Analysis of Antibody–Antigen Interactions in Mixtures Containing Reactive and Nonreactive Components Using Size-Exclusion High-Performance (Pressure) Liquid Chromatography

Sanny, Charles G.; Price, Joseph A.

doi:10.1006/abio.2001.5204

Cited by 7 publications

(4 citation statements)

References 13 publications

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“…The fraction of therapeutic molecules present in the antivenom preparation ([Ab ther ]) can be calculated using the Equation (Ab ther ) = (ED 50 /(M ave toxins))/((Ab)/M Ab ), where (M ave toxins) is the average molecular mass of the venom toxins, (Ab) is the total protein concentration (mg/mL) of the antivenom preparation, and M Ab is the molecular mass of the active antivenom’s molecules (158 kDa for IgG, 100 kDa for (F(ab’) 2 , 50 kDa for Fab). Here, analysis of size-exclusion HPLC (SEC) elution profiles of increasing venom:antivenom ratio mixtures [37,38,39,40] have been applied to quantify stable (EchiTAb-Plus-ICP ® antibody-( B. arietans toxin)) complexes. SE-HPLC elution profiles are mechanistically easy to interpret and mathematically modeled using dose-response functions based on the law of mass action.…”

Section: Resultsmentioning

confidence: 99%

“…To fulfill these recommendations, the second generation antivenomics protocol has been updated to include the determination of the maximal binding capacity of the immunoaffinity column for the different toxins of a venom and the quantification of the fraction of toxin-specific antibodies present in the antivenom. SE-HPLC elution profile analysis has been previously used as a rapid means of separating and quantifying stable antibody (Ab)-antigen (Ag) complexes from unbound reactants [38,39,40]. SE-HPLC has the advantage over other chromatographic methods in that it can be performed under conditions that preserve the native structure and binding characteristics of venoms and antivenoms.…”

Section: Concluding Remarks and Perspectivesmentioning

confidence: 99%

See 1 more Smart Citation

Third Generation Antivenomics: Pushing the Limits of the In Vitro Preclinical Assessment of Antivenoms

Plá

Rodríguez

Calvete

2017

Toxins

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Second generation antivenomics is a translational venomics approach designed to complement in vivo preclinical neutralization assays. It provides qualitative and quantitative information on the set of homologous and heterologous venom proteins presenting antivenom-recognized epitopes and those exhibiting impaired immunoreactivity. In a situation of worrying antivenom shortage in many tropical and sub-tropical regions with high snakebite mortality and morbidity rates, such knowledge has the potential to facilitate the optimal deployment of currently existing antivenoms and to aid in the rational design of novel broad specificity antidotes. The aim of the present work was to expand the analytical capability of the immunoaffinity second-generation antivenomics platform, endowing it with the ability to determine the maximal binding capacity of an antivenom toward the different toxins present in a venom, and to quantify the fraction of venom-specific antibodies present in a given antivenom. The application of this new platform, termed third generation (3G) antivenomics, in the preclinical evaluation of antivenoms is illustrated in this paper for the case of antivenom EchiTAb-Plus-ICP® reactivity towards the toxins of homologous (B. arietans) and heterologous (N. melanoleuca) venoms.

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Section: Resultsmentioning

confidence: 99%

Section: Concluding Remarks and Perspectivesmentioning

confidence: 99%

Third Generation Antivenomics: Pushing the Limits of the In Vitro Preclinical Assessment of Antivenoms

Plá

Rodríguez

Calvete

2017

Toxins

View full text Add to dashboard Cite

show abstract

“…Although several elegant techniques have been developed for the study of antigen-antibody/peptide-protein interactions, such as surface plasmon resonance [1][2][3][4][5], ELISA [6][7][8][9], isothermal calorimetry [10,11], and microarrays [12][13][14][15][16], high-performance size-exclusion chromatography [17][18][19][20] etc., they require complex and expensive instruments, hindering ease of operation and desired separation. Therefore, a simple and rapid method is urgently needed to effectively Article Related Abbreviations: FL, fluorescence detection; FAM-DYKD, FAM-DYKDHDGDYKDHDIDYKDDDDK; M2, monoclonal anti-FLAG M2 antibody detect and monitor the binding process between antibodies and peptides.…”

