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Support/FAQ
Absorbance
The lyophilized calibrator vial may have been reconstituted in a too large volume (more than indicated on the vial label).
The general absorbance level is determined by the concentration of Conjugate. The Conjugate working solution should be prepared directly before use and mixed thoroughly.
– If the amount of Conjugate added to the Conjugate working solution is lower than indicated on the vial, then the general absorbance level will drop.
– If using other tubes for Conjugate dilution, than the ones provided in the kit, absorbances may drop significantly.
– If incubating the ELISA plate at a lower shaking frequency than indicated in the protocol, general absorbance levels will drop. But furthermore, the kinetics of the calibrator vs the Sample binding to the plate differs. If the shaking frequency is lower than 800 rpm, the absorbance will drop for both calibrator and Sample, but the relative levels between the two will be different. This means a high risk of falsely elevated Sample read-outs.
Antibodies
This information is not publicly available. The public information on the epitope is published in: Hybridoma and Hybridomics, 2004, Vol. 21, No. 1. “Monoclonal Antibodies Selective for Low Molecular Weight Neurofilaments“, By Niklas Norgren, Jan-Erik Karlsson, Lars Rosengren and Torgny Stigbrand.
Assay protocol and parameters
Yes, this is very important. With a lower shaking frequency, there’s a risk of generating falsely high sample read-outs (NF-L levels). If incubating the ELISA plate at a lower shaking frequency than indicated in the protocol, general absorbance level will drop. But furthermore, the kinetics of the Calibrator vs the Sample binding to the plate differ. If the shaking frequency is lower than 800 rpm, the absorbance will drop for both Calibrator and Sample, but the relative level between the two will be different resulting in falsely high Sample read-outs.
For the CE labelled NF-light® ELISA kit for CSF samples, the protocol is also available in Swedish, Norwegian, Danish, French, German, Spanish, Italian, Czech, and Portugese.
The protocol for both the NF-light™ Serum ELISA and the RUO NF-light™ ELISA for CSF samples is available in English only.
Calibrator and calibrator curve
The calibrator in the CE labelled NF-light® ELISA for CSF samples is based on NFL purified from certified and healthy bovine sources. The calibrator is non-hazardous, non-infectious and is not likely to disseminate agents of diseases or infectious diseases to domestic animals, wild life or humans.
The calibrator used in the NF-light™ Serum ELISA, as well as in the RUO NF-light™ ELISA for CSF samples, is based on recombinant full-length human NF-L.
Yes, we strongly advise to always run duplicates (of both Calibrator points and Samples). By always running duplicates, you are more likely to obtain reliable data.
Yes, the dilution factor must be compensated for. The NF-L concentration in the original sample is calculated by multiplying the measured value by the applied dilution factor (at least 2 for the NF-light ELISAs for CSF samples and at least 4 for the NF-light™ Serum ELISA).
No. The Calibrator vials are lot specific. Each Calibrator lot is calibrated together with one particular set of reagent lots to generate the correct absorbance level in relation to particular Quality Control Samples with known NF-L concentrations. This means that if a Calibrator vial is used with the “wrong” lots of kit components, the resulting NF-L levels are not reliable.
Each Calibrator lot is uniquely calibrated to a particular set of NF-light ELISA kit components. The calibration is done by adjusting the calibrator curve absorbance in relation to the absorbance of several Quality Control samples with known NF-L concentration. The adjustments are made by increasing or decreasing the Calibrator reconstitution volume for each lot. This is also the reason why Calibrator vials can only be used together with a specific NF-light ELISA Kit lot.
For the NF-light® ELISA kit for CSF samples, the immunogenic NF-L concentration of the Calibrator vial is 5000 pg/ml.
For the NF-light™ Serum ELISA, the immunogenic NF-L concentration is 500 pg/mL.
Lot numbers
Samples and sample types
The NF-light® ELISA is only meant for CSF samples. Analysing other samples may cause false positives.
The NF-light™ Serum ELISA is developed for serum samples. Plasma analyses have not been properly evaluated and we strongly advise against using the assay for anything other than serum.
For plasma analyses, we offer the anti NF-L monoclonal antibody-pair as stand-alone products. These can be used to set up an in-house assay on a high-sensitivity platform such as the Quanterix’s Simoa.
The antibodies in the NF-light ELISAs recognize NF-L from human, macaque, bovine, mouse and rat sources.
For all NF-light ELISA kits (articles 10-7001, 10-7002, 20-8002), 16 wells are required for the calibrator curve and blank samples, with an additional two wells required for the included control sample. That leaves room for 39 samples in duplicate.
All kits include two calibrator vials and two control sample vials, allowing for division into two separate runs.