title: Calculation of the affinity constant KASS for solid phase antigen. A model system using monoclonal antibodies against the cat allergen Fel d I.
abstract: In this report a procedure is described to calculate the affinity constant of an antibody for solid-phase Ag. KSP (KASS solid phase) was defined as the reciprocal of the concentration of Ag required for half saturation of the Ab-binding sites, extrapolated to an infinitely small concentration of Ab (semi-saturation plot). Using this procedure, the affinity of IgE antibodies can be measured without interference from 'invisible' IgG antibodies. As a model system, mAbs against the cat allergen Fel d I were used. Serial dilutions of Fel d I-Sepharose were incubated with serial dilutions of mAb Fd1a and Fd1b, with or without rabbit antibodies as 'invisible' antibodies. The Ab-binding capacity of Sepharose-coupled Fel d I was low: 4.15% and 2.13% of the expected value for mAb Fd1a and Fd1b, respectively, and this must be taken into account when calculating KSP. The K values of mAb Fd1a and Fd1b, calculated from the gamma axis intercept of the semi-saturation plot, were 85 (pmol/test)-1 and 65 (pmol/test)-1 respectively. Using the semi-saturation plot, KSP of mAb Fd1a was not affected by the presence of rabbit antibodies against Fel d I, confirming the applicability of the procedure for measuring the KSP of IgE in patient sera. For one cat-allergic patient the KSP of IgE and IgG4 for Fel d I were calculated and found to be 62 (pmol/test)-1 and 147 (pmol/test)-1 for IgE and IgG4 respectively.
authors.affiliation: Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
authors.forename: F J
authors.initials: FJ
authors.lastname: van Milligen
authors.forename: L
authors.initials: L
authors.lastname: van Etten
authors.forename: R C
authors.initials: RC
authors.lastname: Aalberse
mesh_terms: D000485:Allergens / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D002415:Cats; D006023:Glycoproteins; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology; D007136:Immunoglobulins / Q000032:analysis*; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D011852:Radioallergosorbent Test; D011863:Radioimmunoassay; D012685:Sepharose
chemical_list: D000485:Allergens; D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D007136:Immunoglobulins; D012685:Sepharose; C059296:Fel d 1 protein, Felis domesticus