NF-light® ELISA FAQ
|Samples and sample types||Which assay should I use for serum, plasma and CSF analyses?||
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.
|Samples and sample types||Which species does the two NF-light ELISAs work on?||The antibodies in the NF-light ELISAs recognize NF-L from human, macaque, bovine, goat, mouse, and rat sources.|
|Samples and sample types||How many samples can be analyzed using one NF-light ELISA kit?||
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.
|Absorbance||Higher absorbance for the calibrator curve than expected.||The lyophilized calibrator vial may have been reconstituted in a too small volume (less than indicated on the vial label).|
|Absorbance||Lower absorbance for the calibrator curve than expected.||The lyophilized calibrator vial may have been reconstituted in a too high volume (more than indicated on the vial label).|
|Absorbance||Higher than expected absorbance of both the calibrator curve and the samples.||The amount of Conjugate Concentrate added to prepare the Conjugate working solution may have been too high.|
|Absorbance||Lower than expected absorbance of both the calibrator curve and the samples.||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.
|Absorbance||The sample absorbance is higher than the highest standard point in the calibrator curve.||The concentration of NF-L in the sample may be too high. Dilute the sample more and analyze it again.|
|Absorbance||The sample absorbance is lower than the lowest calibrator point in the calibrator curve.||The concentration of NF-L in the sample may be too low to quantify in the ELISA.|
|Absorbance||Why do I need to measure the absorbance at two wavelengths?||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.|
|Absorbance||Should I subtract the background absorbance from my measurement data?||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).|
|Calibrator and calibrator curve||Does the Calibrator contain human NFL?||
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.
|Calibrator and calibrator curve||Do I have to add Calibrator points in duplicate?||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.|
|Calibrator and calibrator curve||Do I need to compensate for the dilution of the sample when determining the final concentration?||
In the NF-light® ELISA for CSF samples, you should not compensate for the dilution factor. The 1+1 dilution of the sample (according to the assay protocol) is already compensated for in the assay. This is done by including that same dilution in the Standard reconstitution volume, meaning that the actual immunogenic NF-L concentration of the Standard vial is 5000 pg/ml. So the direct NF-L concentration read-out from the curve is the NF-L concentration of the original sample.
In the NF-light™ Serum ELISA, you should compensate for the sample dilution by multiplying the sample read-out with the dilution factor.
|Calibrator and calibrator curve||Can I use a leftover Calibrator vial from an earlier NF-light ELISA lot for an analysis on a newer lot?||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.|
|Calibrator and calibrator curve||Why should Calibrator vials from different kit lots be dissolved in different volumes of Diluent?||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.|
|Calibrator and calibrator curve||What is the NFL concentration of the Calibrator?||
For the NF-light® ELISA kit for CSF samples, the immunogenic NF-L concentration of the Calibrator vial is 5000 pg/ml. When it is used in accordance with the NF-light® ELISA protocol, the 1+1 dilution of the sample is already taken into account which is why the actual highest point of the curve is 10 000 pg/ml.
For the NF-light™ Serum ELISA, the immunogenic NF-L concentration is 500 pg/mL.
|Antibodies||Do the antibodies cross-react with other neuronal proteins?||There is no reported cross-reactivity to NF-M or NF-H, and adverse interference by other factors haven’t been observed.|
|Antibodies||What sort of antibodies are in the kit?||The antibody pair in the assay are both monoclonal mouse IgG1k.|
|Antibodies||What is the sequence of the epitope on the NFL protein that the antibodies bind to?||This information is not publically 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||Is it important to incubate the plate with a shaking frequency of 800 rpm?||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.|
|Assay protocol and parameters||Should I really incubate the TMB in daylight or should I cover it up?||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.|
|Assay protocol and parameters||Are there available protocols in other languages than English?||
For the NF-light® ELISA kit for CSF samples, the protocol is also available in Swedish, Norwegian, Danish, French, German, Spanish, Italian and Portugese.
The NF-light™ Serum ELISA protocol is currently only available in English.