Sensitive assay design for detection of anti-drug antibodies to biotherapeutics that lack an immunoglobulin Fc domain

AbstractToday the evaluation of unwanted immunogenicity is a key component in the clinical safety evaluation of new biotherapeutic drugs and macromolecular delivery strategies. However, the evolving structural complexity in contemporary biotherapeutics creates a need for on-going innovation in assay designs for reliable detection of anti-drug antibodies, especially for biotherapeutics that may not be well-suited for testing by a bridging assay. We, therefore, initiated systematic optimization of the direct binding assay to adapt it for routine use in regulatory-compliant assays of serum anti-drug antibodies. Accordingly, we first prepared a SULFO-TAG labeled conjugate of recombinant Protein-A/G to create a sensitive electrochemiluminescent secondary detection reagent with broad reactivity to antibodies across many species. Secondly, we evaluated candidate blocker-diluents to identify ones producing the highest signal-to-noise response ratios. Lastly, we introduced use of the ratio of signal responses in biotherapeutic-coated and uncoated wells as a data transformation strategy to identify biological outliers. This alternative data normalization approach improved normality, reduced skewness, and facilitated application of a parametric screening cut point. We believe the optimized direct binding assay design employing SULFO-TAG labeled Protein-A/G represents a useful analytical design for detecting serum ADA to biotherapeutics that lack an immunoglobulin Fc domain.

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Measurement of platelet-associated IgG in animal models of immune and nonimmune thrombocytopenia

Blood ◽

10.1182/blood.v69.5.1294.1294 ◽

1987 ◽

Vol 69 (5) ◽

pp. 1294-1299 ◽

Cited By ~ 18

Author(s):

J Arnott ◽

P Horsewood ◽

JG Kelton

Keyword(s):

Platelet Count ◽

Immune Thrombocytopenia ◽

Binding Assay ◽

Protein A ◽

Rabbit Model ◽

Adenosine Diphosphate ◽

Staphylococcal Protein A ◽

Specific Finding ◽

Direct Binding ◽

Survival Studies

Abstract Platelet-associated IgG (PAIgG) is elevated in idiopathic thrombocytopenic purpura (ITP), but it also is elevated in other thrombocytopenic disorders traditionally considered to be nonimmune. Consequently it is possible that elevated PAIgG is a nonspecific finding secondary to thrombocytopenia. To study this issue we developed a rabbit model of immune and nonimmune mediated thrombocytopenia. The mechanism of the thrombocytopenia was validated by platelet survival studies. Immune thrombocytopenia was produced by injection of antirabbit platelet serum that was raised in guinea pigs. Nonimmune aregenerative thrombocytopenia was produced by irradiation of the animals; nonimmune consumptive thrombocytopenia was produced by injection of adenosine diphosphate (ADP). PAIgG was measured in a direct binding assay using 125I-labeled staphylococcal protein A (SpA). Washed platelets from normal, nonthrombocytopenic rabbits bound an average of 81 molecules of SpA per platelet (81 +/- 168, mean +/- 2 SD, n = 39). Infusion of the antiplatelet antiserum produced thrombocytopenia with a rise in PAIgG that was closely correlated with the level of PAIgG (r = 0.86, n = 12). The thrombocytopenia was consumptive, as shown by a very short platelet life span using 111In- labeled platelets. In contrast, both nonimmune thrombocytopenic states resulted in an equal or greater drop in the platelet count but no change in the level of PAIgG. The animals with aregenerative thrombocytopenia had normal or only moderately reduced platelet life spans; however, in every animal the level of PAIgG was not different from the nonthrombocytopenic controls, irrespective of the platelet count. Similarly, the level of PAIgG was unchanged in those rabbits with nonimmune consumptive thrombocytopenia following infusion of ADP (82 +/- 55 molecules of SpA per platelet, mean +/- SD, n = 6). These studies indicate that elevated PAIgG is a specific finding of immune thrombocytopenia and is not secondary to thrombocytopenia itself. Indirectly these results support our hypothesis that immune mechanisms contribute to more thrombocytopenic disorders than was once thought likely.

