Deconvolution of antibody affinities and concentrations by non-linear regression analysis of competitive ELISA data

Stevens, Fred J.; Bobrovnik, S. A.

doi:10.1016/j.jim.2007.08.007

Cited by 19 publications

(29 citation statements)

References 25 publications

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“…of FVIII-binding antibodies, differentiated for IgG subclasses 1-4 and Ig isotypes IgM and IgA, were determined using a competition-based affinity ELISA platform, as described by Hofbauer et al 11 Briefly, the affinity assessment was based on the availability of antibodies for binding to FVIII-coated ELISA plates after competition with FVIII of different molar concentrations in solution. Data for apparent affinities (K A [M 21 ]) were derived from nonlinear regression modeling of competition ELISA DOD, as described by Stevens et al 12 Antibodies without conclusive apparent affinity were considered unspecific and excluded from analysis. Further details of the test principle of the competitionbased affinity ELISA platform are provided in supplemental Methods.…”

Section: Immunologic Analysismentioning

confidence: 99%

Anti–factor VIII IgA as a potential marker of poor prognosis in acquired hemophilia A: results from the GTH-AH 01/2010 study

Tiede

Hofbauer²,

Werwitzke

et al. 2016

Blood

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Section: Immunologic Analysismentioning

confidence: 99%

Anti–factor VIII IgA as a potential marker of poor prognosis in acquired hemophilia A: results from the GTH-AH 01/2010 study

Tiede

Hofbauer²,

Werwitzke

et al. 2016

Blood

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“…Traditionally, neither label-free biosensor technologies nor competitive immunoassay technologies have allowed the assessment of low-affinity antibodies in a mixture with high-affinity antibodies when both antibody populations recognize the same antigen. Bobrovnik et al 12 and Stevens et al 13 introduced a novel approach that combines competition-based enzyme-linked immunosorbent assay (ELISA) technology with nonlinear regression modeling of the ELISA graphs to facilitate the analysis of 2 antibody clusters with low and high apparent affinity in the same sample. We applied this approach to the analysis of FVIII-specific antibodies present in human plasma samples and established a competition-based ELISA platform that combines the advantages of low sample consumption, no need for antibody purification, the potential to assess apparent affinities of low-affinity and high-affinity antibody clusters in the same sample, and the ability to specify apparent affinities of individual antibody isotypes and IgG subclasses.…”

Section: Introductionmentioning

confidence: 99%

Affinity of FVIII-specific antibodies reveals major differences between neutralizing and nonneutralizing antibodies in humans

Hofbauer

Whelan

Hirschler

et al. 2015

Blood

113

121

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Key Points• A new technology is presented to assess apparent affinities of FVIII-specific antibodies, differentiated for isotypes and IgG subclasses.• Affinities of FVIII-specific antibodies in patients with FVIII inhibitors are up to 100-fold higher than in patients without inhibitors.Recently, we reported that distinct immunoglobulin (Ig) isotypes and IgG subclasses of factor VIII (FVIII)-specific antibodies are found in different cohorts of patients with hemophilia A and in healthy individuals. Prompted by these findings, we further investigated the distinguishing properties among the different populations of FVIII-specific antibodies. We hypothesized that the affinity of antibodies would discriminate between the neutralizing and nonneutralizing antibodies found in different study cohorts. To test this idea, we established a competition-based enzyme-linked immunosorbent assay technology to assess the apparent affinities for each isotype and IgG subclass of FVIIIspecific antibodies without the need for antibody purification. We present a unique data set of apparent affinities of FVIII-specific antibodies found in healthy individuals, patients with congenital hemophilia A with and without FVIII inhibitors, and patients with acquired hemophilia A. Our data indicate that FVIII-specific antibodies found in patients with FVIII inhibitors have an up to 100-fold higher apparent affinity than that of antibodies found in patients without inhibitors and in healthy individuals. High-affinity FVIII-specific antibodies could be retrospectively detected in longitudinal samples of an individual patient with FVIII inhibitors 543 days before the first positive Bethesda assay. This finding suggests that these antibodies might serve as potential biomarkers for evolving FVIII inhibitor responses. (Blood. 2015;125(7):1180-1188

