Indirect Enzyme-linked Immunoabsorbent Assay Research Articles

Occurrence and genetic diversity of hemoplasmas in beef cattle from the Brazilian Pantanal, an endemic area for bovine trypanosomiasis in South America

Hemotropic mycoplasmas (hemoplasmas) are Gram-negative bacteria that parasitize the erythrocyte surface of a wide variety of mammals. The present study aimed at investigating the occurrence of hemoplasmas in beef cattle in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis in South America. Additionally, the objective of this study was to characterize molecularly the genotypes of the found hemoplasmas. For this purpose, blood and serum samples of 400 beef cattle were collected from five properties in Corumbá, Nhecolândia sub-region, Mato Grosso do Sul, in Midwest Brazil. Blood samples underwent DNA extraction and standard 16S rRNA gene-based PCR assays for hemoplasmas. The sequences obtained were submitted to phylogenetic inferences, distance analysis, and genotype diversity. The Indirect Enzyme-Linked Immunoabsorbent Assay (iELISA) indicated the presence of anti-Trypanosoma vivax IgG antibodies in 89.75% of the animals sampled, confirming the endemicity of said agent in the studied region. Among the 400 bovine blood samples tested, 2.25% (9/400) were positive for hemoplasmas in cPCR. The phylogenetic analysis of the obtained sequences confirmed the presence of 'Candidatus Mycoplasma haemobos' and Mycoplasma wenyonii DNA in 0.5% (2/400) and 1.75% (7/400) animals, respectively. Five genotypes of M. wenyonii and one of 'Candidatus M. haemobos' were detected among the sequenced amplicons. The present study showed low molecular occurrence of haemoplasmas in beef cattle sampled in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis. Despite of the conservation of the 16S rRNA gene, there was considerable diversity of hemoplasma genotypes infecting the sampled beef cattle.

Anti-p-benzoquinone antibody level as a prospective biomarker to identify smokers at risk for COPD.

Background and objectiveIdentification of smokers having predisposition to COPD is important for early intervention to reduce the huge global burden of the disease. Using a guinea pig model, we have shown that p-benzoquinone (p-BQ) derived from cigarette smoke (CS) in the lung is a causative factor for CS-induced emphysema. p-BQ is also derived from CS in smokers and it elicits the production of anti-p-BQ antibody in humans. We therefore hypothesized that anti-p-BQ antibody might have a protective role against COPD and could be used as a predictive biomarker for COPD in smokers. The objective of this study was to compare the serum anti-p-BQ antibody level between smokers with and without COPD for the evaluation of the hypothesis.MethodsSerum anti-p-BQ antibody concentrations of current male smokers with (n=227) or without (n=308) COPD were measured by an indirect enzyme-linked immunoabsorbent assay (ELISA) developed in our laboratory. COPD was diagnosed by spirometry according to Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines.Results and discussionA significant difference was observed in the serum anti-p-BQ antibody level between smokers with and without COPD (Mann–Whitney U-test =4,632.5, P=0.000). Receiver operating characteristic (ROC) curve analysis indicated that the ELISA had significant precision (area under the curve [AUC] =0.934, 95% confidence interval [CI]: 0.913–0.935) for identifying smokers with COPD from their low antibody level. The antibody cutoff value of 29.4 mg/dL was constructed from the ROC coordinates to estimate the risk for COPD in smokers. While 90.3% of smokers with COPD had a low antibody value (≤29.4 mg/dL), the majority (86.4%) of smokers without COPD had a high antibody value (≤29.4 mg/dL); 13.6% of current smokers without COPD having an antibody level below this cutoff value (odds ratio [OR] =59.3, 95% CI: 34.15–101.99) were considered to be at risk for COPD.Conclusion and future directionsOur results indicate that serum anti-p-BQ antibody level may be used as a biomarker to identify asymptomatic smokers at risk for COPD for early intervention of the disease.

