The Bosterbio OneStep Human AFP Pre-Coated ELISA (Enzyme-Linked Immunosorbent Assay) kit is a solid phase immunoassay specially designed to measure Human AFP with a 96-well strip plate that is pre-coated with antibody specific for AFP. The detection antibody is a HRP conjugated antibody specific for AFP. The capture antibody is monoclonal antibody from mouse, and the detection antibody is monoclonal antibody from mouse. The kit is analytically validated with ready to use reagents. To measure Human AFP, add standards and samples to the wells, then add the HRP conjugated detection antibody. Wash away the unbounded protein and HRP conjugated detection antibody. TMB is substrate to HRP and will be catalyzed to produce a blue color product, which changes into yellow after adding acidic stop solution. Upon addition of the substrate, the density of the yellow product is linearly propotional to Human AFP in the sample. Read the density of the yellow product in each well using a plate reader, and benchmark the sample wells' readings against the standard curve to determine the concentration of Human AFP in the sample. For more information on assay principle, protocols, and troubleshooting tips, see Boster's ELISA Resource Center at https://www.bosterbio.com/elisatechnical-resource-center.
Product Name | Human alpha Fetoprotein OneStep ELISA Kit (AFP) |
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SKU/Catalog Number | EK7020 |
Description | Sandwich High Sensitivity ELISA kit for Quantitative Detection of Human AFP. 96wells/kit, with removable strips. |
Cite This Product | Human alpha Fetoprotein OneStep ELISA Kit (AFP) (Boster Biological Technology, Pleasanton CA, USA, Catalog # EK7020) |
Validated Species | Human |
Application | ELISA *Our Boster Guarantee covers the use of this product in the above tested applications. **For positive and negative control design, consult "Tissue specificity" under Protein Target Info. |
Cross Reactivity | There is no detectable cross-reactivity. |
Pack Size | 96wells/kit, with removable strips. |
Sensitivity | 3 ng/ml *Sensitivity, or Lower Limit of Detection (LLD), is the minimum level of target protein the ELISA assay can detect. We measure 20 blank wells and if the O.D. value is 2 standard deviations higher than the blanks' average O.D. the sample can be deemed positive. |
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Assay Range | 10-400 ng/ml *This assay range is determined using common samples. For samples with low target protein concentrations, users can adjust the standard curve to extend the lower limit of assay range. |
Sample Type | Serum and Plasma(heparin, citrate) *The above listed samples are the ones valided with the assay. If you do not see your sample of interest listed, as long as there is enough level of target protein present in the sample, this Picokine? ELISA kit should detect it. **For protocol and tips regarding preparing your sample of interest, please check our ELISA sample preparation guide. |
Storage | Store the kit at 2°C to 8°C. Keep microwells sealed in a dry bag with desiccants. The reagents are stable until expiration of the kit. Do not expose reagent to heat, sun, or strong light. Avoid multiple freeze-thaw cycles(Shipped with wet ice.) |
Description | Quantity | Volume | Buffers |
Anti-Human AFP Pre-coated 96-well strip microplate | 1 | 8 strips of 12 wells | Anti-AFP monoclonal antibody, Polystyrene micro-well plate |
Human AFP Standards(S1~S6) | 6 | lml | AFP(10, 20, 50, 100, 200, 400) ng/ml, 0.02M PBS, 20% new-born calf serum, 0.1%Proclin-300, from natural protein |
HRP Conjugated anti-Human AFP antibody | 1 | 7ml | HRP Conjugated anti-Human AFP antibody, 0.02M PBS, 20% new-born calf serum, 0.01% azophloxine, 0.1%Proclin-300, from mouse monoclonal antibody |
Controls | 2 | 1ml | L(24.2-45) H(101.6~188.8) ng/ml, 0.02M PBS, 20% new-born calf serum, 0.1%Proclin-300, from natural protein |
20X Wash Buffer Concentrate | 1 | 15ml | Phosphorous salts |
Color Developing Reagent A | 1 | 7ml | Urea hydrogen peroxide |
Color Developing Reagent B | 1 | 7ml | TMB |
Stop Solution | 1 | 7ml | Dilute sulphuric acid |
Plate Sealers | 2 | Piece |
1. Microplate Reader capable of reading absorbance at 450nm.
2. Automated plate washer (optional)
3. Pipettes and pipette tips capable of precisely dispensing 0.5 ?l through 1 ml volumes of aqueous solutions. Multichannel pipettes are recommended for large amount of samples.
