Myeloperoxidase ELISA 试剂盒

(1) Prior to use allow all reagents to come to room temperature. Reagents from different kit lot numbers should not be combined or interchanged.
(2) ELISA Wash Concentrate must be diluted to working solution prior use. Please see REAGENTS section for details.
(3) Reconstitute assay standard by adding 2.0 mL of deminerialized water to standard vial. Separately, reconstitute controls by adding 1.0 mL of deminerialized water to control vials. Allow the standard and controls to sit undisturbed for 5 minutes, and then mix well by inversions or gentle vortexing. Make sure that all solid is dissolved completely prior to use. These reconstituted standard and controls may be stored at 2?8 C for up to 3 days or at ?10 C or below for long-term storage. Do not exceed 3 freeze-thaw cycles.
(4) Dilute the reconstituted standard concentrate 1:4 using the MPO sample diluent to obtain a level six standard by mixing the concentrated MPO standard (Cat. No. 30601) with MPO sample diluent (Cat. No. 30612). For example: mix 300 L of concentrated MPO standard with 900 L of the MPO sample diluent. Continue diluting standards down to level two as it is shown below. Level one standard is the MPO sample diluent.
(5) Place a sufficient number of myeloperoxidase antibody coated microwell strips in a holder to run human myeloperoxidase standards, controls and unknown samples in duplicate.
(6) Test Configuration
(7) Prepare Tracer Antibody working solution by 1:21 fold dilution of the Myeloperoxidase Tracer Antibody (Cat. No. 30606) by adding the tracer antibody into the Tracer Antibody Diluent (Cat. No. 30605). Following is a table that outlines the relationship of strips used and antibody mixture prepared. NOTE: the tracer antibody should be prepared just prior to the end of the first incubation cycle.

Each serum and/or plasma sample has to be diluted 1:5 using MPO Sample Diluent for dilution. For example: 50 μL of human serum or plasma diluted in 200 μL of the MPO Sample Diluent will yield a 1:5 dilution and a sufficient sample amount for myeloperoxidase measurement in duplicate.

(1) Add 100 µL of Standards, Controls and diluted patient samples into the designated microwells.
(2) Seal the plate wells securely, cover with foil or other material to protect from light, and rotate on an ELISA plate shaker (small orbit radius) for 90 5 minutes at 400 to 450 rpm.
(3) Just prior to the end of the incubation time, dilute the proper amount of Tracer Antibody for the assay.
(4) Wash each well five times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
(5) Add 100 µL of above Tracer Antibody to each well.
(6) Seal the plate wells securely, cover with foil or other material to protect from light, and rotate on an ELISA plate shaker (small orbit radius) for 45 minutes 5 minutes at 400 to 450 rpm.
(7) Wash each well five times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
(8) Add 100 µL of ELISA HRP Substrate into each of the wells.
(9) Cover the plate with aluminum foil or other material to avoid exposure to light. Incubate plate static, at room temperature for 20 minutes.
(10) Immediately add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
(11) Read the absorbance at 405 nm with reference filter at 620 nm or 650 nm.

It is recommended to use a point-to-point or 4-parameter standard curve fitting.
1. Calculate the average absorbance for each pair of duplicate test results.
2. Subtract the average absorbance of the level 1 standard (0 ng/mL) from the average absorbance of all other readings to obtain corrected absorbance.
3. The standard curve is generated by the corrected absorbance of all standard levels on the ordinate against the standard concentration on the abscissa using point-to-point or log-log paper. Appropriate computer assisted data reduction programs may also be used for the calculation of results. The serum/plasma human myeloperoxidase concentrations for the controls and the patient samples are read directly from the standard curve using their respective corrected absorbance.