Development and Application of an ELISA Assay Using Excretion/Secretion Proteins from Epimastigote Forms of T. cruzi (ESEA Antigens) for the Diagnosis of Chagas Disease.

An indirect enzyme-linked immunoabsorbent assay (ELISA) for Trypanosoma cruzi was developed using epimastigote secretion/excretion proteins (ESEA antigens) obtained from axenic culture supernatants. A panel of 120 serum samples from subjects with confirmed Chagas disease (n = 50), healthy controls (n = 50), and patients with other parasitic diseases (n = 20) was used to evaluate the new ESEA-based ELISA (ELISAESEA). This new test had excellent sensitivity (98%) and acceptable specificity (88%). Cross-reactivity was observed largely in sera from subjects with Leishmania and Ascaris infections. Using Western blotting and epimastigotes from two distinct T. cruzi isolates, several polypeptide bands with molecular masses ranging from 50 to 220 kDa were detected in pooled chagasic sera. However, the band pattern for each isolate was different. These data suggest that an inexpensive and technically simple ELISA based on ESEA antigens is a promising new tool for the diagnosis of Chagas disease.

Sorologia e sobrevivência do vírus do amarelo letal do mamoeiro

O vírus do amarelo letal do mamoeiro (Papaya lethal yellowing virus, PLYV) é responsável por uma das principais doenças do mamoeiro (Carica papaya) no Nordeste brasileiro. O PLYV pode ser transmitido através do solo, água, instrumento de corte e mãos contaminadas. A presente pesquisa teve como objetivo estudar as características biológicas, sorológicas e físicas de um isolado do vírus e avaliar sua sobrevivência em tecido seco infetado. O PLYV foi detectado por "Enzyme linked immunosorbent assay" (ELISA) indireto e isolado em mudas de mamoeiro, através da inoculação mecânica. A sobrevivência do PLYV em folhas e raízes secas de mamoeiro infetado foi avaliada por sorologia e inoculação em plantas sadias. A presença do vírus foi detectada em folhas e raízes de mamoeiro secas, indicando que o vírus pode permanecer ativo em restos de cultura por até 120 dias. A purificação do PLYV permitiu a obtenção de 309.5 mg de vírus/kg de folha e o anti-soro obtido mostrou-se altamente específico, com títulos de 1:128 em dupla difusão em Agar e 1:1.024.000 em Elisa indireto. Estudos das propriedades físicas do PLYV em mamoeiro revelaram um ponto de inativação térmica (PIT) em torno de 80 ºC, um ponto máximo de diluição (PMD) de ac. 10-6 e uma longevidade in vitro (LIV) acima de 50 dias.

Production and Identification of High Affinity Monoclonal Antibodies Against Pesticide Carbofuran

Abstract To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people's health and environmental protection, the hapten 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl]-amino]-butanoic acid (BFNB) of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier (BFNB-bovine serum albumin, BFNB-BSA) conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay (ELISA), based on BFNB-ovoalbumin conjugates (BFNB-OVA). Purified monoclonal antibody (McAb) was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line (5D3) secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1:2.048 × 10 3 and 1:1.024 × 10 6, respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 10 9 L mol −1, with an IC 50 value of 1.18 ng mL −1 and a detection limit of 0.01 ng mL −1. Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized (4.59 × 10 −4% for 2,3-dihydro-2,2-dimethyl-7-benzofuranol and less than 3.0 × 10 −4% for others). The prepared McAb had a very high affinity and specificity, and it could be used to develop ELISA for rapid determination of carbofuran.

