Direct measurement of antibody affinity distribution by Haften-inhibition enzyme immunoassay

Nieto, Alberto; Gayà, Antoni; Jansà, Margarida; Moreno, Carlos; Vives, Jordi

doi:10.1016/0161-5890(84)90070-1

Cited by 128 publications

(75 citation statements)

References 18 publications

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“…As indicated in Figure 1, these two peptides had different binding affinities to PK99H in the competitive ELISA assay. The apparent association constants (K,) were calculated from the 150 values as described by Nieto et al (1984). The IS0 values (peptide concentration required for 50% inhibition of PK99H binding to PAK pili) for both peptides showed that a 1,500-fold higher concentration of P A 0 peptide was required to attain the same in- …”

Section: Resultsmentioning

confidence: 99%

“…If a single alaninesubstituted peptide showed a decrease in its ability to inhibit the binding of PK99H to PAK pili, the side chain Table 1. K , is the apparent association constant of monoclonal antibody PK99H for the corresponding peptide analog, which is calculated by the formula KO = l/150 (Nieto et al, 1984).…”

Section: Pk99h Epitopementioning

confidence: 99%

“…The reaction was stopped by the addition of 125 pL of 4 mM sodium azide, and the absorbance at 405 nm was determined by using a Titertek Multiskan Plus MK I1 microplate reader (Flow Lab Inc., McLean, Virginia). The apparent association constant (K,) of the monoclonal antibody for each peptide analog can be calculated by the formula K, = 1/Zs0 as described by Nieto et al (1984). By utilizing the K, values of the peptide analogs (Ks) and the native sequence ( K N ) , one can determine the loss of binding free energy (A ( AG)) of each peptide analog as compared with the native peptide through the following procedure: A(AG) = RT ln(KN/Ks) (Bhattacharyya&Brewer, 1988).…”

Section: Competitive Elisamentioning

confidence: 99%

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Antigen‐antibody interactions: Elucidation of the epitope and strain‐specificity of a monoclonal antibody directed against the pilin protein adherence binding domain of Pseudomonas aeruginosa strain K

et al. 1992

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The C-terminal region of Pseudomonas aeruginosa strain K (PAK) pilin comprises both an epitope for the strainspecific monoclonal antibody PK99H, which blocks pilus-mediated adherence, and the adherence binding domain for buccal and tracheal epithelial cells. The PK99H epitope was located in sequence 134-140 (Asp-Glu-Gln-PheIle-Pro-Lys) by using a single alanine replacement analysis on the 17-residue synthetic peptide corresponding to the PAK C-terminal sequence 128-144. Indeed, a 7-residue peptide corresponding to this sequence was shown to have a similar binding affinity to that of the native conformationally constrained (disulfide bridged) 17-residue peptide. This epitope was found to contain two critical residues (PheI3' and LysI4O) and one nonessential residue ( G l r~'~~) .Interestingly, the peptide, Phe-Ile-Pro-Lys, which constitutes the four most important side chains for antibody binding did not bind to PK99H. It was of interest to investigate the structural basis of the strainspecificity of PK99H utilizing naturally occurring pilin sequences. Therefore, all different residues found in the sequence corresponding to the PK99H epitope of the four other strains (PAO, CD4, K122-4, and KB7) were substituted one at a time in the PAK sequence and the changes in binding affinity of these analogs to the antibody PK99H were determined by competitive ELISA. The strain-specificity of PK99H for strains PAO, K122-4, and KB7 can be explained by the accumulated sequence changes in these strains, and at least two amino acid changes were required to explain the strain-specificity of PK99H. Similarly, cross-reactivity of PK99H with CD4 can be explained by the fact that there was only one side chain responsible for decreasing binding affinity compared to the PAK sequence.

