 |
|
 |
ENZYME IMMUNOASSAY KIT
(Range:0-500 ng/ml)
It is recommended that plasma sample be diluted with EIA buffer at 1/16 ~ 1/32 to performance EIA assay.
Introduction
This Enzyme Immunoassay kit is designed to detect a specific peptide and its related peptides based on the principle of "competitive" enzyme immunoassay.
Principle of enzyme immunoassay with this kit
The immunoplate in this kit is pre-coated with secondary antibody and
the nonspecific binding sites are blocked. The secondary antibody can bind
to the Fc fragment of the primary antibody (peptide antibody) whose Fab
fragment will be competitively bound by both biotinylated peptide and peptide
standard or targeted peptide in sample.
The biotinylated peptide is able
to interact with streptavidin-horseradish peroxidase (SA-HRP) which catalyzes
the substrate solution composed of 3,3',5,5'-tetramethylbenzidine (TMB)
and hydrogen peroxide to produce a blue colored solution. The enzyme-substrate
reaction is stopped by hydrogen chloride (HCl) and the solution turns to
yellow. The intensity of the yellow is directly proportional to the amount
of biotinylated peptide-SA-HRP complex but inversely proportional to the
amount of the peptide in standard solutions or samples. This is due to the
competitive binding of the biotinylated peptide and the peptide in standard
solutions or samples to the peptide antibody (primary antibody).
A standard
curve of a peptide with known concentration can be established accordingly.
The peptide with unknown concentration in samples can be determined by extrapolation
to this standard curve.
CAUTION: INVESTIGATIONAL DEVICE. LIMITED BY LAW TO INVESTIGATIONAL USE. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
Kit Materials
1. 20 x assay buffer concentrate (50 ml).
2. 96 well immunoplate (1 plate).
3. Acetate plate sealer (APS), (3 pieces).
4. Primary antiserum (rabbit anti-peptide IgG) (1 vial).
5. Standard peptide (1 ug).
6. Biotinylated peptide (1 vial).
7. Streptavidin-horseradish peroxidase (SA-HRP) (30 ul).
8. Substrate solution (12 ml).
9. 2N HCl (15 ml).
10. Assay diagram (1 sheet).
11. General protocol (1 book).
Note:
Blood collection protocol and extraction procedure for plasma is provided for your information. (Materials for extraction not included).
Phoenix Pharmaceuticals guarantees that its products conform to the information contained in this publication. The purchaser must determine the suitability of the product for its particular use.
Assay Procedure1. Carefully read this protocol before performing assay.
2. Dilute the assay buffer concentrate with 950 ml deionized or HPLC grade water. This assay buffer will be used to reconstitute all of the other compounds in this kit and the extract of plasma samples.
3. Rehydrate standard peptide with 1 ml assay buffer, vortex. The concentration of this stock solution is 1,000 ng/ml.
4. Prepare peptide standard solutions as follows:
 |
|
 |
|||||||||||||||||||||||||||
|
|||||||||||||||||||||||||||||
 |
 |
 |
6. Rehydrate biotinylated peptide with 5 ml of assay buffer, vortex.
7. Leave well A-1 empty as Blank.
8. Add 50 ul assay buffer into well B-1 as Total Binding.
9. Add 50 ul of the prepared peptide standard solutions from #5 to #1 (reverse order of serial dilution) into the wells from C-1 to G-1 respectively.
10. Add 50 ul samples into their designated wells.
11. Add 25 ul rehydrated primary antiserum into each well except the Blank well.
12. Add 25 ul rehydrated biotinylated peptide into each well except the Blank well.
13. Seal the immunoplate with acetate plate sealer (APS).
14. Incubate the immunoplate for 2 hours at room temperature.
15. Centrifuge the SA-HRP vial provided in this kit (500-1,000 r.p.m., 15 seconds, 4°C) and pipet 12 ul SA-HRP into 12 ml assay buffer to make SA-HRP solution, vortex.
16. Remove APS from the immunoplate
17. Discard contents of wells.
18. Wash each well (except the Blank) with 300 ul assay buffer, discard the buffer and blot dry the plate. Repeat 5 times.
19. Add 100 ul SA-HRP solution into each well except the Blank well.
20. Reseal the immunoplate with APS.
21. Incubate for 1 hour at room temperature.
22. Wash and blot dry the immunoplate 6 times with the assay buffer as described above.
23. Add 100 ul substrate solution provided in this kit into each well including the Blank well.
24. Reseal the immunoplate with APS.
25. Incubate for 1 hour at room temperature.
26. Add 100 ul 2N HCl into each well (including the Blank) to stop the reaction. Go to the next step within 20 minutes.
27. Clean the immunoplate bottom with 70% ethanol.
28. Remove APS and load the immunoplate onto a Microtiter Plate Reader.
29. Read absorbance O.D. at 450 nM. The O.D. reading for the TB (well B-1) is usually around 0.8-1.5.
Calculation of Results
Plot the standard curve on semi-log graph paper. Known concentrations of standard peptide and its corresponding O.D. reading is plotted on the log scale (X-axis) and the linear scale (Y-axis) respectively. The standard curve shows an inverse relationship between peptide concentrations and the corresponding O.D. absorbances. As the standard concentration increases, the intensity of the yellow color, and in turn the O.D. absorbance, decreases.
The concentration of peptide in a sample is determined by plotting the sample's O.D. on the Y-axis, then drawing a horizontal line to intersect with the standard curve. A vertical line dropped from this point will intersect the X-axis at a coordinate corresponding to the peptide concentration in the unknown sample.
Summary of Assay Protocol
Storage
Store the kit at 2-4°C upon receipt. The kit will be stable for 6 months. The kit should be equilibrated to room temperature before assay.
Blood Withdrawal:
Collect blood samples into the Lavender Vacutaner tubes (#VT6450) which contain EDTA and can collect 7 ml blood/tube. Gently rock the Lavender Vacutaner tubes several times immediately after collection of blood for anti-coagulation.
Transfer the blood from the Lavender Vacutaner tubes to centrifuge tubes containing aprotinin (0.6 TIU/ml of blood) and gently rock for several times to inhibit the activity of proteinases. Centrifuge the blood at 1,600 x g for 15 minutes at 4°C and collect the plasma. Plasma kept at -70°C may be stable for one month.
It is recommended that plasma sample be diluted with EIA buffer at 1/16 ~ 1/32 to performance EIA assay.
References
1. Enzyme Immunoassay Techniques, An Overview. Porstmann, T. and Kiessig, S.T. Journal of Immunological Methods, 150 (1992) 5-21. 2. Amplification Systems in Immunoenzymatic Techniques, Avrameas, S. Journal of Immunological Mehtods, 150 (1992)23-32.
LIMITED BY FEDERAL LAW TO INVESTIGATIONAL USE
FOR RESEARCH ONLY
NOT FOR USE IN DIAGNOSTIC PROCEDURES