Use Of Enzyme-linked Immunosorbent Assay Research Articles

Diagnostic value of an antibody enzyme-linked immunosorbent assay using affinity-purified antigen in an area endemic for melioidosis.

Melioidosis, an infection caused by Burkholderia pseudomallei, is endemic in southeast Asia. The septicemic form of melioidosis is the leading cause of death from nonhospital-acquired septicemia in the northeastern part of Thailand. A major factor that contributes to the high mortality is the delay in isolation and identification of the causative organism. The present study was undertaken to evaluate the use of enzyme-linked immunosorbent assays based on an immunoaffinity-purified antigen for detecting specific IgG and IgM antibodies to this organism as a rapid serodiagnostic method for melioidosis. The diagnostic value of these tests was evaluated in an actual clinical situation in an area endemic for melioidosis. The specificity of specific IgG test (82.5%) and the specific IgM test (81.8%) were significantly better than that of the indirect hemagglutination (IHA) test (74.7%). The sensitivity of the specific IgG assay (85.7%) was higher than that of the IHA test (71.0%) and the specific IgM test (63.5%). Specific IgG antibody was detected in a majority of septicemic melioidosis (87.8%), as well as in localized forms (82.6%). The specific IgG test was also better than the specific IgM test and the IHA test in identifying acute melioidosis cases in the first five days after admission. In addition, the IgG antibody level to this antigen remained high over a period of more than five years in those who had recovered from melioidosis and remained clinically free of the disease. These results indicate that the detection of specific IgG antibody is clinically useful for the diagnosis of acute melioidosis in an endemic area.

Antigens for serological diagnosis of ovine footrot

An antigen extracted from Dichelobacter nodosus with potassium thiocyanate (KSCN) is currently used in enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of ovine footrot, but the test lacks specificity in mature sheep. Other antigens were therefore evaluated for use in this test. Structural components of the cell envelope of D. nodosus including outer membrane, cytoplasmic membrane, lipopolysaccharide and pilus and extracellular proteases were purified from cultured D. nodosus while recombinant membrane proteins, protease and pilus antigens were also evaluated. Many antigenic components of D. nodosus participated in reactions in ELISA that were not specific for infection with D. nodosus and apart from pilus, none of the antigens resulted in improved specificity of the ELISA. Using a positive-negative cut-off to yield sensitivity of 70%, ELISA using pili from cultured D. nodosus serogroup A had a specificity of 98.3% compared with 89.7% for the ELISA with KSCN-extract as antigen ( P < 0.001). Recombinant pili morphogenetically expressed in Pseudomonas aeruginosa were unsuitable for use in ELISA due to copurification of Pseudomonas antigens to which apparently healthy sheep directed antibodies. The application of ELISA with D. nodosus pilus as antigen in footrot control programs is discussed.

Use of enzyme-linked immunosorbent assay to measure thrombin-antithrombin III complexes in horses with colic

Abstract Objectives To evaluate new ELISA for measurement of thrombin-antithrombin III (TAT) concentration, and to correlate the values to other tests of hemostasis in horses with colic. Design Plasma TAT concentration and 8 other hemostasis analytes were measured in horses with colic at hospital admission and during the next 4 days. Retrospectively, data were analyzed by outcome, broad-category diagnosis, and clinical management, and for correlation between TAT and other assays. Animals 100 horses with colic. Procedure Plasma samples were evaluated for TAT, fibrinogen, and fibrin degradation products concentrations; antithrombin III (ATIII), protein C, α2-antiplasmin, and plasminogen activities; prothrombin time (PT); and activated partial thromboplastin time. Results Changes were indicative of a hypercoagulable state, most severe in nonsurviving horses, characterized by increased TAT concentration; decreased ATIII, protein C, and plasminogen activities; and increased PT. Nonsurvivors had significantly increased TAT concentration compared with that in survivors, without regard to sample collection time; however, compared over time, TAT was significantly increased only at admission. Highest TAT concentration was in nonsurvivors with inflammatory intestinal lesions. There was significant negative correlation between TAT and ATIII, protein C, α2-antiplasmin, and plasminogen values, and significant positive correlation between TAT and PT, and fibrin degradation products values. Conclusions Plasma TAT reflects the current state of coagulation system activation and is a good assay for early diagnosis of the hypercoagulable state in horses with the most severe forms of colic. Clinical Relevance Measurement of equine TAT provides further information to characterize the hypercoagulable state in horses to aid in case management. (Am J Vet Res 1996;57:456–462)