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Section: Resultsmentioning

confidence: 99%

Section: Pk99h Epitopementioning

confidence: 99%

Section: Competitive Elisamentioning

confidence: 99%

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Antigen‐antibody interactions: Elucidation of the epitope and strain‐specificity of a monoclonal antibody directed against the pilin protein adherence binding domain of Pseudomonas aeruginosa strain K

et al. 1992

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“…This was done by using high (22 pg/ml) and low (0.75 pg/ml) concentrations of IgG as precoat. In theory, higheravidity RF will selectively bind to the lower concentration of IgG (antibody excess), whereas RF of all affinities will bind to the higher concentration of precoat IgG (antigen excess) (10). For these observations of RF binding avidity in the ELISA to be valid, it was critical that IgGl and IgG3 coated the microtiter plates to a similar extent, using equivalent coating concentrations.…”

Section: Resultsmentioning

confidence: 99%

Differential reactivity of rheumatoid synovial cells and serum rheumatoid factors to human immunoglobulin g subclasses 1 and 3 and their CH3 domains in rheumatoid arthritis

Robbins

Benisek

Benjamini

et al. 1987

Arthritis & Rheumatism

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19S IgM rheumatoid factors (RF) are polyclonal autoantibodies that may play an important pathogenic role in sustaining inflammatory synovitis in rheumatoid arthritis (RA). RF in RA have reactivity for as‐yet‐uncharacterized antigenic determinants in IgG Fc. We hypothesized that qualitative differences might exist between some of these RF molecules, and that differences such as reactivity and affinity might characterize more pathogenic RF molecules. Previous observations in our laboratory indicate that RF produced by rheumatoid synovial cells (RSC) have greater reactivity with human IgG and IgG3 subclass, in contrast to serum RF, which has greater reactivity with rabbit IgG and human IgG1. These observations were made using a complement‐dependent RF plaque‐forming cell assay. The purpose of this study was to validate and extend those observations. Therefore, we examined the reactivity of RSC and serum RF with human and rabbit IgG and the reactivity and avidity of RSC‐RF for IgG1 and IgG3 molecules and Fab, F(ab')2, and pFc' fragments thereof in a solid‐phase enzyme immunoassay. In particular, we found: 1) RSC‐RF had at least twice as much reactivity with human IgG as with rabbit IgG; 2) serum RF had approximately equal reactivity with human and rabbit IgG; 3) RSC‐RF had greater reactivity and avidity for IgG3 and IgG3 pFc' than for IgG1; and 4) RSC‐RF was nonreactive with Fab or F(ab')2 from either IgG1 or IgG3. These results suggest that the major antigenic determinant for RSC‐RF resides in the CH3 domain of the IgG3 molecule. Precise characterization of this epitope may provide further insight into the etiology and pathogenesis of RA.

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“…The concentration of LPS resulting in 50% inhibition of the antibody binding was determined as described by Reed and Muench [20]. The affinity constants of monoclonal antibodies determined by this method have been shown to be in close agreement with the intrinsic affinity constant measured by Farr assay and fluorescence quenching assays [21]. Calculations of the molar concentrations were based on the assumed molecular weight of 10,000 for LPS as described by Schwartzer et al [22].…”

Section: Methodsmentioning

confidence: 97%

Affinity Constants of Naturally Acquired and Vaccine-Induced Anti-Pseudomonas aeruginosa Antibodies in Healthy Adults and Cystic Fibrosis Patients

Bruderer

Cryz

Schaad

et al. 1992

Journal of Infectious Diseases

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Direct measurement of antibody affinity distribution by Haften-inhibition enzyme immunoassay

Cited by 128 publications

References 18 publications

Antigen‐antibody interactions: Elucidation of the epitope and strain‐specificity of a monoclonal antibody directed against the pilin protein adherence binding domain of Pseudomonas aeruginosa strain K

Antigen‐antibody interactions: Elucidation of the epitope and strain‐specificity of a monoclonal antibody directed against the pilin protein adherence binding domain of Pseudomonas aeruginosa strain K

Differential reactivity of rheumatoid synovial cells and serum rheumatoid factors to human immunoglobulin g subclasses 1 and 3 and their CH3 domains in rheumatoid arthritis

Affinity Constants of Naturally Acquired and Vaccine-Induced Anti-Pseudomonas aeruginosa Antibodies in Healthy Adults and Cystic Fibrosis Patients

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