Biochemical characterization and molecular cloning of a plasminogen activator proteinase (LV-PA) from bushmaster snake venom

The protein (LV-PA) from bushmaster ( Lachesis muta muta) venom is a serine proteinase which specifically activates the inactive proenzyme plasminogen. LV-PA is a single chain glycoprotein with an apparent molecular mass of 33 kDa that fell to 28 kDa after treatment with N-Glycosidase F (PNGase F). Approximately 93% of its protein sequence was determined by automated Edman degradation of various fragments derived from a digestion with trypsin. A cDNA library of L. m. muta was constructed to generate expressed sequence tags (ESTs) and the plasminogen activator precursor cDNA was sequenced. The complete amino acid sequence of the enzyme was deduced from the cDNA sequence. LV-PA is composed of 234 residues and contains a single asparagine-linked glycosylation site, Asn-X-Ser, bearing sugars that account for ∼ 10% of the enzyme's total molecular mass of 33 kDa. The sequence of LV-PA is highly similar to the plasminogen activators (PAs) TSV-PA from Trimeresurus stejnegeri venom and Haly-PA from Agkistrodon halys. Furthermore, the mature protein sequence of LV-PA exhibits significant similarity with other viperidae venom serine proteinases which affect many steps of hemostasis, ranging from the blood coagulation cascade to platelet function. The Michaelis constant (Km) and the catalytic rate constant (kcat) of LV-PA on four chromogenic substrates were obtained from Lineweaver–Burk plots. In addition, we used an indirect enzyme-linked immunoabsorbent assay (ELISA) to explore the phylogenetic range of immunological cross-reactivity (using antibodies raised against LV-PA) with analogous serine proteinases from two viperidae venoms and mammals.

Development of an indirect enzyme-linked immunoabsorbent assay using a monoclonal antibody to identify Ictalurus sp. fillets

The deceptive marketing of imported basa, Pangasius bocourti (Sauvage), fillets as catfish has resulted in serious economic losses to the channel catfish, Ictalurus punctatus (Rafinesque), industry in the US. The similar appearance of channel catfish and basa fillets created a need for a rapid method to differentiate uncooked, cooked and/or marinated channel catfish fillets from basa fillets and other fish products. A monoclonal antibody (MAb) specific for a 36.8 kDa channel catfish fillet protein was produced and characterized by an indirect enzyme-linked immunoabsorbent assay (ELISA) and Western blotting. This MAb was used to develop an indirect ELISA specific for a fillet protein unique to fish of the genus Ictalurus. Using this ELISA, 100% of raw and cooked channel catfish fillets were correctly identified and differentiated from other fish in a single-blind study. These results show that the indirect ELISA using MAbs specific for unique Ictalurus sp. fillet proteins is a rapid and sensitive method for the identification of raw and cooked catfish fillets.

Resolution of isoforms of mutalysin II, the metalloproteinase from bushmaster snake venom

Mutalysin II, a zinc endopeptidase possessing direct-acting fibrinolytic activity has been previously purified from bushmaster ( Lachesis muta muta) snake venom. We now report a method to isolate two isoforms of natural mutalysin II (mut IIa and mut IIb) using chromatographies on Sephacryl S-200, CM Sepharose CL 6B and Sephadex G-50. The two proteins are monomeric non-glycosylated proteinases with similar molecular masses of 23 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tryptic peptide mapping of the two native enzymes suggested a large degree of structural similarity. Both isoforms showed high similarity in all enzymatic properties using fibrinogen, fibrin and dimethylcasein as substrates. Thus, the specific fibrinolytic activity was estimated as 12±1.04 and 11.5±1.02 U/μg for mut IIa and mut IIb, respectively. The antigenic cross-reactivity of both isoforms was examined using rabbit hyperimmune serum or immunoglobulin G anti-mut IIa assays on immunodiffusion microscope slides, indirect enzyme-linked immunoabsorbent assay and western blots. From these experiments it was concluded that the two metalloproteinases mut IIa and mut IIb share identical antigenic structures. Since the stability of mutalysin II is dependent upon the presence of zinc, we examined the EDTA sensitivity of the isoforms of mutalysin II. Thus, the IC 50 values (concentration of EDTA to produce 50% inhibition of dimethylcasein hydrolysis) for mut IIa is 180 μM and 165 μM for mut IIb.