Observations on the use of ELISA for detection of Babesia bigemina specific antibodies

An indirect enzyme-linked immunosorbent assay (ELISA) was evaluated to study the cause of the high level of background reactions which hinders the application of ELISA as a field diagnostic test for Babesia bigemina. Different blockers to improve the specificity of the ELISA were compared. The use of soya milk (25%), gelatine (2.5%) and chicken serum (2%) did not significantly improve the specificity of the test. It was noted that the presence of fibrinogen contributed to the positive ELISA results more than the presence of B. bigemina specific antigen. This conclusion was confirmed by testing bovine fibrinogen as a host protein antigen in ELISA which strongly responded against B. bigemina positive control sera. It is suggested that application of ELISA for B. bigemina is still unreliable until a more purified Babesia-specific antigen or specific monoclonal antibodies are available.

Use of enzyme-linked immunosorbent assay and radioimmunoassay to determine serum and urine dexamethasone concentrations in Thoroughbreds after intravenous administration of the steroid

Abstract Objective To develop a simple and sensitive ELISA for detection of dexamethasone in horse serum and urine. Sample Population Blood and urine samples from 3 Thoroughbred mares. Procedure A dexamethasone oxime was prepared and conjugated to hemocyanin, bovine serum albumin and to horseradish peroxidase. One- and two-step double-antibody ELISA methods, as well as a radioimmunoassay method, were performed. The one-step ELISA was used to test urine from 3 Thoroughbred mares injected with 5 mg of dexamethasone, IV. Results The ELISA could detect dexamethasone in the range of 0.01 to 50 ng/ml, with intra- and interassay variations of 8.92 and 9.42%, respectively. Serum dexamethasone concentration reached a peak of 20 to 35 ng/ml 15 minutes after steroid administration and decreased to 1 ng/ml in 2.5 hours. Urine dexamethasone concentration 18 to 50 ng/ml 1 to 2 hours after drug administration and decreased to 1 ng/ml at 10 hours. Conclusion The developed assay is sensitive as well as simple for detecting dexamethasone in horse serum and urine, and is comparable to radioimmunoassay. Clinical Relevance This method can be useful for screening samples from racehorses, because it is sensitive and does not require sample preparation or sophisticated equipment.

Plasma P selectin (GMP-140) and glycocalicin are elevated in preeclampsia and eclampsia: Their significances

OBJECTIVE: We measured the concentrations of plasma P selectin (or GMP-140) and glycocalicin in preeclamptic and eclamptic women. Correlations between these two parameters and blood pressures, platelet counts, or plasma thrombin-antithrombin complex values were evaluated. STUDY DESIGN: By use of enzyme-linked immunosorbent assays we measured the plasma GMP-140 and glycocalicin levels in normal pregnancies ( n = 10) and preeclamptic ( n = 10) and eclamptic ( n = 20) pregnancies. The glycocalicin index was calculated as follows: (glycocalicin × [250 × 10 6/ml])/(Individual platelet counts). Correlations between plasma GMP-140, glycocalicin, glycocalicin index values, blood pressures, platelet counts, and plasma thrombin-antithrombin complex values were analyzed. RESULTS: Plasma GMP-140 levels were found to be significantly elevated in preeclamptic ( p < 0.0005) and eclamptic cases ( p < 0.0001) compared with normotensive controls. Plasma glycocalicin ( p = 0.01, 0.007) and glycocalicin index ( p = 0.005, 0.002) values were also markedly elevated in preeclamptic and eclamptic patients compared with normal pregnant patients. Significant correlations between platelet counts or plasma thrombin-antithrombin complex levels and their corresponding plasma GMP-140 and glycocalicin and glycocalicin index values have been found in preeclamptic and eclamptic cases. However, blood pressures had correlations with GMP-140, glycocalicin, and glycocalicin index values in eclamptic cases. CONCLUSIONS: We demonstrated an elevation of plasma GMP-140 and platelet glycocalicin in preeclampsia and eclampsia. This study also reflects the usefulness of glycocalicin as a marker of platelet activation or turnover and endothelial dysfunction in these diseases. (A M J O BSTET G YNECOL 1996;174:272-7.)

