Reaction of monoclonal antibodies with plasma membrane proteins after binding on nitrocellulose: Renaturation of antigenic sites and reduction of nonspecific antibody binding

Birk, Horst‐Walter; Koepsell, Hermann

doi:10.1016/0003-2697(87)90360-5

Cited by 116 publications

(34 citation statements)

References 30 publications

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“…The eluted antibodies were immediately neutralized by addition of 1M Tris HCl, pII 8.8, and concentrated in Centricon 100 microconcentrators (Amicon, Danvers, MA) for further analysis by IIF and immunoblotting. SDS-PAGE induces the denaturation of proteins (38,39), and several procedures for renaturing proteins before or after transfer to membranes have been proposed (40)(41)(42)(43)(44)(45)(46). We adapted some of these methods in trying to renature NuMA-2 antigen (see below) and to obtain immunoblot reactivity.…”

Section: Methodsmentioning

confidence: 99%

Two major autoantigen—Antibody systems of the mitotic spindle apparatus

Andrade

Chan

Peebles

et al. 1996

Arthritis & Rheumatism

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Objective. To characterize human autoantigenantibody systems related to the mitotic poles and spindles.Methods. Thirty-seven human sera with autoantibodies staining mitotic poles and spindles in indirect immunofluorescence (IIF) studies were further characterized by immunofluorescence on mitotic cells and by immunoblotting and immunoprecipitation. Clinical diagnoses meeting the American College of Rheumatology criteria were based on chart review and interview with the corresponding physicians.ResuUs. Two autoantibody systems reactive with mitotic poles and spindles were defined. Type 1 nuclear mitotic apparatus (NuMA-1) antibodies were identified in the serum of 30 patients. Interphase cells showed a fine, speckled, nuclear staining, while mitotic cells had bright staining of the rim of the centrosomes and light staining of the spindles proximal to the centrosomes. In telophase, the staining shifted from the centrosomes to the reforming nuclei. On immunoblotting, antiNuMA-1 sera reacted with a 210-kd protein. The reactivity of these sera was identified (with the aid of reference antibodies) as the previously described NuMA antigen-antibody system. Clinical information was available for only 17 of the 30 patients with antiNuMA-1; of these, 17 (53%) had clinical and lip biopsy findings that met the criteria for Sjiigren's syndrome.Publication 7668-MEM from The Scripps Research Institute.

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Section: Methodsmentioning

confidence: 99%

Two major autoantigen—Antibody systems of the mitotic spindle apparatus

Andrade

Chan

Peebles

et al. 1996

Arthritis & Rheumatism

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“…The former enable investigation of allergens in their native form, whereas the denaturing conditions of the latter (SDS gel electrophoresis) include a stronger risk of not presenting native IgE-binding epitopes, possibly causing detection failures. [79] and [80] RAST assays with crude allergen extracts allowed, for the first time, estimation of sIgE serum concentrations, [81], [82] and [83] but the accuracy of in vitro sIgE determinations is highly variable. [54], [84] and [85] This is, for example, due to lacking standardization of the test agents, as well as to instability of some of the respective allergenic proteins.…”

Section: Quantification Of Ige Antibodiesmentioning

confidence: 99%

Potential, pitfalls, and prospects of food allergy diagnostics with recombinant allergens or synthetic sequential epitopes

