Double-blotting: a solution to the problem of non-specific binding of secondary antibodies in immunoblotting procedures

Different types of enzyme-linked immunosorbent assays (ELISA) have been widely used to control food safety and quality. To develop an accurate and reproducible ELISA, false immunodetection results caused by non-specific binding (NSB) and cross-reaction must be prevented. During the case study of sandwich ELISA development for the detection of porcine hemoglobin (PHb), several critical factors leading to NSB and cross-reaction were found. First, to reduce the NSB of the target analyte, the selection of microplate and blocker was discussed. Second, cross-reactions between enzyme-labeled secondary antibodies and sample proteins were demonstrated. In addition, the function of (3-aminopropyl)triethoxysilane (APTES) was evaluated. Overall, this study highlights the essence of both antibody and assay validation to minimize any false-positive/negative immunodetection results.

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Referencing cross-reactivity of detection antibodies for protein array experiments

F1000Research ◽

10.12688/f1000research.7668.1 ◽

2016 ◽

Vol 5 ◽

pp. 73 ◽

Cited By ~ 3

Author(s):

Darragh Lemass ◽

Richard O'Kennedy ◽

Gregor S. Kijanka

Keyword(s):

Specific Binding ◽

Cross Reactivity ◽

Protein Array ◽

Igg Antibody ◽

Western Blots ◽

Protein Arrays ◽

Rabbit Igg ◽

Human Proteins ◽

Antibody Repertoires ◽

Secondary Antibodies

Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires requires a platform-dependent, lot-to-lot validation of secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human proteins. Probing protein arrays with secondary antibodies in absence of chicken serum revealed non-specific binding to 61 distinct human proteins. The cross-reactivity of the tested secondary detection antibodies points towards the necessity of platform-specific antibody characterisation studies for all secondary immunoreagents. Secondary antibody characterisation using protein arrays enables generation of reference lists of cross-reactive proteins, which can be then excluded from analysis in follow-up experiments. Furthermore, making such cross-reactivity lists accessible to the wider research community may help to interpret data generated by the same antibodies in applications not related to protein arrays such as immunoprecipitation, Western blots or other immunoassays.

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Quantitative analysis of albumin uptake and transport in the rat microvessel endothelial monolayer

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00152.2002 ◽

2003 ◽

Vol 284 (1) ◽

pp. L187-L196 ◽

Cited By ~ 170

Author(s):

Theresa A. John ◽

Stephen M. Vogel ◽

Chinnaswamy Tiruppathi ◽

Asrar B. Malik ◽

Richard D. Minshall

Keyword(s):

Specific Binding ◽

Fluid Phase ◽

Scatchard Analysis ◽

Albumin Binding ◽

Endothelial Monolayer ◽

Site Model ◽

Specific Binding Sites ◽

High K ◽

Secondary Antibodies ◽

Uptake And Transport

We determined the concentration dependence of albumin binding, uptake, and transport in confluent monolayers of cultured rat lung microvascular endothelial cells (RLMVEC). Transport of 125I-albumin in RLMVEC monolayers occurred at a rate of 7.2 fmol · min−1 · 106cells−1. Albumin transport was inhibited by cell surface depletion of the 60-kDa albumin-binding glycoprotein gp60 and by disruption of caveolae using methyl-β-cyclodextrin. By contrast, gp60 activation (by means of gp60 cross-linking using primary and secondary antibodies) increased 125I-albumin uptake 2.3-fold. At 37°C, 125I-albumin uptake had a half time of 10 min and was competitively inhibited by unlabeled albumin (IC50= 1 μM). Using a two-site model, we estimated by Scatchard analysis the affinity ( K D) and maximal capacity (Bmax) of albumin uptake to be 0.87 μM ( K D1) and 0.47 pmol/106 cells (Bmax1) and 93.3 μM ( K D2) and 20.2 pmol/106 cells (Bmax2). At 4°C, we also observed two populations of specific binding sites, with high ( K D1 = 13.5 nM, 1% of the total) and low ( K D2 = 1.6 μM) affinity. On the basis of these data, we propose a model in which the two binding affinities represent the clustered and unclustered gp60 forms. The model predicts that fluid phase albumin in caveolae accounts for the bulk of albumin internalized and transported in the endothelial monolayer.