4. Deionized or distilledwater.
5. 500ml graduated cylinders.
6. Test tubes for dilution.
You can check the tissue specificity below for information on selecting positive and negative control.
Gene Name | AFP |
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Uniprot ID | P02771 |
Reseach Areas | Cancer, Cardiovascular, Developmental Biology, Organogenesis, Serum Proteins, Tumor Antigens, Tumor Biomarkers| |
*if product is indicated to react with multiple species, protein info is based on the human gene.
Click the images to enlarge.
Human AFP ELISA Kit standard curve
1. For Research Use Only. Not for use in diagnostic procedures.
2. For Laboratory use.
3. Not for Internal or External Use in Humans or Animals.
4. There should be no eating or drinking within work area.
5. Always wear gloves and a protective lab coat.
6. No pipetting should be done by mouth. Handle all specimens and reagents as potentially infectious and biohazardous.
7. Do not add sodium azide to samples as preservative.
8. Do not use external controls containing sodium azide.
9. Use disposable pipette tips to avoid contaminating chromogenic substrate reagent. Discard reagent if it turns blue.
10. Do not pour chromogenic substrate back into container after use.
11. Do not freeze reagents.
12. Do not mix reagents from different kit lot numbers.
13. Keep reagents out of direct sunlight.
14. Handle stop reagent with care, since it is corrosive.
15. Bring all reagents to room temperature.
16. Viscous forensic samples should always be diluted in phosphate buffered saline or distilled water prior to pipetting.
17. Ensure the bag containing the micro-plate strips and desiccant is sealed well, if only a few strips are used.
1. Collect blood specimens and separate the serum immediately.
2. Typically, specimens may be stored refrigerated at (2-8°C) for 5 days. If storage time exceeds 5 days, store frozen at (-20. C) for up to one month.
3. Avoid multiple freeze-thaw cycles.
4. Prior to assay, frozen sera should be completely thawed and mixed well.
5. Do not use grossly lipemic specimens.
Prepare 1X Wash buffer by adding the contents of the bottle (25 ml, 20X) to 475 ml of distilled or deionized water. Store at room temperature (20-25. C).
Prior to assay, allow reagents to stand at room temperature. Gently mix all reagents before use.
1. Place the desired number of coated strips into the holder
2. Pipette 25ul of AFP standards, control and patient's sera.
3. Add 100ul of Anti-AFP-Biotin Reagent to all wells and mix for 20-30 seconds.
4. Cover the plate and incubate for 30 minutes at room temperature (20-25. C).
5. Remove liquid from all wells. Wash wells three times 300ul with 1X wash buffer. Blot on absorbent paper towels.
6. Add 100 ul of the Anti-AFP- Enzyme conjugate to all wells. Cover and incubate for 30 minutes.
7. Remove liquid from all wells. Wash wells three times 300ul with 1X wash buffer. Blot on absorbent paper towels.
8. Add 100ul of TMB substrate to all wells.
9. Incubate for 15 minutes at room temperature.
10. Add 50ul of stop solution to all wells. Shake the plate gently to mix the solution.
11. Read absorbance on ELISA Reader at 450 nm within 15 minutes after adding the stopping solution.
The standard curve is constructed as follows:
1. Check AFP standard value on each standard vial. This value might vary from lot to lot. Make sure you check the value on every kit. See example of the standard attached.
2. To construct the standard curve, plot the absorbance for the hCG standards (vertical axis) AFP standard concentrations in ng/ml (horizontal axis) on a linear graph paper. Draw the best curve through the points.
3. Read the absorbance for controls and each unknown sample from the curve. Record the value for each control or unknown sample.
1. Do not use sodium azide as preservative. Sodium azide inhibits HRP enzyme activities.
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