Anti-actin specificity of human smooth muscle antibodies in chronic active hepatitis.

Thirty sera reacting by IFL technique in titres greater than or equal to 100 with smooth muscle fibres of rat stomach, rat renal glomeruli, and with the membrane region of thyroid cells were randomly chosen among sera sent in for routine testing of tissue antibodies. All sera but one were found to be derived from patients with chronic active hepatitis. The smooth muscle and other relevant cell staining were abolished after absorption of sera with actin, prepared from rabbit skeletal muscle and found to be homogeneous by SDS gel-electrophoresis and by electron microscopy. The actin anti-bodies were purified by precipitation of sera with F-actin and elution of the precipitates at acid pH. The purified antibodies stained all tissues in the same way as the original sera. In double immunodiffusion tests all thirty sera gave precipitation with actin. Thus, it was concluded that these broad-reacting SMA are directed against actin. The finding of high-titred SMA is of diagnostic value and supports the clinical diagnosis of active chronic hepatitis. In addition, anti-actin antibodies eluted from human sera are a suitable tool for studying actin-containing cellular structures.     

Detection and quantitation of anti-neutrophil cytoplasm antibodies in Wegener's granulomatosis by ELISA using affinity-purified antigen

Autoantibodies directed against cytoplasmic components of neutrophil granulocytes and monocytes (ACPA) have previously been described as a disease-specific marker for Wegener's granulomatosis (WG). We have developed an ELISA for determining and quantifying ACPA using an affinity-purified antigen preparation. The antigen was purified from supernatants of human neutrophils stimulated with phorbol ester to induce degranulation, by means of affinity chromatography with naturally occurring human autoantibodies. The established ELISA was sufficiently specific and sensitive, and the ACPA concentrations obtained with it correlated significantly with the ACPA titres determined by an indirect immunofluorescence technique (Spearman's rank correlation coefficient = 0.85, P less than 0.001, n = 105 WG patients). This ELISA provides precise ACPA quantitation and should prove valuable for monitoring disease activity in WG.     

Anti-actin antibodies revealed by counter-immunoelectrophoresis. Relation to smooth muscle antibodies and bile canalicular antibodies.

In investigations by counter-immunoelectrophoresis, anti-actin antibodies were found in 59% of patients with chronic hepatitis and in 8% of patients with non-hepatic diseases and normal blood donors. Anti-actin antibodies were found more frequently in patients with hepatitis and IgG smooth muscle antibodies than in other groups of diseases and normal subjects with IgG smooth muscle antibodies. Anti-actin antibodies showed no correlation with bile canalicular antibodies.     

Detection of Campylobacter species in foods by indirect competitive ELISA using hen and rabbit antibodies

The competitive enzyme immunoassays for detection of Campylobacter jejuni, C. coli and C. fetus subsp. fetus have been developed. Rabbit and hen immunoglobulins were prepared for these purposes. The working conditions of ELISAs, such as the concentrations of immunoreactants, incubation temperatures and time, and the composition of the substrate have been established. The detection limits were in the range 5.0 10⁴–3.2 10⁶ cfu/ml. The application of chemiluminescent substrates did not result in any significant improvement of the assay's detectability and sensitivity. Prepared antibodies showed rather high specificity and cross-reactivity profiles, and both rabbit and hen immunoglobulins were similar. Only IgY to C. jejuni cross-reacted with seven strains of C. jejuni and two other Campylobacter spp.

A limited number of naturally and artificially contaminated food samples were tested. The results obtained by means of an enzyme immunoassay were compared with those obtained from PCR or commercially available Singlepath® Campylobacter GLISA-Rapid Test. Poultry products were naturally contaminated with Campylobacters. The wild species were identified as C. jejuni and C. coli.     

Detection of Campylobacter species in foods by indirect competitive ELISA using hen and rabbit antibodies

The competitive enzyme immunoassays for detection of Campylobacter jejuni, C. coli and C. fetus subsp. fetus have been developed. Rabbit and hen immunoglobulins were prepared for these purposes. The working conditions of ELISAs, such as the concentrations of immunoreactants, incubation temperatures and time, and the composition of the substrate have been established. The detection limits were in the range 5.0 10⁴-3.2 10⁶ cfu/ml. The application of chemiluminescent substrates did not result in any significant improvement of the assay's detectability and sensitivity. Prepared antibodies showed rather high specificity and cross-reactivity profiles, and both rabbit and hen immunoglobulins were similar. Only IgY to C. jejuni cross-reacted with seven strains of C. jejuni and two other Campylobacter spp.

A limited number of naturally and artificially contaminated food samples were tested. The results obtained by means of an enzyme immunoassay were compared with those obtained from PCR or commercially available Singlepath® Campylobacter GLISA-Rapid Test. Poultry products were naturally contaminated with Campylobacters. The wild species were identified as C. jejuni and C. coli.     

