Frequently asked questions

 

Topic
Question/Query
Answer
Samples and sample types Does the NF-light® ELISA work on serum or plasma samples? No, the assay is only meant for NFL-measurements in CSF (cerebrospinal fluid). The sensitivity of the NF-light® ELISA isn’t high enough for serum analyses, and the interference from heterophilic antibodies may result in falsely high results.
  Which species does the NF-light® ELISA work on? The antibodies in the NF-light® ELISA assay reacts well with NFL from human, macaque, bovine, goat, mouse and rat.
  How many samples can be analyzed using one NF-light® ELISA kit? The format is a regular 96-well plate, and 14 wells are needed for the Standard curve. If analyzing the samples in duplicate, one kit is enough for 41 samples. The kit contains 2 Standard vials to enable the kit to be used on two separate occasions. When doing two separate runs, the maximum number of samples is 34.
 
Absorbance Higher absorbance for the standard curve than expected. The lyophilized standard vial may have been reconstituted in a too small volume (less than indicated on the vial label).
  Lower absorbance for the standard curve than expected. The lyophilized standard vial may have been reconstituted in a too high volume (more than indicated on the vial label).
  Higher than expected absorbance of both the standard curve and the samples. The amount of Conjugate Concentrate added to prepare the Conjugate working solution may have been too high.
  Lower than expected absorbance of both the standard 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 Standard vs the Sample binding to the plate differs. If the shaking frequency is lower than 800rpm, the absorbance will drop for both Standard and Sample, but the relative levels between the two will be different resulting in falsely high Sample read-outs.
  The sample absorbance is higher than the highest standard point in the standard curve. The concentration of NFL in the sample may be too high. Dilute the sample more and analyze it .
  The sample absorbance is lower than the lowest standard point in the standard curve. The concentration of NFL in the sample may be too low to quantify in the ELISA. Levels lower than 100pg/ml can not be quantified using the NF-light® ELISA.
  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.
  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. Also, by not subtracting the background, it will be easier to spot any inconsistencies in your data-set due to contaminating factors.
 
Standard and Standard curve Does the Standard contain human NFL? No, the Standard is based on NFL purified from certified and healthy bovine sources. The Standard is non-hazardous, non-infectious and is not likely to disseminate agents of diseases or infectious diseases to domestic animals, wild life or humans.
  Do I have to add Standard points in duplicate? Yes, we strongly advice to always run duplicates (of both Standard points and Samples). By always running duplicates, you are more likely to obtain reliable data.
  Do I need to compensate for the dilution of the sample when determining the final concentration? No. 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 NFL concentration of the Standard vial is 5000 pg/ml. So the direct NFL concentration read-out from the curve is the NFL concentration of the original sample.
  Can I use a leftover Standard vial from an earlier NF-light® ELISA lot for an analysis on a newer lot? No. The Standard vials are lot specific. Each Standard 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 Standard vial is used with the “wrong” lots of kit components, the resulting NF-L levels are not reliable.
  Why should Standard vials from different kit lots be dissolved in different volumes of Sample Diluent? Each Standard lot is uniquely calibrated to a particular set of NF-light® ELISA kit components. The calibration is done by adjusting the Standard curve absorbance in relation to the absorbance of several Quality Control CSF samples with known NF-L concentration. The adjustments are made by increasing or decreasing the Standard reconstitution volume for each lot. This is also the reason why Standard vials can only be used together with it’s “own” NF-light® ELISA Kit lot.
  What is the NFL concentration of the Standard? The immunogenic NFL concentration of the Standard vial is 5000 pg/ml, when it is used in accordance with the NF-light® ELISA protocol. But with the 1+1 dilution of the sample taken into account, the actual highest limit of the curve is 10 000 pg/ml.
 
Antibodies Does the antibodies cross-react with other neuronal proteins? There is no cross-reactivity to NF-M or NF-H, and adverse reactivities to other proteins haven’t been observed.
  What sort of antibodies are in the kit? The antibody pair in the assay are both monoclonal mouse IgG1k
  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, the sample read-outs (NFL-levels) will be too high. 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 Standard vs the Sample binding to the plate differs. If the shaking frequency is lower than 800rpm, the absorbance will drop for both Standard and Sample, but the relative levels between the two will be different resulting in falsely high Sample read-outs.
  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.
  Are there available protocols in other languages than English? The protocols are also available in Swedish, Norwegian, Danish, French, German, Spanish and Italian. All protocols can be downloaded from here.

 

 

Protocol ELISA NF-light ELISA®


Step 1 Add Sample

step-2

Step 2 Add tracer

step-3

Step 3 Add conjugate

step-4

Step 4 Add substrate

step-5

  1. Dilute the CSF samples with equal amount (1+1) of sample diluent to a total minimumvolume of 210 μL. The standards reconstituted and diluted according to the standard dilution table are ready to use (i.e. no further dilution should be made).
  2. Wash the wells to be used with wash buffer (3×300 μL). The wash buffer added could be either aspirated or removed by knocking the plate against absorbing material immediately before next washing cycle.
  3. Add 100 μL of each standard and sample in duplicate. Incubate 1 hour at room temperature (20-25°C) with agitation (800 rpm).
  4. Wash the wells with wash buffer (3×300 μL), see point 2.
  5. Add 100 μL of freshly diluted tracer (biotin anti-NF-L) antibody to each well. Incubate 45 minutes at room temperature (20-25°C) with agitation (800 rpm).
  6. Wash the wells with wash buffer (3×300 μL), see point 2.
  7. Add 100 μL of newly diluted conjugate (streptavidin-HRP) to each well. Incubate 30 minutes at room temperature (20-25°C) with agitation (800 rpm).
  8. Wash the wells with wash buffer (3×300 μL), see point 2.
  9. Add 100 μL of TMB to each well. Incubate 15 minutes at room temperature (20-25°C) with agitation (800 rpm).
  10. Add 50 μL of stop reagent to each well and read the absorbance at 450 nm (reference wavelength 620-650 nm).