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“…In addition, these methods were improved by us [10], and a new method was proposed for estimating affinity of two antibodies, which are found in the studied mixture [11]. The use of our method is very important, if the affinities of two studied antibodies differ greatly from each other.…”

Section: Methodsmentioning

confidence: 99%

“…in TBS or in TBS + protamine. In addition, our method allows determining the relation between concentrations of high affinity and low affinity reagents in the studied samples [11].…”

Section: Methodsmentioning

confidence: 99%

“…For this purpose the curves of the dependences of free PRIGs concentration in solution with different concentration of antigen when the equilibrium of reaction is established, were compared with theoretical curves for similar interaction of the reagents according to equation (5) [11] and the quantities of both equilibrium constant for high and low affinity PRIGs were calculated by the method of linear regression using computer program Origin 9. The relations between concentrations of high and low affinity PRIGs in TBS or in TBS + protamine were also determined as relations between the values of A 01 и A 02 : ,…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Kinetic parameters of polyreactive immunoglobulins interaction with antigens in the presence of protamine

Bobrovnik¹,

Demchenko²,

Комісаренко³

2016

Ukr.Biochem.J

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The discovered earlier phenomenon of the enhancment of polyreactive immunoglobulines (PrIGs) [1,2], that PRIGs binding to antigens strongly depended (resulted in increase or decrease) on the properties of the incubation medium. For example, the presence in the incubation media either Tween 20 or 1-anilino-8-naphthalenesulfonate significantly inhibi ted PRIGs binding to antigens. In contrast, several proteins, which had positive charge at neutral pH in aqueous solutions, were able to increase PRIGs reactivity and stimulate PRIGs-antigen interac tion. Some of such proteins were salmon caviar protamine and hen egg lysozyme. As we have found, the protamine much stronger increased PRIGs interaction with antigens than lysozyme [1,2]. In addition, it was determined, that albeit Tween 20 weakened the PRIGs interaction with antigens. We could observe the opposite effect in the presence of protaminetogether with protamine Tween 20 increased the PRIGs binding to antigens more efficiently, than the same concentration of protamine in the absence of Tween 20.Since more detailed study of this effect could shed light on the details of the mechanism of PRIGs interaction with antigens, we decided to investigate how protamine optimal dozes influenced the rate constant of PRIGs binding to the immobilized on the plate antigen in the presence of Tween 20, as well as on the affinity of PRIGs binding to the antigen in solution. The results of this investigation are described in this paper. materials and methodsAntigen, PrIGs, and eLISA. We used bovine serum albumin (BSA) from Sigma, USA as an antigen. Protamine from the salmon caviar was from Sigma, USA. Mice normal serum was used as a pool of PRIGs. In our preliminary investigations it was shown that this serum had high level of PRIGs activi ty (not published).The quantity of immunoglobulins bound to the absorbed antigen on the plate in different conditions was determined by enzyme-linked immunosor bent assay (ELISA). For this purpose, the plates with PRIGs bound to immobilized antigen were incubated with goat anti-mouse IgG antibodies conjugated with peroxidase (Sigma, USA) during 60 min at 4 ºС. The unbound conjugates were carefully washed away, and peroxidase substrate -the solution of orthophenilen diamine (1 mg/ml) in 0.01 M phosphate buffer, pH 5.0 and 0.003% Н 2 О 2 solution were added .

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Deconvolution of antibody affinities and concentrations by non-linear regression analysis of competitive ELISA data

Cited by 19 publications

References 25 publications

Anti–factor VIII IgA as a potential marker of poor prognosis in acquired hemophilia A: results from the GTH-AH 01/2010 study

Anti–factor VIII IgA as a potential marker of poor prognosis in acquired hemophilia A: results from the GTH-AH 01/2010 study

Affinity of FVIII-specific antibodies reveals major differences between neutralizing and nonneutralizing antibodies in humans

Kinetic parameters of polyreactive immunoglobulins interaction with antigens in the presence of protamine

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