive polypeptides (A-I, A-II, B, C-I, C-II, C-III, D and arginine-rich apolipoprotein) reacted negatively with antibodies to intact or EDTA-extracted ghosts. The EDTA and Triton X-100 extracts of erythrocyte ghosts gave no reaction with monospecific antibodies to serum apolipoproteins and their constitutive polypeptides. 4. Ghosts dissolved 2% sodium dodecyl sulfate gave positive immunoprecipitin lines with antisera to alpha- and beta-lipoproteins. However, the sodium dodecyl sulfate solution in concentrations greater than 0.1% also formed precipitin lines with antisera to the same lipoproteins. 5. These results do not support the suggestion (Langdon, R.G. (1974) Biochim. Biophys. Acta 342, 213-228) that serum apolipoptoteins are integral protein constituents of human erythrocyte ghosts. The immunoprecipitin lines observed in the latter study might have been due to the presence of trace amounts of serum lipoproteins loosely attached to the cellular surfaces or, more probably, resulted from nonspecific interactions between the proteins and the sodium dodecyl sulfate used as the solubilizing agent

Apoproteins↗
PubMed1977

Evidence suggesting the presence of common antigenic determinant between dynein and tubulin.

The present experiments showed that the guinea pig antiserum prepared against the main polypeptides of 14 S dynein from Tetrahymena cilia reacted with sea urchin sperm flagellar dynein and with bovine brain high molecular weight protein to give rise to a precipitin line confluent with that formed between the antiserum and Tetrahymena dynein. Furthermore, it was found that this antiserum also reacted with tubulins from Tetrahymena cilia, sea urchin sperm flagella and bovine brain to give rise to the confluent precipitin line. Among muscle proteins, only actin preparation from rabbit skeletal muscle reacted with the anti-Tetrahymena dynein serum, whereas neither rabbit skeletal muscle myosin, chicken skeletal muscle tropomyosin nor chicken skeletal muscle troponin reacted with the antiserum. These results suggest that dynein and tubulin and probably actin share an antigenic determinant regardless of different protein species and of different animal species. The common antigenic determinant was detected only when the proteins denatured with urea/sodium dodecyl sulfate/beta-mercaptoethanol/N-ethylmaleimide were used, but it was not detected at all when the native proteins were used. This implies that a certain common antigenic determinant which is involved in the precipitin line formation exists in the primary structures of dyneins and tubulins and probably actin, and is hidden inside the tertiary structures of the native protein molecules.

Adenosine Triphosphatases↗
PubMed1981

Immunological cross-reactivity between chicken slow skeletal and ventricular muscle myosin.

Antibodies elicited in rabbits against chicken slow skeletal anterior latissimus dorsi and ventricular myosin were analyzed by double immunodiffusion for their ability to react with homologous and heterologous antigen at different stages of immunization (1--12 months). Each anti myosin antiserum formed a single, strong precipitin line with its immunogen after short time of immunization. This reaction was specific for myosin heavy chains as determined by GEDELISA (gel electrophoresis derived enzyme lined immunosorbent assay) test. In rabbits injected with ventricular myosin after long time of immunization a second, fainter precipitin line has generally been observed. The antigenic determinants responsible for this precipitin line have been localized on the light myosin subunits. By comparing the two types of anti myosin antisera with heterologous antigen we have obtained evidence for partial immunological cross-reactivity between slow skeletal and ventricular muscle myosins. In particular, all anti ventricular myosin antisera displayed a marked immunological reactivity with anterior latissimus dorsi myosin whereas most of anti anterior latissimus dorsi myosin antisera showed absence of reciprocity. By means of immunofluorescence and immunoabsorption techniques both common and unique slow skeletal and ventricular antigenic determinants have been demonstrated.

Animals↗
PubMed1977

Evaluation of counter immuno-electrophoresis crossed electro-immunodiffusion and agar gel diffusion for immunodiagnosis of human hydatid disease.