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Referencing cross-reactivity of detection antibodies for protein array experiments

F1000Research ◽

10.12688/f1000research.7668.2 ◽

2017 ◽

Vol 5 ◽

pp. 73

Author(s):

Darragh Lemass ◽

Richard O'Kennedy ◽

Gregor S. Kijanka

Keyword(s):

Reference Lists ◽

Specific Binding ◽

Cross Reactivity ◽

Protein Array ◽

Igg Antibody ◽

Western Blots ◽

Protein Arrays ◽

Human Proteins ◽

Antibody Repertoires ◽

Secondary Antibodies

Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires necessitates the use of extensively validated secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human proteins. Probing protein arrays with secondary antibodies in absence of chicken serum revealed non-specific binding to 61 distinct human proteins. Despite the identified non-specific binding, the tested antibodies are well suited for use in protein array experiments as the cross-reactive binding partners can be readily excluded from further analysis. The evident cross-reactivity of the tested secondary detection antibodies points towards the necessity of platform-specific antibody characterisation studies for all secondary immunoreagents. Furthermore, secondary antibody characterisation using protein arrays enables the generation of reference lists of cross-reactive proteins, which can be then marked as potential false positives in follow-up experiments. Providing such cross-reactivity reference lists accessible to the wider research community may help to interpret data generated with the same antibodies in applications not only related to protein arrays such as immunoprecipitation, Western blots or other immunoassays.

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Specific Binding of Secondary Antibodies to Neurons in Rat Brainstem Sections

Microscopy and Microanalysis ◽

10.1017/s1431927605503362 ◽

2005 ◽

Vol 11 (S02) ◽

Author(s):

J Pflugheber ◽

S Casella ◽

J S Erlichman ◽

T Budd

Keyword(s):

Specific Binding ◽

Rat Brainstem ◽

Secondary Antibodies

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Ultrastructural Localization of Antigens by Capillary Action Immunocytochemistry

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162661 ◽

1996 ◽

Vol 54 ◽

pp. 40-41

Author(s):

László G. Kömüves

Keyword(s):

San Francisco ◽

Colloidal Gold ◽

Specific Binding ◽

Ultrastructural Localization ◽

Ultrastructural Level ◽

Uranyl Acetate ◽

Adhesive Tape ◽

Distilled Water ◽

Capillary Action ◽

Rabbit Igg

Light microscopic immunohistochemistry based on the principle of capillary action staining is a widely used method to localize antigens. Capillary action immunostaining, however, has not been tested or applied to detect antigens at the ultrastructural level. The aim of this work was to establish a capillary action staining method for localization of intracellular antigens, using colloidal gold probes.Post-embedding capillary action immunocytochemistry was used to detect maternal IgG in the small intestine of newborn suckling piglets. Pieces of the jejunum of newborn piglets suckled for 12 h were fixed and embedded into LR White resin. Sections on nickel grids were secured on a capillary action glass slide (100 μm wide capillary gap, Bio-Tek Solutions, Santa Barbara CA, distributed by CMS, Houston, TX) by double sided adhesive tape. Immunolabeling was performed by applying reagents over the grids using capillary action and removing reagents by blotting on filter paper. Reagents for capillary action staining were from Biomeda (Foster City, CA). The following steps were performed: 1) wet the surface of the sections with automation buffer twice, 5 min each; 2) block non-specific binding sites with tissue conditioner, 10 min; 3) apply first antibody (affinity-purified rabbit anti-porcine IgG, Sigma Chem. Co., St. Louis, MO), diluted in probe diluent, 1 hour; 4) wash with automation buffer three times, 5 min each; 5) apply gold probe (goat anti-rabbit IgG conjugated to 10 nm colloidal gold, Zymed Laboratories, South San Francisco, CA) diluted in probe diluent, 30 min; 6) wash with automation buffer three times, 5 min each; 7) post-fix with 5% glutaraldehyde in PBS for 10 min; 8) wash with PBS twice, 5 min each; 9) contrast with 1% OSO4 in PBS for 15 min; 10) wash with PBS followed by distilled water for5 min each; 11) stain with 2% uranyl acetate for 10 min; 12) stain with lead citrate for 2 min; 13) wash with distilled water three times, 1 min each. The glass slides were separated, and the grids were air-dried, then removed from the adhesive tape. The following controls were used to ensure the specificity of labeling: i) omission of the first antibody; ii) normal rabbit IgG in lieu of first antibody; iii) rabbit anti-porcine IgG absorbed with porcine IgG.