Use of ELISA to Rapidly Screen Hazelnut for Resistance to Eastern Filbert Blight

A rapid and accurate screening system was developed to more rapidly identify resistance to eastern filbert blight based on an indirect enzyme-linked immunosorbent assay (ELISA) of greenhouse-inoculated hazelnut. Polyclonal antibodies were obtained from rabbits following immunization with antigens from pure cultures of Anisogramma anomala, the pathogen. One-thousand-fold dilution of the antiserum produced positive reactions to 1.7 x 10 5 dilutions of A. anomala-infected hazelnut tissue extracts, but did not react to 1.7 x 10 2 dilutions of healthy hazelnut tissue extracts. Symptomless plants infected by A. anomala were detected by the indirect ELISA 3 to 5 months after inoculation, an improvement over the 13- to 27-month incubation period required for susceptible genotypes to develop external symptoms of infection (cankers). ELISA was more sensitive and efficient than conventional microscopic assays (i.e., visualizing A. anomala mycelium in hand-sectioned plant tissue) in both healthy and infected extracts. The screening system was tested on selected progenies from populations segregating for a single, dominant resistance gene. ELISA detected 100% of the infections while microscopic examination detected only 36% of the infected samples. ELISA rapidly and reliably identifies hazelnut progeny with a gene conferring a high level of resistance derived from the cv. Gasaway.

Enhanced proliferation and IL-2 secretion by lung lymphocytes from HIV-infected subjects.

Human immunodeficiency virus (HIV)-positive patients frequently develop a CD3+/CD8+ cytotoxic T cell lymphocytic alveolitis. This could occur through in situ expansion of lung lymphocytes. We evaluated lung and blood lymphocyte proliferation in asymptomatic HIV-infected individuals by measuring spontaneous and cytokine-induced tritiated thymidine incorporation. Interleukin (IL)-2 and IL-4 secretion was determined with the use of enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation techniques. Spontaneous proliferation by lung lymphocytes from HIV-positive patients was significantly greater than that of normal volunteers. Proliferation was confined to the CD8+ lymphocyte subset. Over time, spontaneous proliferation declined unless autologous alveolar macrophages (AM) were added, suggesting AM were providing additional stimulatory signals to lung lymphocytes. Lung and blood lymphocytes proliferated in response to IL-2 but not IL-4. Lymphocytes in HIV-infected lung spontaneously produced and secreted more IL-2 than either normal lung lymphocytes or autologous blood lymphocytes. IL-4 production was not detectable in either group. These findings support the hypothesis that lymphocytic alveolitis in asymptomatic HIV-positive patients results from IL-2-dependent in situ proliferation of CD3+/CD8+ cytotoxic T cells.

Mycoplasma pneumoniae and Chlamydia pneumoniae in pediatric community-acquired pneumonia

We evaluated 260 previously healthy children ages 3 through 12 years who had clinical signs and symptoms of pneumonia, radiographically confirmed. Patients were randomized 1:1 to a 10-day course of either clarithromycin suspension 15 mg/kg/day divided twice a day or erythromycin suspension 40 mg/kg/day divided twice a day or three times a day. Evidence of infection with Chlamydia pneumoniae was detected in 28% (74) of patients: 13% (34) by nasopharyngeal culture and 18% (48) by serology with the microimmunofluorescence assay. Evidence of infection with Mycoplasma pneumoniae was detected in 27% (69) of patients: 20% (53) by nasopharyngeal culture or polymerase chain reaction and 17% (44) by serology with the use of enzyme-linked immunosorbent assay. Serologic confirmation of infection was observed in 23% (8) and 53% (28) of patients with bacteriologically detected C. pneumoniae and M. pneumoniae, respectively. Treatment with clarithromycin vs. erythromycin, respectively, yielded the following outcomes: clinical success 98% (121 of 124) vs. 95% (105 of 110); radiologic success 98% (109 of 111) vs. 94% (92 of 110); and eradication by pathogen, C. pneumoniae 79% (15 of 19) vs. 86% (12 of 14) and M. pneumoniae 100% (9 of 9) vs. 100% (4 of 4). Adverse events were primarily gastrointestinal occurring in almost one-fourth of patients in both groups, and were mild to moderate in severity. Clarithromycin and erythromycin were similarly effective and safe for the treatment of radiographically proved, community-acquired pneumonia in children older than 2 years old.(ABSTRACT TRUNCATED AT 250 WORDS)

