Hi Glenn,
I have used the Immundiagnostik kit for anti infliximab antibodies. It is based on the same principle as the in house assay that I have ie infliximab as the coating antigen
, incubation with patient serum and the detection step is enzyme conjugated infliximab. I have biotinylated the infliximab. The commercial kit uses a peroxidise label. In this assay format any bound immunoglobulin that can capture more than a single infliximab molecule is part of the readout. The assay is most informative when the serum sample is collected at the time of lowest drug concentration (ie just prior to the next infusion). As I mentioned earlier I compared it with the in house assay I set up and had concordance in 83.6% of samples. Of the 16% that were discordant two thirds were close to the cut – off. The commercial assay uses a 1:10 dilution of the patient serum which is a low dilution in comparison to a lot of ELISAs. The read out for both the in house and the commercial assay is semi quantitative (ie relative to a cut –off).
, incubation with patient serum and the detection step is enzyme conjugated infliximab. I have biotinylated the infliximab. The commercial kit uses a peroxidise label. In this assay format any bound immunoglobulin that can capture more than a single infliximab molecule is part of the readout. The assay is most informative when the serum sample is collected at the time of lowest drug concentration (ie just prior to the next infusion). As I mentioned earlier I compared it with the in house assay I set up and had concordance in 83.6% of samples. Of the 16% that were discordant two thirds were close to the cut – off. The commercial assay uses a 1:10 dilution of the patient serum which is a low dilution in comparison to a lot of ELISAs. The read out for both the in house and the commercial assay is semi quantitative (ie relative to a cut –off).
This is the details from the Immundiagnoistik kit insert:
"This Enzyme Immuno Assay is a sandwich assay for the determination of free
antibodies against therapeutic antibodies (anti-TNF therapeutic
antibodies, e.g. REMICADE) in serum samples. In a first incubation step, the
free anti-therapeutic antibodies from the sample are bound to the
therapeutic antibodies (e.g. REMICADE) coated on the plate. To remove all
unbound substances, a washing step is carried out. In a further incubation
step, Peroxidase-labeled therapy antibody is added. After another washing
step, to remove all unbound substances, the solid phase is incubated with
the substrate, Tetramethylbenzidine (TMB). An acidic stop solution is then
added. The colour converts to yellow. The absorbance of the color
compound is determined photometrically at 450 nm. The intensity of the
color is directly proportional to the amount of bound anti-therapeutic
antibodies (e.g. anti-REMICADE) from the sample. The results are evaluated
by a cut off value."
Regards
Chris Bundell PhD (UWA)
Clinical Immunology
PathWest Laboratory Medicine
QEII Medical Centre
Locked Bag 2009
Nedlands
Western Australia 6009
Tel: (08) 9346 1844
Fax: (08) 9346 3160