Techniques of counter immuno-electrophoresis (CIEP), crossed electro-immunodiffusion (CEID) and agar gel diffusion (AGD) were used for diagnosis of hydatid disease. Sera were obtained from suspected patients before surgery and the antigens used were either human hydatid fluid (HHF) or sheep hydatid fluid (SHF). Out of the 29 suspected cases, 21 were confirmed by recovering the cysts from lung (ten), liver (nine), liver and spleen (two). The experimental results showed that precipitin line(s) with CIEP were obtained with the sera of all 21 confirmed patients using HHF and of 13 using SHF. With CEID, all 19 sera tested demonstrated precipitin patterns with HHF. In the AGD test precipitin lines were obtained in the sera of 15 patients using HHF and of six using SHF. None of the sera from 22 patients suffering from illnesses other than hydatidosis or of the sera from 34 apparently normal individuals gave positive results with CIEP when either HHF or SHF was used as the source of antigen. The CIEP test correlated well with CEID. It is, therefore, suggested that CIEP, which is a rapid and less sophisticated test, can be applied for the diagnosis of hydatid disease and in epidemiological surveys.

Counterimmunoelectrophoresis↗
PubMed1985

Use of ELISA for measuring the extent of serological cross-reactivity between plant viruses.

The degree of antigenic relatedness between two plant viruses is commonly expressed by a serological differentiation index (SDI) which corresponds to the average number of two-fold dilution steps separating homologous from heterologous precipitin titers. Results obtained with several tobamo- and tombusviruses indicated that the indirect form of the enzyme-linked immunosorbent assay (ELISA) can also be used for calculating SDI values. This was achieved by comparing the antiserum dilutions that lead to the same absorbance measurements (for instance 1.0) when homologous and heterologous viruses are assayed by ELISA. SDI values calculated from ELISA were similar to those obtained from precipitin tests. Because of its greater sensitivity, ELISA is able to quantify weak cross-reactions that are not detectable by precipitin tests.

Antigens, Viral↗
PubMed1989

Soluble antigens from culture-grown Besnoitia besnoiti endozoites.

Soluble antigens from Besnoitia besnoiti cell culture-grown endozoites, obtained either by hypotonic lysis or by freeze-thawing and ultrasonication (FTS) of the organisms, were detected by the agar gel immunodiffusion method. Each antigenic preparation yielded 1-4 precipitin lines when reacted with the corresponding rabbit hyperimmune serum, while no reaction was observed with Besnoitia-positive sera from naturally infected cattle. Soluble exoantigens released by viable Besnoitia endozoites into the supernatant of infected cell cultures formed two precipitin lines with rabbit anti-FTS hyperimmune serum and appeared as positively charged protein in immunoelectrophoresis. The precipitin lines observed were parasite specific since no reaction occurred with either lysates of normal Vero cells or with supernatants from non-infected cell cultures.

Animals↗
PubMed1986

Comparative antigenic analysis of extracellular proteins of Bacteroides nodosus isolated from virulent and benign ovine footrot.

Antigens in the extracellular protein (ECP) complexes of Bacteroides nodosus, isolated from sheep with either benign or virulent footrot, were studied by immunoelectrophoresis (IEP). Rabbit antisera against ECP from virulent and benign strains, were used in homologous and heterologous crossed IEP. Four precipitin peaks unique to the virulent strain, and five peaks unique to the benign strain were identified. In an attempt to characterize the different antigens in ECP, rabbit antisera were raised against an outer membrane protein (OMP, mol. wt. 35 000 daltons), pili and various proteases of virulent and benign strains of B. nodosus. No precipitin band was observed when ECP from both B. nodosus strains were reacted against anti-OMP and anti-pilus antisera. However, single precipitin bands unique to one protease from the benign strain and one protease from the virulent strain were identified. The results suggest that specific antigens other than proteases or pili are important in determining whether a B. nodosus isolate is virulent or benign.

Animals↗
PubMed2003

Hypersensitivity pneumonia-nonspecific interstitial pneumonia/fibrosis histopathologic presentation: a study in diagnosis and long-term management.