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Measurement of platelet-associated IgG in animal models of immune and nonimmune thrombocytopenia

Blood ◽

10.1182/blood.v69.5.1294.bloodjournal6951294 ◽

1987 ◽

Vol 69 (5) ◽

pp. 1294-1299 ◽

Cited By ~ 1

Author(s):

J Arnott ◽

P Horsewood ◽

JG Kelton

Keyword(s):

Platelet Count ◽

Immune Thrombocytopenia ◽

Binding Assay ◽

Protein A ◽

Rabbit Model ◽

Adenosine Diphosphate ◽

Staphylococcal Protein A ◽

Specific Finding ◽

Direct Binding ◽

Survival Studies

Platelet-associated IgG (PAIgG) is elevated in idiopathic thrombocytopenic purpura (ITP), but it also is elevated in other thrombocytopenic disorders traditionally considered to be nonimmune. Consequently it is possible that elevated PAIgG is a nonspecific finding secondary to thrombocytopenia. To study this issue we developed a rabbit model of immune and nonimmune mediated thrombocytopenia. The mechanism of the thrombocytopenia was validated by platelet survival studies. Immune thrombocytopenia was produced by injection of antirabbit platelet serum that was raised in guinea pigs. Nonimmune aregenerative thrombocytopenia was produced by irradiation of the animals; nonimmune consumptive thrombocytopenia was produced by injection of adenosine diphosphate (ADP). PAIgG was measured in a direct binding assay using 125I-labeled staphylococcal protein A (SpA). Washed platelets from normal, nonthrombocytopenic rabbits bound an average of 81 molecules of SpA per platelet (81 +/- 168, mean +/- 2 SD, n = 39). Infusion of the antiplatelet antiserum produced thrombocytopenia with a rise in PAIgG that was closely correlated with the level of PAIgG (r = 0.86, n = 12). The thrombocytopenia was consumptive, as shown by a very short platelet life span using 111In- labeled platelets. In contrast, both nonimmune thrombocytopenic states resulted in an equal or greater drop in the platelet count but no change in the level of PAIgG. The animals with aregenerative thrombocytopenia had normal or only moderately reduced platelet life spans; however, in every animal the level of PAIgG was not different from the nonthrombocytopenic controls, irrespective of the platelet count. Similarly, the level of PAIgG was unchanged in those rabbits with nonimmune consumptive thrombocytopenia following infusion of ADP (82 +/- 55 molecules of SpA per platelet, mean +/- SD, n = 6). These studies indicate that elevated PAIgG is a specific finding of immune thrombocytopenia and is not secondary to thrombocytopenia itself. Indirectly these results support our hypothesis that immune mechanisms contribute to more thrombocytopenic disorders than was once thought likely.

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Synthetic Strategies for the Modification of Diclofenac

Synlett ◽

10.1055/s-0036-1588858 ◽

2017 ◽

Vol 28 (15) ◽

pp. 1984-1989 ◽

Cited By ~ 1

Author(s):

Rudolf Schneider ◽

Stephan Schmidt ◽

Sven Hanelt ◽

Carsten Canitz ◽

Holger Hoffmann ◽

...

Keyword(s):

Amino Acid ◽

Solid Phase ◽

Free Acid ◽

Protein A ◽

Sensor Applications ◽

Protein Conjugates ◽

Lysine Residues ◽

Sufficient Distance ◽

Direct Binding

For many heterogeneous sensor applications as well as the synthesis of hapten antigens to produce antibodies, protein conjugates of the target substance are essential. A requirement is that the target substance already offers or is modified to contain a functionality that allows for coupling to a protein, that is, an amino acid residue. Ideally, to avoid shielding of the compound by the carrier protein, a sufficient distance to the protein surface should be provided. With its carboxyl function diclofenac (DCF) allows for direct binding to lysine residues after in situ synthesis of the NHS ester. One problem is that diclofenac as free acid tends to autocondensation, which results in low yields. Here we describe the ‘insertion’ of a C6 spacer via synthesis of the amide with 6-aminohexanoic acid. To carry out the reaction in solution, first the methyl ester of the amino acid had to be produced. Due to otherwise low yields and large cleaning efforts, solid-phase synthesis on Fmoc Ahx Wang resin is recommended. The crude product is mainly contaminated by cleavage products from the resin which were removed by chromatography. The structure of the highly pure hapten was completely determined by nuclear magnetic resonance (NMR) spectroscopy.

