Standardization of the homogeneous mobility shift assay protocol for evaluation of anti-infliximab antibodies. Application of the method to Crohn's disease patients treated with infliximab.
BACKGROUND
The availability of a quantitative method to measure anti-infliximab (IFX) antibodies (ATI) would facilitate the implementation of therapeutic drug monitoring in clinical decision-making. Our aim was to standardize the homogeneous mobility shift assay (HMSA) used in the measure of ATI levels.


METHODS
In this prospective longitudinal multicenter study, 50 IFX-treated Crohn's disease (CD) patients were followed up for 54weeks. During this period 360 human serum samples were analysed. Monomeric ATI levels were measured by a quantitative HMSA-method using an anti-IFX calibrator. IFX trough levels measured by ELISA were correlated with ATI levels.


RESULTS
Using HMSA and a pure anti-idiotypic monoclonal antibody specific for IFX (anti-IFX calibrator), we measured the levels of monomeric ATI generated in Crohn's disease patients treated with IFX. Anti-IFX calibrator allowed to quantify monomeric antibodies against IFX with a low limit of quantification (3nM). The threshold level of ATI in order to classify the immunogenicity of the patients was 10nM. We observed that 24% (12/50) of IFX-treated patients developed ATI (>10nM) during the observation period (54weeks). Serum concentration of ATI higher than 10nM dramatically increased the probability (OR=51.1; 95% CI: 20.4-128.0; p<0.0001) of presenting low levels of IFX (⩽1.5nM) in serum, as observed in some CD patients treated with standard doses of the drug.


CONCLUSIONS
The HMSA-method described here allows an accurate quantification of ATI concentration in international units (IU) and therefore it could be useful in the study of the relationship between ATI concentration, infliximab level and the clinical response to the drug.
Systems Biology Department, Universidad de Alcalá and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Alcalá de Henares, Spain.
B
B
Hernández-Breijo
Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP) and CIBEREHD, Madrid, Spain.
M
M
Chaparro
Systems Biology Department, Universidad de Alcalá and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Alcalá de Henares, Spain.
D
D
Cano-Martínez
Hospital de Fuenlabrada, Madrid, Spain.
I
I
Guerra
Hospital la Fe, Valencia and CIBEREHD, Spain.
M
M
Iborra
Hospital de Galdakano, Vizcaya, Spain.
J L
JL
Cabriada
Hospital de Donostia, Guipúzcoa, Instituto Biodonostia, UPV/EHU and CIBEREHD, Spain.
L
L
Bujanda
Hospital Clínico San Carlos, and IdISSC, Madrid, Spain.
C
C
Taxonera
Hospital Reina Sofía, Instituto Maimónides de Investigación Biomédica de Córdoba, Universidad de Córdoba, Córdoba, Spain.
V
V
García-Sánchez
Hospital Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Madrid, Spain.
I
I
Marín-Jiménez
Hospital Clínico Universitario de Santiago, Santiago de Compostela, Spain.
M
M
Barreiro-de Acosta
Hospital Universitario Puerta de Hierro, Madrid, Spain.
I
I
Vera
Hospital Universitario la Paz, Madrid, Spain.
M D
MD
Martín-Arranz
Hospital Universitario Ramón y Cajal, Madrid, Spain.
F
F
Mesonero
Hospital General de Alicante, Alicante, Spain.
L
L
Sempere
Hospital Clínico Universitario "Lozano Blesa", IIS Aragón, Zaragoza and CIBEREHD, Spain.
F
F
Gomollón
Hospital de Manises, Valencia, Spain.
J
J
Hinojosa
Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP) and CIBEREHD, Madrid, Spain.
J P
JP
Gisbert
Systems Biology Department, Universidad de Alcalá and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Alcalá de Henares, Spain. Electronic address: luis.gonzalez@uah.es.
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LG
Guijarro
D000906:Antibodies / Q000097:blood*; D003424:Crohn Disease / Q000097:blood / Q000188:drug therapy*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D006801:Humans; D000069285:Infliximab / Q000627:therapeutic use*; D011446:Prospective Studies
D000906:Antibodies; D000069285:Infliximab
Anti-drug antibodies; Crohn’s disease; HMSA; Infliximab