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LOT NUMBER GR38174 ORDER NUMBER 953609 DESCRIPTION OF THE PROBLEM No signal or weak signal. When used as a positive control/external standard in a sandwich ELISA specific to human ApoB, the samples do not differ from the blank negative control, regardless of protein concentration (ranging from 4.14-265ug/dL for a kit with a detection range of 2-259ug/dL). SAMPLE Human serum and/or plasma. PRIMARY ANTIBODY AlerCHEK, Inc., Anti-human ApoB DETECTION METHOD TMB/peroxide substrate POSITIVE AND NEGATIVE CONTROLS USED Positive controls: AlerCHEK ApoB standard and Abcam ApoB protein purified from human plasma. Negative control: AlerCHEK specimen diluent without any added sample or protein ANTIBODY STORAGE CONDITIONS Supplied at 0.82mg/mL in 200mM Tris, 10mM SDS, pH 8.25. Diluted 1:100 to 820ug/dL with 1x PBS, pH 7.4 (no alternative dilution instructions were provided) Separated into ~150uL aliquots and stored at -20C. TYPE OF ELISA Sandwich ELISA specific to human Apolipoprotein B. COATING WELL Manufacturer plate pre-coated with proprietary antibody mix (AlerCHEK, Inc.) BLOCKING CONDITIONS N/A SECONDARY ANTIBODY AlerCHEK, Inc., HRP conjugated affinity purified goat anti-ApoB HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? At this point, we have simply repeated the run with the kit-supplied standards and raw plasma/serum alongside the Abcam ApoB protein to verify that positive results are attainable with the kit. Only the Abcam ApoB protein has failed repeatedly. We were hoping to use the raw protein as in-house standards and are unclear as to why they would not work with an apparently appropriate ELISA kit.
Asked on Oct 24 2011
Thank you for bringing this to our attention. The product, apolipoprotein B protein, has not, to our knowledge, been tested in ELISA. The information we have from the laboratory that provides the protein to us is as follows: "ApoB-100 (MW 413kDa) is isolated from other smaller apoproteins (ApoC’s with MW 6-10kDa; ApoE with MW 33kDa) by gel filtration chromatography. The presence or absence of these smaller apoproteins is tested by Ouchterlony immunodiffusion using the corresponding antibody. The presence of ApoB-100 is demonstrated by the formation of an immunoprecipitin line between apoB100 and antibody raised against highly purified LDL." Can you tell me the catalogue number of the ELISA kit? I am wondering if the kit is incompatible with this protein, or if we can conclude that the protein is defective.
Answered on Oct 24 2011