Support/FAQ

Absorbance

The lyophilized calibrator vial may have been reconstituted in a too small volume (less than indicated on the vial label).

The lyophilized calibrator vial may have been reconstituted in a too large volume (more than indicated on the vial label).

The amount of Conjugate Concentrate added to prepare the Conjugate working solution may have been too high.

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.

The concentration of NF-L in the sample may be too high. Dilute the sample more and analyze it again.
The concentration of NF-L in the sample may be too low to quantify in the ELISA.
The measurement at 450nm measures the yellow color in the well which is proportional to the NF-L amount loaded in the well. The reference measurement (620-650nm) will detect any inconsistencies in the plastic well bottom. Such inconsistencies will affect (increase) the 450nm measurements as well, so by subtracting the reference data from the 450nm-data, you ensure that the results are based solely on the reactions occurring in the well.
No. When subtracting the background (absorbances in the wells that contain only sample diluent and no NF-L) the data may be skewed by the lower absorbance data approaching zero. Concentration determinations should always be based on the 450nm absorbances minus the reference absorbances (620-650nm).

Antibodies

There is no reported cross-reactivity to NF-M or NF-H, and adverse interference by other factors haven’t been observed.
The antibody pair in the assay are both monoclonal mouse IgG1k.

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.
Yes, it should be incubated in daylight. The plate does not have to be kept in the dark or be covered when TMB has been added.

For the NF-light® ELISA kit for CSF samples, the protocol is also available in Swedish, Norwegian, Danish, French, German, Spanish, Italian, Czech, and Portugese.

The NF-light™ Serum ELISA protocol is currently only available in English.

Calibrator and calibrator curve

The calibrator in the 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 in the NF-light™ Serum ELISA is based on recombinant human full-length 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.

The way of calculating the original concentration of a sample was recently updated for the NF-light® ELISA for CSF samples. For lots up to and including 70845, the dilution factor of the sample should not be compensated for since a 2-time dilution is already accounted for in the assay. However, for all lots with lot numbers above 70845, the NF-L concentration in the original sample is obtained by multiplying the read-out with the applied dilution factor (at least 2 for the CSF-ELISA).

In the NF-light™ Serum ELISA, you should always compensate for the sample dilution by multiplying the sample read-out with the dilution factor.

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

No, the product performance is unchanged. Two numbers have been added at the end of the lot number in order to differentiate between specific assembly occasions. This means that lot 12345-01 and lot 12345-02 are both from the same product lot but different kit assembly occasions.

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.

The format of both NF-light ELISA kits is a regular 96-well plate.

For the NF-light® ELISA for CSF-samples, 14 wells are needed for the calibrator curve. If analyzing the samples in duplicate, one kit is enough for 41 samples.

The NF-light™ Serum ELISA requires 16 wells for its calibrator curve, and 2 more for the included Control sample. That leaves room for 39 duplicate serum samples.

Both kits contain two calibrators enabling them to be divided into two separate runs.

If you have additional questions please contact UmanDiagnostics technical support.

Download the NF-light® ELISA protocols

The most recent instructions for use are available for direct down load here if you are in need of an older instruction please contact UmanDiagnostics.

NF-light Serum ELISA RUO

NF-light CSF ELISA RUO

NF-light CSF ELISA CE

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