Lesions of the Respiratory Tract Associated With the Finding of Anti-Neutrophil Cytoplasmic Autoantibodies With a Perinuclear Staining Pattern

To characterize the clinicopathologic spectrum of respiratory tract involvement in patients with positive results of immunofluorescence microscopy for anti-neutrophil cytoplasmic autoantibodies with a perinuclear staining pattern (p-ANCA) and to assess the clinical value of p-ANCA testing. We retrospectively reviewed the medical records of all patients at Mayo Clinic Rochester in whom p-ANCA were detected by indirect immunofluorescence microscopy during 1992. Additional target antigen identification was performed with use of enzyme-linked immunosorbent assays for antibodies against myeloperoxidase (MPO) and proteinase 3. We summarized the clinical findings in MPO-positive and MPO-negative patients. Sera were positive for p-ANCA in 42 of 2,381 patients (1.8%). In 13 of these 42 patients (31%), the respiratory tract was involved. Twelve patients had chest roentgenographic abnormalities, including a diffuse alveolar filling pattern (N = 8), a diffuse interstitial pattern (N = 2), and a combined interstitial and alveolar pattern (N = 2); three others had nasal inflammation. Ten of 16 sera tested were positive for MPO, and proteinase 3 antibodies were detected in 1 specimen. All patients with alveolar hemorrhage were positive for MPO antibodies. Testing for p-ANCA by immunofluorescence microscopy discloses a wide range of clinical disorders distinct from the main cytoplasmic-staining ANCA-associated disease--namely, Wegener's granulomatosis. In particular, the respiratory tract is affected much less frequently. Further evaluation of positive p-ANCA immunofluorescence test results by enzyme-linked immunosorbent assay to determine whether MPO is the target antigen is necessary to obtain clinically useful information from this test.

The use of enzyme-linked immunosorbent assay for the quantitation of Enhydrina schistosa (common sea snake) venom: N.-H. Tan, K.-K. Lim and M. I. Nik Jaafar (Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia)

The use of enzyme-linked immunosorbent assay for the quantitation of Enhydrina schistosa (common sea snake) venom: N.-H. Tan, K.-K. Lim and M. I. Nik Jaafar (Department of Biochemistry, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia)

Association of Mycoplasma pneumoniae antigen with initial onset of bronchial asthma.

Although mycoplasmal airway infection frequently exacerbates bronchial asthma, the cause of the initial onset of asthma remains unclear at present. In this report, we describe a patient in whom a previous acute mycoplasmal respiratory infection led to an initial onset of bronchial asthma. One month after the onset of the illness, cough and wheezing appeared. Pulmonary function studies revealed an airway obstructive dysfunction. Oral administration of bronchodilators resulted in a marked improvement of the asthmatic symptoms. An airway hyperresponsiveness to methacholine was demonstrated even 2 yrs after the initial onset of the illness, and IgE antibody specific to Mycoplasma pneumoniae was detected in the serum by use of enzyme-linked immunosorbent assay. An immediate skin test for M. pneumoniae was positive in addition to multiple positive skin tests. A bronchial inhalation challenge test with M. pneumoniae antigen also yielded a positive result. We conclude that the effects of mycoplasmal respiratory infections on the airway are multifactorial and involve a complex interplay of airway inflammation and IgE-mediated hypersensitivity.