Section: Introductionmentioning

confidence: 99%

A novel in‐capillary assay for dynamically monitoring fast binding between antibody and peptides using CE

Wang

Qiu

You

et al. 2018

J of Separation Science

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Nowadays, despite numerous techniques available for the detection of antibody–peptide binding, sensitivity and detection limit inflow still remains a major challenge. Herein, we report a strategy for the detection of binding inflow of monoclonal anti‐FLAG M2 antibody and 5,6‐carboxyfluorescein‐labeled FLAG tag in capillary. Antibody and peptides were sequentially injected into the capillary where they mixed and bound together. Capillary electrophoresis with fluorescence detection was employed to monitor the binding process, and the results showed that the efficacy of the antibody–peptide binding in capillary (in‐capillary assay) is affected by the stoichiometry. Compared to the out‐capillary assay, this novel assay showed different KD values and required a very short detection time, thus showing great potential for rapid detection as well as other applications. Additionally, our novel assay can be used to investigate the stability of the antibody–peptide complex. The addition of excess DYKDDDDK or TAMRA‐DYKDDDDK, with similar binding priorities with M2, can leads to dissociation of the complex with a two‐step mechanism including dissociation and association. We believed that our developed method can be extended to monitor wide range of other biomolecule interactions.

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“…These methods often require immobilized antigen or antibody with operations that are relatively complex and involved. Others take advantage of high‐performance size‐exclusion chromatography (HPSEC) to purify antigens and study antigen‐antibody complexes . It is, however, difficult to distinguish between monovalent and multivalent complexes due to their similarities in molecular mass, shape, and surface hydrophobicity.…”

Section: Introductionmentioning

confidence: 99%

Investigation of multivalent interactions between conjugate of quantum dots with c‐Myc peptide tag and the anti‐c‐Myc antibody by capillary electrophoresis with fluorescence detection

Wang

Yang

Liu

et al. 2016

J of Separation Science

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Herein, we report an assay for detecting the binding of a multivalent peptide and antibody within a capillary with the use of fluorescence coupled capillary electrophoresis. Quantum dots and a c-Myc tag containing peptide EQKLISEEDLG H were injected sequentially and formed a multivalent quantum dot-EQKLISEEDLG H assembly within the capillary. The efficiency of the quantum dot-peptide self-assembly was affected by the peptide/quantum dot molar ratio, sampling time, and interval time. Finally, the binding of the monoclonal anti-c-Myc antibody and the multivalent quantum dot-EQKLISEEDLG H ligand was studied using an in-capillary assay. The microscopic dissociation constant for the self-assembly of monoclonal anti-c-Myc antibody and quantum dot-EQKLISEEDLG H was determined to be 14.1 μM with a stoichiometry of the peptide-antibody complex of 1.7 determined after fitting this to the Hill equation. This method can be further extended to detect a wide range of biomolecule-biomolecule binding interactions.

show abstract

Analysis of Antibody–Antigen Interactions in Mixtures Containing Reactive and Nonreactive Components Using Size-Exclusion High-Performance (Pressure) Liquid Chromatography

Cited by 7 publications

References 13 publications

Third Generation Antivenomics: Pushing the Limits of the In Vitro Preclinical Assessment of Antivenoms

Third Generation Antivenomics: Pushing the Limits of the In Vitro Preclinical Assessment of Antivenoms

A novel in‐capillary assay for dynamically monitoring fast binding between antibody and peptides using CE

Investigation of multivalent interactions between conjugate of quantum dots with c‐Myc peptide tag and the anti‐c‐Myc antibody by capillary electrophoresis with fluorescence detection

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