Objective: To test if we can use the ELISA to detect serum NfL levels in GBS patients. This ELISA is valided to be used only in CSF, and its measuring reange is 100pg/ml – 10.000pg/ml. As we have tested our GBS samples by SIMOA and some of the serum samples have >100pg/ml, we want to test if the ELISA can detect those levels.
Materials: NF-light ELISA from UmanDiagnostics Ref 10-7001
Preparations:
All assay reagents should be brought to room temperature prior to use.
Preparation of wash buffer; Dilute the total content of one 10x Wash buffer concentrate (10xWASH) bottle with deionized water to a final volume of 400 mL. Diluted unused wash buffer can be stored at room temperature and should be used within two months. The 10x Wash buffer concentrate can appear opalescent due to high salt concentration (no effect on assay performance).
Preparation of standard dilution series (calibrators): Reconstitution and preparation of standard dilution series should be performed directly before use. Label 6 micro-tubes, one for each standard point (that is 5 000 pg/mL, 2 500 pg/mL, 1 000 pg/mL, 500 pg/mL, 100 pg/mL and 0 pg/mL). The highest standard point (10 000 pg/mL) is obtained by reconstituting one vial of lyophilized Standard (STAND) with the volume of sample diluent (SAMDIL) indicated on the bottle label. Vortex briefly and keep in room temperature.
Running the assay:
1. Dilute the samples with equal amount (1+1) of Sample diluent (SAMDIL) to a total minimum volume of 210 mL. The standards reconstituted and diluted according to the standard dilution table are ready to use (i.e. no further dilution should be made).
NOTE: We have made to tests: one diluted 1+1 and the other without make the dilutions.
2. Wash the wells to be used with Wash buffer (3×300 mL). 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 mL of each Standard and sample in duplicate. Incubate 1 hour at RT with agitation (800 rpm).
4. Wash the wells with Wash buffer (3×300 mL), see point 2.
5. Directly before use, dilute the concentrated Tracer (50x TRAC) 1:50 with Sample diluent (SAMDIL). Mix thoroughly by inverting the tube or by vortexing. Add 100 mL of freshly diluted Tracer antibody to each well. Incubate 45 minutes at RT with agitation (800 rpm).
6. Wash the wells with Wash buffer (3×300 mL), see point 2.
7. Directly before use, dilute the concentrated Conjugate (CONJ) in the supplied Sarstedts 15 mL tube according to the vial label with Conjugate diluent (CONDIL). Mix thoroughly by inverting the tube or by vortexing. Add 100 mL of newly diluted Conjugate to each well. Incubate 30 minutes at RT with agitation (800 rpm). Important information: Use only the supplied 15 mL tube when preparing the conjugate solution.
8. Wash the wells with Wash buffer (3×300 mL), see point 2.
9. Add 100 mL of TMB to each well. Incubate 15 minutes at RT with agitation (800 rpm).
10. Add 50 mL of Stop reagent (STOP) to each well and read the absorbance at 450 nm (reference wavelength 620-650 nm).
RESULTS: The results were here.
The results did not correlate with sNfL measured by SIMOA. We have done the calibration curve with Excel and with “MyAssays” program, similar results with good calibration curves (R2>0.9898).
The comparison between both and sNfL by SIMOA were here.