Overexpression and simple purification of human immunodeficiency virus-1 gag epitope derived from a recombinant antigen in E. coli and its use in ELISA

To develop a test for diagnosis of human immunodeficiency virus-1 (HIV-1) exposure sensitivity, a part of the gag gene was cloned and expressed in Escherichia coli, using expression vectors containing a trp promoter. The immunoreactivity of recombinant protein was determined using HIV-1 specific antibodies in a Western blot analysis. The recombinant plasmid, pYHCgag3, gag gene was fused to the trpE′ gene linked to the hydroxylamine (HA) cleavage recognition sequence which was induced to overexpress a core antigen (gag a.a. 121–398 from plasmid BH10) as fusion protein in the form of insoluble inclusion body. Recombinant gag was purified by a simple single step purification procedure. After partial purification of inclusion bodies and subject to the HA-cleavage treatment, gag protein was further purified to homogeneity using DEAE-Sepharose chromatography. The purified core antigen offered reliable results with high sensitivity and specificity for identification of HIV-1 antibodies when tested in the enzyme-linked immunosorbent assay (ELISA). These results suggest that mass production of recombinant core antigen will provide a valuable resource to HIV-1 serodiagnostics for the screening of large groups of blood donors to prevent HIV-1 infection.

Diagnosis of infections with Shiga-like toxin-producing Escherichia coli by use of enzyme-linked immunosorbent assays for Shiga-like toxins on cultured stool samples

Shiga-like toxin-producing (SLT) Escherichia coli, particularly those belonging to serogroup O157, are responsible for haemorrhagic colitis, haemolytic uraemic syndrome and some cases of gastro-enteritis. The rapid and reliable diagnosis of all these infections is necessary for correct patient management and for epidemiological reasons, but is rarely possible with present methods. We compared the efficacy of two methods, (i) the culture of faeces in broth that contained mitomycin C followed by enzyme-linked immunosorbent assay (ELISA) for SLTs, and (ii) the culture of faeces on sorbitol MacConkey agar (SMA), in the detection of infections caused by SLT-producing E. coli. SLT-producing E. coli O157 strains were isolated on SMA from 42 of 475 faecal samples, but SLTs were detected by ELISA in culture supernates or lysates of 54 of 475 samples. SLT-producing E. coli strains were isolated subsequently from 11 of 12 ELISA-positive, SMA culture-negative samples by a colony blot technique. In four cases, SLT-producing E. coli of serogroups other than O157 were isolated and in seven cases E. coli O157 was isolated in small numbers. The ELISA is a rapid and sensitive technique for the diagnosis of SLT-producing E. coli infection, especially where low numbers of the organism are present in faeces and when the infection is caused by a serogroup other than O157.

Double-sandwich enzyme-linked immunosorbent assay (ELISA) with murine monoclonal antibodies for detection of staphylococcal enterotoxin B.

Three monoclonal antibodies (MAbs) which react specifically with staphylococcal enterotoxin B (SEB) were studied for their suitability for use in ELISA. One of them (MAb B14) worked well as a coating antibody; MAb B12 was shown to be a good probing antibody (conjugated with peroxidase) when ELISA plates were coated with MAb B14. This effective pair of MAbs (B14-B12PO) is able to detect 0.625 ng SEB ml, and to distinguish between SEB and other proteins present in food extracts.

Use of Enzyme-Linked Immunosorbent Assay (ELISA) for Detection and Quantification of Monoclonal Antibodies

The thrust of the present work is the demonstration of the feasibility of using an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of a monoclonal antibody human serum. The antibodies used, 7E3 and D3GP3, have been proposed as platelet receptor blockers, being directed specifically against the platelet membrane receptors (glycoprotein IIb/IIIa) and thus of significance in the management of patients at a very hgih risk of thrombotic occlusion. It is shown that 7E3 is more easily adsorbed than D3GP3 and hence has a higher potential for the stated application.