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Bordetella bronchiseptica Adherence to Cilia Is Mediated by Multiple Adhesin Factors and Blocked by Surfactant Protein A

Infection and Immunity ◽

10.1128/iai.73.6.3618-3626.2005 ◽

2005 ◽

Vol 73 (6) ◽

pp. 3618-3626 ◽

Cited By ~ 29

Author(s):

Jessica A. Edwards ◽

Nathan A. Groathouse ◽

Scott Boitano

Keyword(s):

Binding Assay ◽

Binding Capacity ◽

Protein A ◽

Upper Airway ◽

Surfactant Protein ◽

Bordetella Bronchiseptica ◽

Surfactant Protein A ◽

Dermonecrotic Toxin ◽

Bacterial Adhesins ◽

Tracheal Epithelial

ABSTRACT In the virulent state (Bvg+), Bordetella bronchiseptica expresses adhesins and toxins that mediate adherence to the upper airway epithelium, an essential early step in pathogenesis. In this study, we used a rabbit tracheal epithelial cell binding assay to test how specific host or pathogen factors contribute to ciliary binding. The host antimicrobial agent surfactant protein A (SP-A) effectively reduced ciliary binding by Bvg+ B. bronchiseptica. To evaluate the relative contributions of bacterial adhesins and toxins to ciliary binding, we used mutant strains of B. bronchiseptica in the binding assay. When compared to Bvg+ or Bvg− phase-locked B. bronchiseptica strains, single-knockout strains lacking one of the known adhesins (filamentous hemagglutinin, pertactin, or fimbriae) displayed an intermediate ciliary binding capacity throughout the coincubation. A B. bronchiseptica strain deficient in adenylate cyclase-hemolysin toxin also displayed an intermediate level of adherence between Bvg+ and Bvg− strains and had the lowest ciliary affinity of any of the Bvg+ phase strains tested. A B. bronchiseptica strain that was missing dermonecrotic toxin also displayed intermediate binding; however, this strain displayed ciliary binding significantly higher than most of the adhesin knockouts tested. Taken together, these findings suggest that virulent-state B. bronchiseptica expresses multiple adhesins with overlapping contributions to ciliary adhesion and that host production of SP-A can provide innate immunity by blocking bacterial adherence to the ciliated epithelium.

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Curcumin Reduces the Motility of Salmonella enterica Serovar Typhimurium by Binding to the Flagella, Thereby Leading to Flagellar Fragility and Shedding

Journal of Bacteriology ◽

10.1128/jb.00092-16 ◽

2016 ◽

Vol 198 (13) ◽

pp. 1798-1811 ◽

Cited By ~ 16

Author(s):

Sandhya Amol Marathe ◽

Arjun Balakrishnan ◽

Vidya Devi Negi ◽

Deepika Sakorey ◽

Nagasuma Chandra ◽

...

Keyword(s):

Salmonella Enterica ◽

Binding Assay ◽

Protein A ◽

Intestinal Barrier ◽

Site Directed Mutagenesis ◽

Content Type ◽

Serovar Typhimurium ◽

Flagellar Filament ◽

Fluorescence Binding ◽

Fluorescence Binding Assay

ABSTRACTOne of the important virulence properties of the pathogen is its ability to travel to a favorable environment, cross the viscous mucus barrier (intestinal barrier for enteric pathogens), and reach the epithelia to initiate pathogenesis with the help of an appendage, like flagella. Nonetheless, flagella can act as an “Achilles heel,” revealing the pathogen's presence to the host through the stimulation of innate and adaptive immune responses. We assessed whether curcumin, a dietary polyphenol, could alter the motility ofSalmonella, a foodborne pathogen. It reduced the motility ofSalmonella entericaserovar Typhimurium by shortening the length of the flagellar filament (from ∼8 μm to ∼5 μm) and decreasing its density (4 or 5 flagella/bacterium instead of 8 or 9 flagella/bacterium). Upon curcumin treatment, the percentage of flagellated bacteria declined from ∼84% to 59%. However, no change was detected in the expression of the flagellin gene and protein. A fluorescence binding assay demonstrated binding of curcumin to the flagellar filament. This might make the filament fragile, breaking it into smaller fragments. Computational analysis predicted the binding of curcumin, its analogues, and its degraded products to a flagellin molecule at an interface between domains D1 and D2. Site-directed mutagenesis and a fluorescence binding assay confirmed the binding of curcumin to flagellin at residues ASN120, ASP123, ASN163, SER164, ASN173, and GLN175.IMPORTANCEThis work, to our knowledge the first report of its kind, examines how curcumin targets flagellar density and affects the pathogenesis of bacteria. We found that curcumin does not affect any of the flagellar synthesis genes. Instead, it binds to the flagellum and makes it fragile. It increases the torsional stress on the flagellar filament that then breaks, leaving fewer flagella around the bacteria. Flagella, which are crucial ligands for Toll-like receptor 5, are some of the most important appendages ofSalmonella. Curcumin is an important component of turmeric, which is a major spice used in Asian cooking. The loss of flagella can, in turn, change the pathogenesis of bacteria, making them more robust and fit in the host.

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