Enzyme-Labeling of Antibodies and Their Fragments for Enzyme Immunoassay and Immunohistochemical Staining

Ishikawa, Eiji; Imagawa, Masayoshi; Hashida, Seiichi; Yoshitake, Shinji; Hamaguchi, Yukio; Ueno, Tetsuo

doi:10.1080/15321818308057011

Cited by 534 publications

(326 citation statements)

References 50 publications

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“…Female New Zealand White rabbits were immunized with 200 µg of peptide-KLH conjugate in Freund's complete adjuvant ; booster immunizations with 200 µg of conjugate in Freund's incomplete adjuvant were given on days 14, 28 and 48. Blood was collected from the marginal ear vein starting 7 days after the final injection, and IgG fractions were isolated from the resulting antisera by Na # SO % precipitation and DE52-cellulose ion-exchange chromatography [23]. The antibodies were further purified by affinity chromatography using the appropriate peptide immobilized on CNBractivated Sepharose 4B [20,24].…”

Section: Immunization and Characterization Of Peptide Antibodiesmentioning

confidence: 99%

Substrate access channel topology in membrane-bound prostacyclin synthase

Deng¹,

Huang²,

So³

et al. 2002

Biochem. J.

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Results from our molecular-modelling and site-directed-mutagenesis studies of prostaglandin I # synthase (PGIS) have suggested that the large PGIS cytoplasmic domain is anchored to the endoplasmic reticulum (ER) membrane by the N-terminal segment in a way that orients the substrate access channel opening to face the membrane. To test this hypothesis we have explored the accessibility of the PGIS substrate channel opening to site-specific antibodies. The working three-dimensional PGIS model constructed by protein homology modelling was used to predict surface portions near the substrate access channel opening. Two peptides corresponding to the surface immediately near the opening [residues 66-75 (P66-75) and 95-116 (P95-116)], and two other peptides corresponding to the surface about 10-20 A H (1 A H l 0.1 nm) away from the opening [residues 366-382 (P366-382) and 472-482 (P472-482)] were used to prepare sitespecific antibodies. All four antipeptide antibodies specifically recognized the synthetic segments of human PGIS and recombinant PGIS, as shown by binding assays and Western-blot analysis. The site-specific antibodies were used to probe the

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Section: Immunization and Characterization Of Peptide Antibodiesmentioning

confidence: 99%

Substrate access channel topology in membrane-bound prostacyclin synthase

Deng¹,

Huang²,

So³

et al. 2002

Biochem. J.

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“…14 The MAb was purified on a protein G-sepharose column. Ten milligrams of the purified antibody were covalently linked to horse radish peroxidase (HRPO), as previously described 9 and another 10 mg of the purified antibody were linked to biotin using a biotinylation procedure according to the manufacturer's instructions a . The peroxidase-linked and biotinylated conjugates produced were designated AK13A2 HRPO and AK13A2 bi, respectively.…”

Section: Methodsmentioning

confidence: 99%

Development of a 1-Step Enzyme-Linked Immunosorbent Assay for the Rapid Diagnosis of Bovine Respiratory Syncytial Virus in Postmortem Specimens

Quinting

Robert

Letellier³

et al. 2007

J VET Diagn Invest

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Abstract. Bovine respiratory syncytial virus (BRSV) is associated with severe respiratory disease in cattle. BRSV infection frequently leads to the death of young infected animals. The presence of BRSV in postmortem specimens is routinely detected using indirect immunofluorescence (IIF). However, this technique requires special equipment and considerable expertise. The present paper describes the development of a 1-step ELISA for rapid (1.5 hours) detection of BRSV antigen in organ homogenates. The performance of the new 1-step ELISA was evaluated using bovine postmortem specimens (n 5 108) in comparison with 3 other BRSV diagnostic techniques: indirect immunofluorescence, the Clearview respiratory syncytial virus (RSV) test, and real-time reverse transcriptase polymerase chain reaction (RT-PCR). The relative sensitivity, specificity, and the kappa coefficient of 1-step ELISA, the Clearview RSV electroimmunoassay (EIA), and IIF were calculated, using real-time RT-PCR as the reference test. The new 1-step ELISA was the most sensitive and specific of the 3 tests. Thus, the new 1-step ELISA is a reliable test for detecting BRSV antigen in organ homogenates.

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“…The mouse used for the development of HMFG2 also received cultured milk epithelial cells Arklie et al, 1981;Burchell et al, 1983 (Feller et al, 1985). HMFGI and HMFG2 were directly conjugated to phosphatase making the method a simple one step procedure to minimise the proportion of false positive results (Ishikawa et al, 1983) Representative samples (positive and negative controls) of the same sera were dried down using PBS (pH 7.0) alone and were compared to the sera dried down with the low pH method. It was found that the positive controls were lost when using PBS alone.…”

Section: Methodsmentioning

confidence: 99%

A low pH enzyme linked immunoassay using two monoclonal antibodies for the serological detection and monitoring of breast cancer

Dhokia¹,

Pectasides²,

Self³

et al. 1986

Br J Cancer

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Summary A new, simple and sensitive low pH ELISA method has been developed to measure serum levels of tumour associated antigens detectable by monoclonal antibodies HMFG1 and HMFG2. We examined sera from healthy controls, patients with neoplastic and non-neoplastic conditions of breast, liver and gastrointestinal tract. The majority of patients with metastatic breast cancer had elevated serum antigens (69% HMFG1, 72% HMFG2) compared to healthy controls (6.3% HMFG1, 3.0% HMFG2) or patients with benign breast disease (17% HNFG1, 4% HMFG2). There was no discrimination using these assays between patients with neoplastic and non-neoplastic conditions of liver and gastrointestinal tract. This new method promises to be of value in the assessment and management of patients with breast cancer.

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Enzyme-Labeling of Antibodies and Their Fragments for Enzyme Immunoassay and Immunohistochemical Staining

Cited by 534 publications

References 50 publications

Substrate access channel topology in membrane-bound prostacyclin synthase

Substrate access channel topology in membrane-bound prostacyclin synthase

Development of a 1-Step Enzyme-Linked Immunosorbent Assay for the Rapid Diagnosis of Bovine Respiratory Syncytial Virus in Postmortem Specimens

A low pH enzyme linked immunoassay using two monoclonal antibodies for the serological detection and monitoring of breast cancer

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