In vitro production of specific antibody by equine peripheral blood mononuclear cells using tetanus toxoid as a recall antigen

Anti-tetanus toxoid ( tt) antibody (Ig) levels in the supernatant of cultured, pre-immunised equine peripheral blood mononuclear cells ( pbmc) were measured by an indirect enzyme-linked immunoabsorbent assay ( elisa). Optimal anti- tt Ig production occurred at concentrations of stimulating, purified tt of between 0001 and 0·1 μg ml −1, which varied depending on the cell concentration. Optimal anti- tt Ig production was most consistently produced when the cell concentration was 5 × 10 6 ml −1. At this cell concentration maximal anti- tt Ig was induced using 0·1 μg ml −1 tt. At a cell concentration of 5 × 10 6 ml −1 and a tt concentration of 0·1 μg ml −1 anti- TT Ig was first detectable in supernatant on day 5 of stimulation. Maximal levels of anti- tt Ig were present in the supernatant by day 10. No anti- tt Ig was produced in cultures of PBMC from non-immune animals.

Class-specific antibodies to bovine respiratory syncytial virus in experimentally infected lambs.

Enzyme-linked immunoabsorbent assay (ELISA) was used to titrate virus-specific IgG, IgM and IgA levels in nasal secretions, lung lavage fluids and serum samples sequentially obtained from lambs experimentally infected with bovine respiratory syncytial virus (RSV). Virus-specific IgG and IgM responses were measured by the indirect double antibody sandwich ELISA using anti-bovine RSV monoclonal antibody, as capture antibody, and peroxidase-conjugated anti-sheep IgG and anti-sheep IgM. Virus-specific IgA antibodies were measured by antibody capture assay using anti-sheep IgA (alpha-chain specific) and anti-bovine RSV monoclonal antibodies. Bovine RSV-specific IgM and IgA antibodies were detected in the serum samples within 6 days post-inoculation (p.i.). Virus-specific IgC antibodies appeared in serum samples 4 days later. In nasal secretions, IgA antibodies appeared 7 days p.i. but IgM antibodies were not detected until 12-16 days p.i. In serum samples, IgM titres were predominant for the first 2 weeks p.i. IgC titres becoming predominant thereafter. In nasal secretions and lung lavage fluids, IgA titres were significantly higher than IgM or IgG titres up to 21 days p.i. (0.01).

Isolation of two antigens from the culture filtrates of Mycobacterium tuberculosis and their applications in the laboratory diagnosis of the tuberculous meningitis

Two antigens were isolated from the culture filtrates of H37Ra Mycobacterium tuberculosis by immunoabsorbent affinity chromatography, M. tuberculosis antigen 5 and immunoabsorbent affinity column-purified antigen (IAP). The potential application of these two mycobacterial antigens in the laboratory diagnosis of tuberculous meningitis was evaluated by indirect enzyme-linked immunoabsorbent assay in cerebrospinal fluid specimens. IAP antigen was more sensitive than antigen 5, although antigen 5 was more specific than IAP antigen in detecting tuberculous aetiology. Technical aspects of immunoabsorbent affinity chromatography have been highlighted in this study.

Quantitative determination of rheumatoid factor by an enzyme-labeled immunoassay

The presence and quantity of rheumatoid factor (RF) in human serum were determined by an indirect enzyme-linked immunoabsorbent assay (ELISA). A human rheumatoid factor control serum, standardized against the WHO reference rheumatoid arthritis serum preparation, was used to derive a standard curve in each assay. The results of unknowns were estimated from the standard curve and reported in International Units (IU) per milliliter (ml). The ELISA assay was compared with the bentonite flocculation test. The overall coefficient of correlation between the two assays was 87%; it was 93.3% for patients with rheumatoid arthritis but only 67.5% for patients with undiagnosed conditions. The error of the ELISA assay (coefficient of variation) was generally less than 10% at both high and low concentrations of rheumatoid factor. The quantitative reproducible nature of the assay allows the detection of small variations of rheumatoid factor level and could be useful in the serial evaluation of patients.