Detection of human acid alpha-glucosidase in fibroblasts using monoclonal antibodies in a biotin-avidin amplified ELISA

A sensitive and specific immunological assay for detection of human lysosomal alpha-glucosidase was developed using a mouse monoclonal antibody incorporated into a biotin-avidin amplified ELISA. The immunoassay was more than 60 times more sensitive than the currently used enzymatic assay for alpha-glucosidase activity using a fluorimetric substrate. This methodology provides an alternative approach with increased sensitivity for screening individuals for alpha-glucosidase deficiency.     

Detection of aldrin and dieldrin in egyptian milk samples using a competitive ELISA

A number of milk samples collected in Egypt from different animal species and at different locations were analyzed for the presence of the organochloride pesticides aldrin and dieldrin. A simple competitive enzyme‐linked immunosorbent assay (ELISA) was used for the detection and quantification of aldrin and dieldrin in milk samples from different species: buffalo, cow, goat, sheep and donkey. Pesticides were detected in 62.5% (10/16) of the buffalo milk samples, 73.33% (11/15) of the cows’ milk samples, 25% (3/12) of the goats’ milk samples, 71.42% (5/7) of the sheeps’ milk samples and 66–66% (2/3) of the donkeys’ milk samples.     

Detection of anti-contractile antibodies after cardiac surgery using ELISA assay.

Sera from 196 patients were collected before and after cardiac surgery to measure antibodies against heart tissue and against actin and myosin. In the post-operative period antibodies were found in 87 patients (44%) producing a cross-striated fluorescence pattern in heart tissue. Antibodies against the major contractile proteins were found in 91 patients (46%), anti-actin antibodies in 49 patients (25%) and anti-myosin antibodies in 65 patients (33%). We found a significant correlation (P less than 0.0001) between the antibodies producing a cross-striated fluorescence pattern and antibodies recognizing contractile proteins. These results suggest that contractile proteins evoke an immune response after cardiac surgery and that this response may compromise cardiac function.     

Detection of enterovirus-specific total and polymeric IgA antibodies in serum using a synthetic peptide or heated virion antigen in ELISA

The presence of enterovirus-specific total and polymeric IgA antibodies was assessed in serum from different groups of patients and healthy controls by indirect ELISA using heated virions and synthetic peptide, both enteroviral broad reactive antigens. Total IgA antibody response against a synthetic peptide, representing an enterovirus group-common epitope, was detected in 52% of the patients with an acute enterovirus infection and in 12% of the patients with other infections (P = 0.02). We also found a significant difference (P = 0.005) in the prevalence of peptide IgA antibodies between serum samples collected from blood donors during summer (20%), the prevalent season of enterovirus infections, and winter (6%). A polymeric IgA activity against the peptide was detected in only three patients with an enterovirus infection. In contrast, when a heated coxsackie B5 (coxB5) virus antigen was used, the prevalence of total serum IgA antibodies was not significantly different between patients with an acute enterovirus infection and patients with other infections (71 % vs. 53% respectively; P = 0.3). Also no difference was found between the two groups of blood donors (47% in summer vs. 51% in winter; P = 0.7). However, the prevalence of serum polymeric IgA antibodies against coxsackie B5 antigen was significantly greater (P = 0.02) in patients with an acute enterovirus infection (57%) than in patients with other infections (18%). These findings suggest that the presence of total peptide-IgA or of polymeric coxsackie B5-IgA in serum is a specific marker of acute enterovirus infection. Finally, we show that the total peptide-IgA-and polymeric coxsackie B5-ELISAs may have a diagnostic value for the serodiagnosis of enterovirus infections when they are used in combination with enteroviral IgG-ELISA. © 1994 Wiley-Liss, Inc.     

生物薄膜干涉技术和ELISA法检测抗体药物免疫原性的方法学比较

采用生物薄膜干涉技术(BLI)开发快速检测治疗性单抗(抗EGFR单抗,Cetuximab)的抗抗体(ADAs)的检测方法。本研究利用ForteBio公司开发的Octet系统,在光纤制成的生物传感器底端覆盖生物分子相容层,偶联配体,形成生物膜层。当分析物结合传感器底端偶联的配体时,生物膜层厚度增加,反射光干涉光谱曲线产生可测量的漂移,从而可以实现对分子间相互作用的实时测量,同时以传统的抗体桥ELISA法作为平行对照。SA(Streptavidin)传感器结合生物素化的单抗,用免疫原性试剂孵育血清样品后,利用传感器检测血清样品的ADA。分别应用BLI和ELISA两种方法确定cutpoint,并进行剂量反应曲线的分析。研究结果表明BLI快速分析法具有同ELISA法极其相似的免疫原性检测行为,均能准确检测出筛选分析中的20例正常人血清的基线反应值。同时,BLI法在竞争抑制实验中体现出了很高的特异性。与ELISA相比,BLI技术用于治疗性抗体的临床免疫原性分析,更加省时,快捷,准确,而且可避免低亲和力ADA易受洗板干扰的弊端。因此,BLI技术可能成为抗体药物免疫原性检测的一种新的有效方法。     

Obtention of monoclonal antibodies against human IgG4 using two different immunization strategies: development of a biotin-based ELISA for IgG4 quantitation.