Agonist‐induced lipoxin A4 generation: Detection by a novel lipoxin A4‐ELISA

Lipoxin A4 (LXA4) possesses potent bioactions. To facilitate its detection, an enzyme-linked immunosorbent assay (ELISA) was developed that proved sensitive and selective. Quantitation by ELISA of LXA4 generated from cellular sources strongly correlated (r = 0.99) with values obtained by high-pressure liquid chromatography (HPLC). We used this LXA4-ELISA to examine parameters influencing LXA4 generation from endogenous substrates during human platelet-neutrophil (PLT-PMN) interactions in vitro. Agonist-induced LXA4 production was clearly evident at a PLT-PMN ratio of 10:1, and recombinant human granulocyte/monocyte colony stimulating factor-priming of PMN augmented LXA4 generation 5-6 fold. The chemotactic peptide formylmethionyl-leucyl-phenylalanine, platelet-derived growth factor and arachidonic acid (20:4n-6) each stimulated formation of immunoreactive LXA4 (iLXA4) in these co-incubations. The presence of iLXA4 was also evaluated in vivo in aspirin-sensitive asthmatic patients who, in a randomized, double-blind crossover design, underwent nasal lavage after they each ingested a predetermined threshold dose of aspirin or placebo. Aspirin challenge provoked statistically significant increases in iLXA4 in each patient (P < 0.005). These results validate the use of a solid-phase ELISA for detection of LXA4. Furthermore, the use of this ELISA has allowed the first documentation of iLXA4 formation in human subjects with aspirin-sensitive asthma following specific antigenic challenge.

Barley Yellow Dwarf Virus Multiplication and Host Plant Tolerance in Durum Wheat

AbstractA Canadian PAV‐like isolate of barley yellow dwarf virus (BYDV) was used to infect durum wheat (Triticum durum) cultivars previously identified in field trials involving artificial inoculation as highly sensitive (12 IDSN74), slightly tolerant (La Dulce), and relatively tolerant (Boohai and 12 IDSN227) to BYDV. The cultivars were inoculated in the greenhouse as seedlings, and indexed for virus accumulation by enzyme‐linked immunosorbent assay (ELISA) at various intervals between 3 and 60 days thereafter. Mean ELISA values were somewhat consistent with tolerance levels for 4 durum wheat cultivars, but the use of ELISA to screen for BYDV resistance in durum wheat is not practical. The magnitude of the difference between sensitive and tolerant cultivars for the mean ELISA value is not high enough, and it may be necessary to average readings between 3 and 60 days after inoculation to obtain somewhat meaningful ELISA data. The effect of vector aphid numbers on virus titre and aerial biomass in the sensitive durum wheat cv. Karim was also evaluated. There was no significant effect on virus content in a preliminary trial, but a second trial revealed that more viruliferous aphids per plant resulted in higher ELISA values. Infestation with 32 or 50 viruliferous Rhopalosiphum padi per plant depressed biomass yield below the level observed with 1–10 aphids per plant.

Development of an Enzyme-Linked Immunosorbent Assay for the Serological Diagnosis of Big Liver and Spleen Disease

An enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of big liver and spleen (BLS) disease. The test utilizes a soluble, BLS-specific antigen that can be recovered from the livers of infected hens and that is known to react in the agar gel immunodiffusion (AGID) test. For use in ELISA, the BLS-specific antigen is fractionated by gel filtration chromatography and immobilized on microtiter plates using glutaraldehyde. The ELISA was evaluated using sera from infected and uninfected flocks originating in the United Kingdom and the United States. An ELISA format that incorporated control antigen recovered from the livers of uninfected birds for each serum tested was found to be more sensitive than the AGID test. A less sensitive but more cost-effective format that did not incorporate this control is considered suitable for large-scale flock screening programs.

Enzyme-linked immunosorbent assay for an octapeptide based on a genetically engineered fusion protein.

Traditional chemical means of preparing enzyme-ligand conjugates for use in enzyme-linked immunosorbent assays (ELISAs) lead to the production of multisubstituted enzyme-ligand conjugates with a high degree of variability in the site of ligand attachment. A genetically engineered fusion protein was prepared in order to investigate the feasibility of controlled production of conjugates for use in ELISAs. Specifically, a synthetic octapeptide was fused with bacterial alkaline phosphatase. The resulting enzyme-peptide conjugate is monosubstituted (one peptide per subunit), has a single site of attachment, and results in assays with good response characteristics. The use of such fusion proteins, which combine small analyte peptides with enzyme labels, can lead to a new approach to improved assays for numerous biomolecules, including peptide pharmaceuticals, neurotransmitters, hormones, cell surface antigens, etc.