Easy assessment of the avidity of polyclonal allergen-specific serum antibodies
CLINICAL AND EXPERIMENTAL ALLERGY(2024)
摘要
Introduction: Allergen-specific IgE-blocking IgG antibodies contribute to successful allergen immunotherapy (AIT), however, not much is known about their affinity. Since affinity measurements of polyclonal antibodies in serum are technically challenging we evaluated the applicability of acidic disruption of antibody-allergen complexes by a modified ELISA protocol with monoclonal antibodies (mAbs) specific for the relevant major allergens Betv1 and Mald1. Then, AIT-induced blocking and non-blocking Mald1-specific antibodies in sera from individuals with or without reduced apple allergy were compared.Methods: After testing their pH stability coated recombinant allergens were incubated with (i) mAbs diluted in PBS or human serum and (ii) sera from individuals after sublingual administration of Mald1 or Betv1 for 16 weeks. Immune complexes were exposed to buffers in the pH range of 6.4-3.4 and residual antibodies were measured. Avidity indexes (AI), defined as the pH removing 50% of antibodies, were compared to the dissociation constants (K-D) of mAbs determined by surface plasmon resonance.Results: The selected pH range was applicable to disrupt allergen complexes with highly affine mAbs without compromising allergen integrity. AIs of mAbs accorded with K-D values and were unaffected by epitope specificity or the presence of serum proteins. The inter-assay variability was <4% CV. Protective Mald1-specific IgG antibodies from individuals with reduced apple allergy showed a higher collective binding strength than that of the non-protective antibodies of individuals without reduced apple allergy.Conclusion: Acidic disruption of allergen-antibody complexes may be used to estimate the net-binding force of polyclonal serum antibodies and eases the investigation of affinity-related research questions in AIT.
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关键词
acidic elution,affinity,allergen immunotherapy,antibody,avidity
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