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On the Role of Transferrin in 67Ga Uptake by Tumor Cells

Nuklearmedizin ◽

10.1055/s-0038-1629447 ◽

1988 ◽

Vol 27 (04) ◽

pp. 151-153

Author(s):

P. Thouvenot ◽

F. Brunotte ◽

J. Robert ◽

L. J. Anghileri

Keyword(s):

Specific Binding ◽

Subcellular Fractionation ◽

Animal Blood ◽

Tumor Bearing ◽

Cell Structures ◽

The Difference ◽

Sarcoma Cells ◽

In Vitro Uptake

In vitro uptake of 67Ga-citrate and 59Fe-citrate by DS sarcoma cells in the presence of tumor-bearing animal blood plasma showed a dramatic inhibition of both 67Ga and 59Fe uptakes: about ii/io of 67Ga and 1/5o of the 59Fe are taken up by the cells. Subcellular fractionation appears to indicate no specific binding to cell structures, and the difference of binding seems to be related to the transferrin chelation and transmembrane transport differences

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Visualization of von Willebrand Factor Multimers by Enzyme-Conjugated Secondary Antibodies

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642536 ◽

1986 ◽

Vol 55 (02) ◽

pp. 276-278 ◽

Cited By ~ 26

Author(s):

F Brosstad ◽

Inge Kjønniksen ◽

B Rønning ◽

H Stormorken

Keyword(s):

Von Willebrand Factor ◽

Chromogenic Substrate ◽

Agarose Electrophoresis ◽

Secondary Antibodies ◽

Von Willebrand ◽

Willebrand Factor ◽

Beta Galactosidase

SummaryA method for visualization of the multimeric forms of von Willebrand Factor (vWF) in plasma and platelets is described. The method is based upon: 1) Separation of the vWF multimers by SDS-agarose electrophoresis, 2) Subsequent blotting of the vWF multimers onto nitrocellulose, 3) Immunolocalization and visualization of the vWF pattern by the sequential incubation of the blot with a) primary vWF antiserum, b) peroxidase- or beta-galactosidase-conjugated secondary antibodies and a relevant chromogenic substrate.

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Plasminogen-Plasmin System IX. Specific Binding of Tranexamic Acid to Plasmin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647851 ◽

1975 ◽

Vol 33 (03) ◽

pp. 573-585 ◽

Cited By ~ 20

Author(s):

Masahiro Iwamoto

Keyword(s):

Tranexamic Acid ◽

Affinity Chromatography ◽

Dissociation Constant ◽

Factor Xiii ◽

Specific Binding ◽

The Other ◽

Inhibitory Mechanism ◽

Binding Studies ◽

Similar Affinity ◽

Fibrin Structure

SummaryInteractions between tranexamic acid and protein were studied in respect of the antifibrinolytic actions of tranexamic acid. Tranexamic acid did neither show any interaction with fibrinogen or fibrin, nor was incorporated into cross-linked fibrin structure by the action of factor XIII. On the other hand, tranexamic acid bound to human plasmin with a dissociation constant of 3.5 × 10−5 M, which was very close to the inhibition constant (3.6 × 10−5 M) for this compound in inhibiting plasmin-induced fibrinolysis. The binding site of tranexamic acid on plasmin was not the catalytic site of plasmin, because TLCK-blocked plasmin also showed a similar affinity to tranexamic acid (the dissociation constant, 2.9–4.8 × 10−5 M).In the binding studies with the highly purified plasminogen and TLCK-plasmin preparations which were obtained by affinity chromatography on lysine-substituted Sepharose, the molar binding ratio was shown to be 1.5–1.6 moles tranexamic acid per one mole protein.On the basis of these and other findings, a model for the inhibitory mechanism of tranexamic acid is presented.

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Concanavalin A-induced Aggregation of Human Blood Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647887 ◽

1975 ◽

Vol 33 (02) ◽

pp. 354-360 ◽

Cited By ~ 4

Author(s):

Heinrich Patscheke ◽

Reinhard Brossmer

Keyword(s):

Concanavalin A ◽

Binding Capacity ◽

Specific Binding ◽

Blood Platelets ◽

Residual Effect ◽

Independent Effect ◽

Con A ◽

Experimental Conditions ◽

Main Effect ◽

Induced Aggregation

SummaryConcanavalin A (CON A) causes platelets to aggregate. A Ca++-independent effect of CON A could be separated from a main effect which depends on Ca++. The main effect probably is a consequence of the CON A-induced platelet release reaction and therefore is platelet-specific. The weak residual effect observed in the presence of Na2EDTA may be due to a similar mechanism as has been demonstrated for CON A-induced aggregations of several other normal and malignant transformed animal cells.Na2EDTA did not inhibit the carbohydrate-specific binding capacity of CON A. Therefore, Na2EDTA appears not to demineralize the CON A molecules under these experimental conditions.α-methyl-D-glucoside inhibits the Ca++-independent as well as the Ca++-dependent effect of CON A.Pretreatment by neuraminidase stimulated the platelet aggregation induced by CON A. It is possible that removal of terminal sialic acid residues makes additional receptors accessible for the binding of CON A.

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