Recommended buffers, solutions
Note: Do not use sodium azide in these preparations. Sodium azide inactivates the horseradish peroxidase enzyme.
The BD OptEIA Reagent Set B (Cat. No 550534) containing Coating Buffer, Assay Diluent, Substrate Reagents A and B, Stop Solution and 20X Wash Buffer Concentrate is recommended.
1. Coating Buffer - 0.1 M Sodium Carbonate, pH 9.5; 7.13 g NaHCO3, 1.59 g Na2CO3; q.s. to 1.0 L; pH to 9.5. Freshly prepare or use within 7 days of preparation, stored at 2-8°C.
2. Assay Diluent- PBS* with 10% FBS#, pH 7.0. The BD OptEIA Assay Diluent (Cat. No. 555213) is recommended.
*Phosphate-Buffered Saline: 80.0 g NaCl, 11.6 g Na2HPO4, 2.0 g KH2PO4, 2.0 g KCL, q.s. to 10 L; pH to 7.0.
#Fetal Bovine Serum: Hyclone Cat. No. SH30088 (heatinactivated) recommended.
Freshly prepare or use within 3 days of preparation, with 2-8°C storage.
3. Wash Buffer - PBS* with 0.05% Tween-20. Freshly prepare or use within 3 days of preparation, stored at 2-8°C.
4. Substrate Solution - Tetramethylbenzidine (TMB) and Hydrogen Peroxide. The BD OptEIA TMB Substrate Reagent Set (Cat. No. 555214) is recommended.
5. Stop Solution - 1 M H3PO4 or 2 N H2SO4
Additional Materials Required
1. 96-well BD Falcon™ ELISA plates (Cat. No. 353279) are recommended
2. Microplate reader capable of measuring absorbance at 450 nm
3. Precision pipettes
4. Graduated cylinder, one liter
5. Deionized or distilled water
6. Wash bottle or automated washer
7. Log-log graph paper or automated data reduction
8. Tubes to prepare standard dilutions
9. Laboratory timer
10. Plate sealers or parafilm
Specimen Collection and Handling
Specimens should be clear, non-hemolyzed and non-lipemic.
Cell culture supernatants: Remove any particulate material by centrifugation and assay immediately or store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles.
Serum: Use a serum separator tube and allow samples to clot for 30 minutes, then centrifuge for 10 minutes at 1000 x g. Remove serum and assay immediately or store samples at ≤ -20° C. Avoid repeated freeze-thaw cycles.
Plasma: Collect plasma using EDTA as anticoagulant. Centrifuge for 10 minutes at 1000 x g within 30 minutes of collection. Assay immediately or store samples at ≤ -20° C. Avoid repeated freeze-thaw cycles. Citrate or heparin may also be used as anticoagulant if necessary.
Standards Preparation and Handling
1. Reconstitution: After warming lyophilized standard to room temperature, carefully open vial to avoid loss of material. Reconstitute lyophilized standard with appropriate amount of assay diluent to yield a 30 ng/mL stock standard. Allow the standard to equilibrate for at least 15 minutes before making dilutions. Vortex gently to mix.
2. Storage/ handling of reconstituted standard: Use reconstituted standard immediately. Use one vial of standard per 96-well plate.
3. Standards Preparation for Assay:
a. Prepare 30 ng/mL stock standard as indicated in 1. Reconstitution above. Vortex gently to mix.
b. Add 300 μL Assay Diluent to 6 tubes. Label as 15 ng/mL, 7.5 ng/mL, 3.75 ng/mL, 1.875 ng/mL, 0.938 ng/mL, and 0.469 ng/mL.
Working Detector Preparation
1. Determine volume needed for experiment: 100 μL per well.
2. Determine amount of 250X detector antibody and 250X Streptavidin-HRP to add to working detector mix.
3. Add appropriate volumes of 250X detector antibody and 250X Streptavidin-HRP to Assay Diluent to prepare required volume of Working Detector. Do not dilute more Detection Antobody than is needed for your experiment.
4. Add Working Detector to each well as described in the assay procedure.
Note: Working Detector must be prepared within 15 minutes prior to use.
Detailed Assay Procedure
1. Dilute the Capture Antibody 1:250 in Coating Buffer and coat microwells with 100 µL of diluted Capture Antibody per well. Seal plate and incubate overnight at 4C. Do not dilute more Capture Antibody than is needed for your experiment.
2. Aspirate wells and wash 3 times with ≥ 300 μL/well Wash Buffer. After last wash, invert plate and blot on absorbent paper to remove any residual buffer.
3. Block plates with ≥ 200 μL/well Assay Diluent. Incubate at room temperature (RT) for 1 hour.
4. Aspirate/wash as in step 2.
5. Prepare standard and sample dilutions in Assay Diluent. See "Standards Preparation and Handling." Be sure to record the reconstituted standard concentration for future use.
6. Pipette 100 μL of each standard, sample, and control into appropriate wells. Seal plate and incubate for 2 hours at RT.
7. Aspirate/ wash as in step 2, but with 3 total washes.
8. Add 100 μL of Working Detector to each well. Seal plate and incubate for 1 hour at RT.
9. Aspirate/ wash as in step 2, but with 7 total washes.
10. Add 100 μL of TMB Substrate Solution to each well. Incubate plate (without plate sealer) for 30 minutes at room temperature in the dark.
11. Add 50 μL of Stop Solution to each well.
12. Read absorbance at 450 nm within 30 minutes of stopping reaction. If wavelength correction is available, subtract absorbance at 570 nm from absorbance 450 nm.
Assay Procedure Summary
1. Add 100 μL diluted Capture Ab to each well. Incubate overnight at 4° C.
2. Aspirate and wash 3 times.
3. Block plates: 200 μL Assay Diluent to each well. Incubate 1 hr RT.
4. Aspirate and wash 3 times.
5. Add 100 μL standard or sample to each well. Incubate 2 hr RT.
6. Aspirate and wash 3 times.
7. Add 100 μL diluted Working Detector to each well. Incubate 1 hr RT.
8. Aspirate and wash 7 times.
9. Add 100 μL TMB Substrate Solution to each well. Incubate 30 min RT in dark.
10. Add 50 μL Stop Solution to each well. Read at 450 nm within 30 min with λ correction 570 nm.
Calculation of Results
Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from each.
Plot the standard curve on log-log graph paper, with C5b-9 concentration on the x-axis and absorbance on the y-axis. Draw the best fit curve through the standard points.
To determine the C5b-9 concentration of the unknowns, find the unknown's mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the C5b-9 concentration. If samples were diluted, multiply the C5b-9 concentration by the dilution factor.
Computer data reduction may also be employed, utilizing log-log regression analysis.
Cross Reactivity: The following factors were tested in the BD OptEIA assay at ≥ 5 μg/mL and no cross-reactivity (value ≥ 470 pg/mL) was identified.
Purified native human C3, C4, C5, C6, C7, C8 and C9
Limitations of the Procedure
· Samples that generate absorbance values higher than the standard curve should be diluted with Standard Diluent and re-assayed.
· Interference by drug metabolites, soluble receptors, or other binding proteins in specimens has not been thoroughly investigated. The possibility of interference cannot be excluded.
· BD OptEIA Sets are intended for use as an integral unit. Do not mix reagents from different Set batches. Reagents from other manufacturers are not recommended for use in this Set.