Several monoclonal antibodies (MAbs) against human IgG4 have been produced in two fusion experiments. In fusion I, Balb/c mice were conventionally immunized and most of the antibodies obtained recognized IgG class-specific epitope(s). Therefore, a second fusion experiment was performed using IgG4-immunized mice that had been made tolerant to epitopes shared by the four IgG subclasses, and most of the hybridomas derived from this second fusion secreted antibodies specific for IgG4. Five MAbs which were found to be specific for both IgG4 isoallotypes (4a and 4b) were selected from the two fusion experiments. These antibodies define at least two non-overlapping epitopes located on the Fab and Fc regions of IgG4 molecule, respectively. Two MAbs were used to develop a two-site ELISA for IgG4 quantitation, in which one MAb (anti-Fab) was immobilized in the solid phase and the other one (anti-Fc) was biotin-labeled. The assay had a detection limit below 1 ng/ml and a practical working range between 4 and 200 ng/ml. The use of these MAbs should improve our understanding of the role of IgG4 in different clinical conditions.     

Detection of antibodies to human nerve antigens in sera from leprosy patients by ELISA.

Anti-neural antibodies have been implicated to play a role in the pathogenesis of nerve damage in leprosy patients. To find the relationship between anti-neural antibodies and clinical findings, we attempted to detect antibodies against neurofilament-enriched proteins by ELISA in sera from leprosy patients. Of 289 sera from leprosy patients, 74 (25.6%) had significant anti-neural antibodies; in contrast, 1 (5.0%) of 20 tuberculosis patients and 11 (7.1%) of 154 controls were seroreactive to nerve antigen. When clinical types were considered, a significant level of anti-neural IgG antibodies was detectable in 53 (30.1%) of 176 sera from lepromatous patients compared with 21 (18.6%) of 113 sera from tuberculoid patients, indicating that lepromatous patients were more likely to be seropositive to nerve antigens in ELISA. Some of the ELISA-reactive sera showed antibody reactivity with 38-kD, 40-kD and 43-kD nerve antigens in Western blotting analysis. There was no apparent correlation between seroreactivity to nerve antigens and bacterial load in leprosy patients. Although there was no statistical significance, anti-neural antibodies were detectable more often among the patients on chemotherapy than the untreated and among the patients with erythema nodosum leprosum than without. The results, therefore, suggest that anti-neural antibodies are elicited during the course of leprosy and may be associated with the extensiveness of nerve involvement in the patients.     

Rapid, simple and sensitive antigen capture ELISA for the quantitation of myoglobin using monoclonal antibodies

A rapid ELISA method has been developed to quantitate myoglobin in serum. An antigen capture enzyme-linked immunoassay with IgG1 mouse monoclonal antibodies produced by cell clones MGB20-4A1.1 and MGB20-3C1.2 were used for myoglobin detection. These monoclonal antibodies are specific for different epitopes of the myoglobin molecule. Monoclonal antibodies from the hybridoma clone MGB20-4A1.1 were adsorbed to microtiter plate wells. The plates were washed with PBS containing 0.05% Tween 20 and then 20 microliter of standard serum or serum of patients and 200 microliter of peroxidase labeled monoclonal antibodies MGB20-3C1.2 were added to each well. Plates were incubated for 90 min at 37 degrees C and enzyme activity was determined using o-phenylenediamine as a substrate. The ELISA assay described is a rapid and sensitive procedure to assess the quantity of myoglobin within the range 2-1000 ng/ml serum. 120 samples can be tested in 3 h.     

Competition ELISA using a human monoclonal antibody for detection of antibodies against human immunodeficiency virus type 1.

