20210928: PMP2 ELISA in GBS patients

Objective: To detect PMP-2 in serum samples of GBS patients and see if the concentration is detectable and different from healthy controls.

ELISA PMP-2 Kit Catalog number MBS705027 My Biosource

Bring all reagents ans samples to room temperature before use (30 min)

MATERIALS – Reagent preparation:

• Dilute biotin-antibody 100-fold in biotin-antibody diluent (Centrifuge the vial before opening).
• Dilute HRP-avidin 100-fold in HRP-avidin diluent (Centrifuge the vial before opening).
• Preparation of Wash Buffer: 20ml of Wash Buffer concentrate (25x) in 500ml of destilated water.
• Standards: Centrifuge the standar vial 6000rpm 30sec. Reconstitute the standard with 1ml of sample diluent. This reconstitution produces a stock solution of 20ng/ml (S7), mix gently 15 min prior dilutions. Pipette 250ul of sample diluent to each tube (S0-S6) and use stock solution to produce a 2-fold dilution series (250uL to next dilution tube). Sample diluent serves as zero standard (0ng/ml – SO).

PROCEDURE:

1. Prepare all reagents.
2. Add 100uL of standard and sample per well. *Note: In this first assay we use undiluted sera (100uL /well) to see if we can detect PMP2 levels. Cover with the adhesive strip and incubate for 2h at 37ºC.
3. Remove the liquid, don’t wash
4. Add 100uL of biotin-antibody 1x to each well. Cover with a new adhesive strip and incubate for 1h at 37ºC.
5. Aspirate each well and wash, repeating the process two times for a total of 3 washes. 200uL of Wash Buffer and let it stand for 2 mintues. After the last wash, aspirate or decant to remove any remaining wash Buffer. Invert the plate and blot it against clean papel towels.
6. Add 100uL of HRP-avidin 1x to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 hour at 371C.
7. Wash 5 times as in step 5.
8. Add 90uL of TMB substrate to each well. Incubate for 15-30min at 37ºC. Protect from light.
9. Add 50uL of Stop solution to each well.
10. Determine the optical density of each well whitin 5 minutes, using a microplate reader 450nm. Wavelenght correction seted in 540 or 570.

RESULTS: The results were here. We can detect PMP2 levels in some samples, but most samples were out of the curve (detection range of the ELISA: 0.312ng/ml to 20ng/ml). Two healthy controls have high levels of PMP2 in sera but they have a hugh CV.