Steckelbroeck¹,

Ballmer‐Weber²,

Vieths³

2008

Journal of Allergy and Clinical Immunology

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This article aims to critically review developments in food allergy diagnostics with regard to the verification of specific IgE antibodies and the identification of the responsible allergens. Results of IgE-binding tests with food extracts are hampered by cross-reactive proteins, low-quality test agents, or both. Specificity can be increased by defining adequate cutoff values, whereas sensitivity can be improved by using high-quality test agents. IgE-binding tests with purified allergens enabled reliable quantification of allergen-specific IgE titers, with higher levels found in individuals with food allergy compared with individuals without food allergy. However, the overlap in individual test reactivity between allergic and nonallergic subjects complicates interpretation. Recombinant allergens and synthetic sequential epitopes enabled detection of sensitization profiles, with IgE specific to several allergens and substructures now being suggested as markers of severity, persistence, or both. However, high-power quantitative studies with larger numbers of patients are required to confirm these markers. This article aims to critically review developments in food allergy diagnostics with regard to the verification of specific IgE antibodies and the identification of the responsible allergens. Results of IgE-binding tests with food extracts are hampered by cross-reactive proteins, low-quality test agents, or both. Specificity can be increased by defining adequate cutoff values, whereas sensitivity can be improved by using high-quality test agents. IgE-binding tests with purified allergens enabled reliable quantification of allergen-specific IgE titers, with higher levels found in individuals with food allergy compared with individuals without food allergy. However, the overlap in individual test reactivity between allergic and nonallergic subjects complicates interpretation. Recombinant allergens and synthetic sequential epitopes enabled detection of sensitization profiles, with IgE specific to several allergens and substructures now being suggested as markers of severity, persistence, or both.However, high-power quantitative studies with larger numbers of patients are required to confirm these markers. IgE-mediated and non-IgE-mediated syndromes, where IgE-mediated manifestations are responsible for the majority of food-induced, immediate-type, immune-mediated hypersensitivity reactions. 3 The development of an IgE-mediated response to food requires a series of molecular and cellular interactions, which involve antigenpresenting cells, T lymphocytes, and B lymphocytes. [4] and [5] Depending on the route of sensitization, food allergy is the result of either genuine reactivity to comestibles through the gastrointestinal tract (class I food allergens) or secondary sensitization to cross-reactive food allergens as a consequence of the initial reactivity to homologous pollen-related allergens (class II food allergens). [6] and [7] The majority of class I food allergens are heat stable and resistan...

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“…Molecular mass markers (6H; Sigma) were run on each gel. Western blotting analysis was carried out according to the method of Birk and Koepsell (1987). The membranes were blocked using 1 mg/ml polyvinylalcohol (PVA) in PBS and developed using anti-mouse or anti-rabbit horseradish peroxidase conjugate followed by enhanced chemiluminescence (ECL) reagents (Amersham International plc, Amersham, Bucks, UK).…”

Section: Western Blot Analysismentioning

confidence: 99%

Binding characteristics of antibodies to the TSH receptor

Oda

Sanders²,

Roberts³

et al. 1998

Journal of Molecular Endocrinology

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We have used fragments of the TSH receptor (TSHR) expressed in E. coli as glutathione S-transferase fusion proteins to produce rabbit polyclonal antibodies and a panel (n=5) of monoclonal antibodies to the extracellular fragment of the TSHR. The binding characteristics of the antibodies to linear, conformational, glycosylated and unglycosylated forms of the receptor in different assay systems have been investigated.The reactivity of these antibodies with the TSHR was assessed by Western blotting with both native and recombinant human TSHR expressed in CHO cells, immunoprecipitation of 35 S-labelled fulllength TSHR produced in an in vitro transcription/ translation system, immunoprecipitation of 125 I-TSH/TSHR complexes, inhibition of 125 I-TSH binding to the TSHR and fluorescence activated cell sorter (FACS) analysis of binding to CHO-K1 cells expressing the TSHR on their cell surface. Fab fragments of monoclonal antibodies were isolated, labelled with 125 I and used to determine the affinity constants of these antibodies with receptor, bound and free Fab being separated by polyethylene glycol (PEG) precipitation.Rabbit polyclonal and mouse monoclonal antibodies reacted with the TSHR in Western blotting and one monoclonal antibody (3C7) was able to inhibit 125 I-TSH binding to native human TSHR (74% inhibition), recombinant human TSHR (84% inhibition) and porcine TSHR (65% inhibition). Affinity constant values for TSHR monoclonal antibody Fab fragments calculated using Scatchard analysis were about 10 7 M 1 . Four out of five monoclonal antibodies reacted in FACS analysis with TSHR expressed on the surface of CHO-K1 cells. The FACS unreactive monoclonal (3C7) bound well to detergent solubilised TSH receptors and this emphasised the importance of using a combination of FACS analysis and radioactivelylabelled probes in analysis of the TSH receptor.The monoclonal antibodies produced in this study were found to be of relatively low affinity but proved useful for detection of the receptor by Western blotting and by FACS analysis.

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Reaction of monoclonal antibodies with plasma membrane proteins after binding on nitrocellulose: Renaturation of antigenic sites and reduction of nonspecific antibody binding

Cited by 116 publications

References 30 publications

Two major autoantigen—Antibody systems of the mitotic spindle apparatus

Two major autoantigen—Antibody systems of the mitotic spindle apparatus

Potential, pitfalls, and prospects of food allergy diagnostics with recombinant allergens or synthetic sequential epitopes

Binding characteristics of antibodies to the TSH receptor

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