A novel competition ELISA for detection of antibodies against HIV-1 was developed. The assay is based on competition at the single epitope level and utilises a human monoclonal antibody and an E. coli-produced fragment of the transmembrane glycoprotein gp41. The sensitivity of the assay was 100% in tests on 247 serum samples obtained from 219 individuals previously shown to be HIV-1 antibody positive by both conventional indirect ELISA and the immunoblotting test. The patients represented various clinical and immunological stages of HIV-1 infection. Likewise, the specificity of the assay was 100% in tests on 105 serum samples from normal individuals previously tested negative by indirect ELISA. Further, among 105 serum samples selected due to consistent false positive reactions in the indirect ELISA only 2 samples (1.9%) demonstrated false positive reactions in the competition ELISA, i.e. 98.1% specificity. Finally, only 2 of 57 (3.5%) serum samples from HIV-2 infected individuals showed positive reactions in the assay, while 54 (94.7%) had absorbance values similar to the negative controls. These results demonstrate that human monoclonal antibodies may form the basis for highly sensitive and specific assays for detection of antibodies to HIV-1.     

Application of a human monoclonal antibody in a rapid competitive anti-HIV ELISA

The ELISA is the established screening technique for the detection of antibodies directed against HIV. The first generation assays, mostly based on the sandwich principle, employed purified virus from cell culture and gave both false-positive and false-negative results. Sandwich-type assays preferentially detect IgG antibodies, require a high serum dilution and are two-step procedures. In order to detect an immune response as early as possible after infection anti-HIV antibodies of the IgM class should also be measured. To this end a competitive ELISA has been developed using a solid phase-adsorbed recombinant HIV envelope protein and an enzyme-labelled human monoclonal antibody. This detects both IgM and IgG antibodies, the results are available within 1 h and a serum predilution is not necessary.     

Optimization of a competitive ELISA with polyclonal antibodies for quantification of prolamins in foods

The optimization of a sequential competitive ELISA for the quantification of prolamins in foods is described in this article. The assay was developed using polyclonal antibodies obtained by the hyper‐immunization of rabbits with commercial gliadin. The ELISA developed in this way showed a very high degree of detectability (detection limit, 1 ng ml^‐1), as well as the ability to discriminate between prolamins harmful to coeliac individuals from non‐toxic prolamins. The influence of the solvent used for extraction of the samples on the detection capability of the test was also studied. The assay proved to be useful for the evaluation of gliadins in processed foods including meat products. The assay was applied to many types of foods and was compared with a commercial kit approved by the Association of Official Analytical Chemists.     

Detection of porcine interleukin-18 by sandwich ELISA and immunohistochemical staining using its monoclonal antibodies.

We describe here the development of sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunohistochemical staining for porcine interleukin-18 (PoIL-18) and their application to detection of PoIL-18 in vivo. Ten anti-PoIL-18 monoclonal antibodies (mAb), all of which were reactive with recombinant PoIL-18 by Western blotting, were established. Four (2-C-4, 9-H-6, 11-H-5, and 12-C-12) of 10 neutralized the biologic activity of PoIL-18 to induce interferon-y (IFN-gamma) from porcine peripheral blood mononuclear cells (PBMC). Four (2-C-4, 5-F-6, 9-H-6, and 12-C-12) of 10 were shown to be useful in immunohistochemical staining and detected PoIL-18 in Kupffer cells and macrophages in hepatic focal necrosis and macrophages in interstitial pneumonia in piglets with experimental endotoxemia using formalin-fixed, paraffin-embedded sections. A sandwich ELISA was developed using mAb 7-G-8 as a capture antibody and biotinylated mAb 5-C-5 as a detection antibody. This ELISA detected PoIL-18 with a minimum detectable concentration of 20 pg/ml and did not show cross-reactivity against PoIL-1beta, IL-8, IL-12, and IFN-gamma or murine and human IL-18. Using this ELISA, PoIL-18 was detected in the plasma and the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Actinobacillus pleuropneumoniae. The availability of this ELISA and immunohistochemical staining for PoIL-18 may contribute to a further understanding of the role of this cytokine in various porcine immune responses and diseases.     

Detection and quantitation of rotavirus using monoclonal antibody coupled red blood cells: comparison with ELISA

A total of 125 faecal extracts from infants were tested by reverse passive haemagglutination (RPH) using red cells coated with a monoclonal antibody against the major group-specific rotavirus antigen (VP 6). Results were compared with those obtained using a rabbit anti-rotavirus capture, guinea pig anti-rotavirus detector-based ELISA. The specificity of the assay was confirmed by use of 'normal' immunoglobulin coupled red cells and by inhibition with rabbit antiserum. The antibody-coated red cells could be stabilised by treatment with glutaraldehyde and subsequent freeze-drying with no detectable loss of activity even after storage at 45 degrees C for 4 wk. Good correlation was obtained between RPH and ELISA. Purified bovine rotavirus could be detected by RPH down to approximately 10(5) particles in a 25 microliters vol. Similar results were obtained with polyclonal antibody coupled cells and an ELISA using monoclonal antibody. Experiments using subgroup-specific monoclonal antibodies indicated the feasibility of rapid subgroup determination.