BACKGROUND: Nonspecific interstitial pneumonia/fibrosis (NSIP) has been classified a form of idiopathic interstitial pneumonia/fibrosis. We have shown that cases of NSIP without demonstrable serum precipitins may be caused by inhalation of high levels of mold and/or bacteria in closed environments. OBJECTIVE: We report a patient with a clinical and histopathologic diagnosis of NSIP without serum precipitins caused by a microbial contamination in her home. Her case was converted from an acute to an insidious clinical presentation by inadequate remediation. A prolonged avoidance-challenge technique demonstrated that this case of NSIP was a form of hypersensitivity pneumonia that was reversible by effective remediation. METHODS: The patient was identified by compatible signs and symptoms, roentgenographic studies, pulmonary function tests, and a transbronchial lung biopsy. She was further evaluated with a detailed environmental history, serologic tests, and investigation of the home environment. An environmental avoidance and challenge technique was performed to confirm cause and effect and to determine that remediation had been effective. RESULTS: Review of the biopsy showed NSIP and failed to reveal any non-caseating granuloma formation. Investigation of the home revealed a Cladosporium species contamination of the air conditioning system and Penicillium species beneath an entryway carpet. Serum precipitins to commercial antigens of common mold to the south Texas area were negative. Avoidance and challenge techniques confirmed the home as the causative environment in this case of NSIP. The patient has been free of signs and symptoms and has taken no medication for interstitial lung disease over the past 30 months. CONCLUSIONS: Some cases of NSIP may be caused by inhalation of microbial antigen(s) in a closed environment. An environmental challenge technique was an effective method to determine the causative environment and confirm that remediation had been effective. Inadequate remediation may lead to symptomatic improvement, but may convert a patient from an acute to an insidious presenter. The environmental challenge obviates a need for specific challenges to determine specific causation. Remediation of or moving from an environmental contamination to achieve reversibility or prevent progression was the treatment of choice to avoid use of long-term immunosuppressive agents.

Air Pollution, Indoor↗
PubMed1982

Immunodiffusion studies of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density.

Tryptic fragments of bovine nasal-cartilage proteoglycan, fractionated by dissociative density-gradient ultracentrifugation, were made to react by immunodiffusion against antiserum to a hyaluronidase-digest subfraction of cartilage proteoglycan monomer. This reaction produced two families of partly superimposed precipitin lines. One family was restricted to gradient fractions of medium or low buoyant density and included the immunoprecipitation reaction attributed to the hyaluronic acid-binding region of the cartilage proteoglycan monomer. The second family of precipitin lines was present alone in gradient fractions of high buoyant density. Immunodiffusion studies with antisera to relatively homogeneous keratan sulphate-rich and chondroitin sulphate-bearing fragment subfractions isolated from the gradient fraction of highest density indicated that both subfractions contained the antigenic determinants responsible for the second family of precipitin lines. Additional immunodiffusion studies, with the use of multispecific antisera to chondroitinase ABC digest and hyaluronidase digest of proteoglycan monomer, confirmed that the two subfractions shared antigenic determinants, and, in addition, indicated that these determinants were on one molecular species in the keratan sulphate-rich fragment subfraction and divided among at least three in the chondroitin sulphate-bearing fragment subfraction. Although an unprecedentedly large number of cartilage proteoglycan antigens could be recognized with the antisera employed in this cartilage proteoglycan antigens could be recognized with the antisera employed in this study, it was not possible to identify antigenic determinants unambiguously specific for the three structurally and functionally distinct regions of the cartilage proteoglycan monomer.

Animals↗
PubMed1978

Synthesis of cold-insoluble globulin by cultured calf endothelial cells.

Radiolabeled amino acids were incorporated into nondialyzable protein by cultures of endothelial cells derived from calf aorta. Antibody prepared against purified bovine plasma cold-insoluble globulin (CIG) formed a strong precipitin line upon immunodiffusion against 3H-labeled proteins from endothelial cell culture media. This precipitin line formed a line of identity with the precipitin line formed by anti-CIG and purified CIG. Double-antibody immunoprecipitation of 3H-labeled protein showed that 36.3% of the radioactivity was specifically precipitated by the anti-CIG antibody. Gel filtration and polyacrylamide gel electrophoresis of the immunoprecipitate indicated that it contained a single protein species whose molecular weight was consistent with that of CIG isolated from plasma. When cultured cells were examined by indirect immunofluorescence microscopy, the endothelial cells stained specifically with anti-CIG antibody. These data indicate that cultured endothelial cells derived from calf aorta synthesize and secrete a protein antigenically similar to CIG.

Animals↗