Antibody affinity measurements

Azimzadeh, A.; Regenmortel, M.H.V. Van

doi:10.1002/jmr.300030304

Cited by 44 publications

(22 citation statements)

References 66 publications

(51 reference statements)

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“…Various surface effects have been considered as possible explanations for differences in affinities in solid-phase assays [12][13][14]. Coating of protein on microtitre plate wells may change conformation or even lead to denaturation [15].…”

Section: Discussionmentioning

confidence: 99%

Demonstration of non‐linear detection in ELISA resulting in up to 1000‐fold too high affinities of fibronogen binding to integrin αIIbβ3

Tangemann

Engel

1995

FEBS Letters

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To clarify the question as to why different solid-phase assays yield different results in terms of interaction strength, we used fihrinogen binding to immobilized aIIbfl3 integrin as a test system. A classical 'three step' enzyme-linked (ELISA), a 'two step' biotin enzyme-linked streptavidin and a 'one step' radioligand assay were compared under otherwise identical conditions. Only the last assay yielded binding constants comparable to earlier data by total internal reflection fluorescence microscopy while the other assays yielded apparent binding constants 5-to 1000-fold too high. These effects are explained by non-linearity of detection signals.

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Section: Discussionmentioning

confidence: 99%

Demonstration of non‐linear detection in ELISA resulting in up to 1000‐fold too high affinities of fibronogen binding to integrin αIIbβ3

Tangemann

Engel

1995

FEBS Letters

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“…β 3 has been used as a measure of affinity (Azimzadeh and Rongenmortel, 1990) and reciprocal of β 3 was used as the apparent association constant ( K a ). The molecular weight of d‐dimer was assumed to be 228 kD in the calculations.…”

Section: Resultsmentioning

confidence: 99%

Application of Antibody and Fluorophore‐Derivatized Liposomes to Heterogeneous Immunoassays for D‐dimer

Singh

Kilpatrick

Carbonell

1996

Biotechnology Progress

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Small unilamellar liposomes comprised of cholesterol and phospholipids, in which one of the lipids is labeled with a fluorophore, have been covalently functionalized with antibodies. The liposomes were conjugated with thousands of fluorescein molecules and 10-20 monoclonal antibodies per liposome. These bifunctional liposomes were used in a direct (sandwich-type) immunoassay for the detection of thromboembolic disorders by assaying for d-dimer. D-dimer is the final and the smallest proteolytic product in the degradation of cross-linked fibrin by the plasma protein plasmin. The immunoassay using liposomes was compared to a conventional immunoassay that uses a fluor-antibody conjugate. The liposomes, by virtue of having thousands of fluorophores coupled to one liposome in contrast to one or a few reporter molecules in the conventional fluor-antibody conjugate, performed better on two counts: (1) they lowered the detection limit by a factor of 120 and (2) they provided a 1 order of magnitude amplification in signal. The minimum detectable concentration (MDC) of d-dimer was 5.6 ng/mL with the liposomal assay as compared to an MDC of 674 ng/mL with conventional fluor-antibody conjugate. The results of fluorescence assays were also compared with the results obtained by Singh et al. (Biotechnol. Prog. 1995, 11, 333-341) in an enzyme immunoassay developed using liposomes. These results demonstrate the potential of liposomes in lowering detection limits and increasing the sensitivity of immunoassays.

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“…The strength of the affinity is represented by the affinity constant. Equilibrium dialysis is the classical method used to determine the affinity constant, but it is only suitable for small molecule antigen and antibody, and the operations involved are quite complicated [18]. The solid phase non-competitive ELISA method for the determination of affinity constants is not limited by the antigen size and it is easy to operate, but some scholars believed that antigen-antibody reaction kinetics would be changed and the application of the Law of Mass Action would be limited, because the antigen-antibody reaction is transferred from a liquid phase to the solid-liquid interface, caused by the antigen has been coated in microtitre plate [19].…”

Section: Discussionmentioning

confidence: 99%

Screening a Phage Display Library for Two Novel OmpU-Binding Peptides with Adhesion Antagonistic Activity against Vibrio mimicus

et al. 2016

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Vibrio mimicus is a pathogen that causes ascites disease in fish. We have previously demonstrated that the outer membrane protein U (OmpU) is an important adhesin in V. mimicus. Here eight specific OmpU-binding phage clones, which presented three different OmpU-binding peptides (designated P1, P2, P3), were screened from a commercially available phage displayed 12-mer peptide library using rOmpU protein as target. Then, synthetic OmpU-binding peptides were measured for their adhesion antagonistic activity and binding affinity via adhesion inhibition test and non-competitive ELISA, respectively. The results showed that after co-incubated with the mixture of rOmpU and P3, visible green fluorescence could be observed on the epithelioma papulosum cyprinidi (EPC) cells surface; while the EPC cells co-incubated with the mixture of rOmpU and P1/P2 exhibited little green fluorescence. The average adhesion number of V. mimicus 04–14 isolate before and after treatment with peptide was 21.4 ± 1.5, 20.8 ± 0.8 (irrelevant peptide), 20.2 ± 0.5 (P3), 5.1 ± 0.7 (P1) and 3.4 ± 0.8 (P2), respectively. There was a significant decrease in the adhesive level of 04–14 isolate treated with P1/ P2 compared to the untreated isolate (p<0.01). The affinity constants of P1 and P2 were (6.17 ± 0.19) × 108 L/mol and (1.24 ± 0.56) × 109 L/mol, respectively. Furthermore, protective effects of P1 and P2 on grass carps challenged with V. mimicus were preliminary detected. It was found there was delayed death of fish in the groups treated with P1/P2, and the survival rate of challenged fish improved with the increase of the dose of adhesion antagonistic peptide. Taken together, two novel OmpU-binding peptides, which possessed adhesion antagonistic activity, high affinity and a certain degree of antibacterial activity against V. mimicus, were screened and identified.

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Antibody affinity measurements

Cited by 44 publications

References 66 publications

Demonstration of non‐linear detection in ELISA resulting in up to 1000‐fold too high affinities of fibronogen binding to integrin αIIbβ3

Demonstration of non‐linear detection in ELISA resulting in up to 1000‐fold too high affinities of fibronogen binding to integrin αIIbβ3

Application of Antibody and Fluorophore‐Derivatized Liposomes to Heterogeneous Immunoassays for D‐dimer

Screening a Phage Display Library for Two Novel OmpU-Binding Peptides with Adhesion Antagonistic Activity against Vibrio mimicus

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