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Pack Size:96wells/kit, with removable strips.
Validated Species:Human
Sensitivity:3 ng/ml
Assay Range:10-400 ng/ml
Sample Type:Serum and Plasma(heparin, citrate)
Data & Images


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


Product Name Human alpha Fetoprotein OneStep ELISA Kit (AFP)
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.
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.)

Kit Components

Anti-Human AFP Pre-coated 96-well strip microplate18 strips of 12 wellsAnti-AFP monoclonal antibody, Polystyrene micro-well plate
Human AFP Standards(S1~S6)6lmlAFP(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 antibody17mlHRP Conjugated anti-Human AFP antibody, 0.02M PBS, 20% new-born calf serum, 0.01% azophloxine, 0.1%Proclin-300, from mouse monoclonal antibody
Controls21mlL(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 Concentrate115mlPhosphorous salts
Color Developing Reagent A17mlUrea hydrogen peroxide
Color Developing Reagent B17mlTMB
Stop Solution17mlDilute sulphuric acid
Plate Sealers2Piece

Material Required But Not Provided

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 µl 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.

Protein Target Info (Source:

You can check the tissue specificity below for information on selecting positive and negative control.

Gene Name AFP
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.

Human alpha Fetoprotein OneStep ELISA Kit (AFP) Images

Click the images to enlarge.

Human alpha Fetoprotein OneStep ELISA Kit (AFP)
Human alpha Fetoprotein OneStep ELISA Kit (AFP)
Human AFP ELISA Kit standard curve

Typical Data Obtained from Human AFP ELISA Kit


Warnings and Precautions

  • 1. To inspect the validity of experiment operation and the appropriateness of sample dilution proportion, pilot experiment using standards and a small number of samples is recommended.
  • 2. Before using the Kit, spin tubes and bring down all components to the bottom of tubes.
  • 3. Don’t let 96-well plate dry, for dry plate will inactivate active components on plate.
  • 4. Don’t reuse tips and tubes to avoid cross contamination.
  • 5. Avoid using the reagents from different batches together.
  • Sample Preparation and Storage

  • 1. Use a serum separator tube (SST) and allow serum to clot at room temperature for about four hours. Then, centrifuge for 15 min at approximately 1,000 x g. assay immediately or store samples at -20°C.
  • 2. Collect plasma using heparin or citrate as an anticoagulant. Centrifuge for 15 min at approximately 1,000 x g. Assay immediately or store samples at -20°C.
  • 3. Avoid multiple freeze-thaw cycles.
  • 4. Prior to assay, frozen sera should be completely thawed and mixed well.
  • Note: Grossly hemolyzed samples and chylemia samples are not suitable for use in this assay, so the samples should be centrifugated adequately and no hemolysis or granule was allowed.
  • Preparation Before The Experiment

    All reagentsBring all reagents to room temperature (20-25°C) for 30 minutes.
    20X Wash Buffer ConcentratePrepare 1X wash buffer by adding 15 ml of Wash Buffer Concentrate to 285 ml deionized or distilled water to prepare 300 mL of Wash Buffer.

    Assay Procedure

    It is recommended that all reagents and materials be equilibrated to 37°C/room temperature prior to the experiment (see Preparation Before The Experiment if you have missed this information).

  • 1. Prepare all reagents and working standards as directed previously.
  • 2. Remove excess microplate strips from the plate frame and seal and store them in the original packaging.
  • 3. Set Standard wells, Sample wells, Control wells and Blank wells, add 50 μl of the standard, sample, or control per well. At least two replicates of each standard, sample, control or blank is recommended.
  • 4. Add 50 μl of HRP Conjugated anti-Human AFP antibody to each well except for the blank well and mix thoroughly.
  • 5. Cover with plate sealer and incubate for 30 minutes at 37°C.
  • 6. Wash the plate 3 times with the 1x wash buffer.
    a. Discard the liquid in the wells into an appropriate waste receptacle. Then, invert the plate on the benchtop onto a paper towel and tap the plate to gently blot any remaining liquid. It is recommended that the wells are not allowed to completely dry at any time.
    b. Add 300 μl of the 1x wash buffer to each assay well. (For cleaner background incubate for 60 seconds between each wash).
    c. Repeat steps a-b 2 additional times.
  • 7. Add 50μl Color Developing Reagent A and 50μl Color Developing Reagent B to each well and incubate in the dark for 15 minutes at 37°C.
  • 8. Add 50 μl of Stop Solution to each well.
  • 9. Read absorbance on Plate Reader at 450 nm within 15 minutes after adding the stopping solution.
  • Calculation of Results

    Average the duplicate readings for each standard, sample, and control. Subtract the average blank O.D. reading. It is unnecessary to set blank control for dual wavelength plate reader.
    It is recommended that a standard curve be created using computer software to generate a four parameter logistic (4-PL) curve-fit. A free program capable of generating a four parameter logistic (4-PL) curve-fit can be found online at: curve.assay.
    Alternatively, plot the mean absorbance for each standard against the concentration. The measured concentration in the sample can be interpolated by using linear regression of each average relative OD against the standard curve generated using curve fitting software. This will generate an adequate but less precise fit of the data.
    For diluted samples, the concentration reading from the standard curve must be multiplied by the dilution factor.

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    Customer Q&As

    Q: How many samples can I test on one ELISA plate?
    A: On each 96 well plate, we recommend running an 8-point standard curve with duplicate wells. With the remaining 80 wells, 40 samples can be tested in duplicate.
    Q: The volumes of my samples are small, would it be possible to use 50µl/sample?
    A: There are two solutions for insufficient sample volume.
    1. Dilute the samples with the provided sample diluent buffer into 100ul.
    2. If 50ul of sample is added into a well, add 50ul of standard solution.
    Q: Will this kit work on tissue samples?
    A: For tissue homogenates, endogenous biotin should be considered. Endogenous biotin in tissue homogenates might introduce false positive signal. Please run the test as described below to confirm if there is Endogenous biotin in the tissue lysate samples using the ELISA kit.
    • Add tissue homogenates into the wells and then add ABC and TMB without adding any biotinylated detection antibody to see if any signal will be observed.
    • If no signal is produced, then you can work on the tissue sample by using the kit.
    Q: Is it possible to dilute 10X TBS stock that is 250mM Tris Base/Tris-Hcl, 27 mM KCl, and 1.37M NaCl to be used as the wash buffer?
    A: You can dilute the 10X TBS stock to 1XTBS and test the pH value of the 1xTBS.
    The 1XTBS can be used if the pH value falls in the range of 7.2-7.6.
    Q: Would your kits work normally with samples from Wistar Rat?
    A: Our kits would work normally with samples from Wistar Rat.
    Q: How can I store the dissolved wash buffer for a longer period of time?
    A: You can prepare a concentrated wash buffer (25X) which can be stored at 4°C for 1-3 months.
    Q: Does the TMB color developing agent contain H2O2?
    A: Yes, the TMB color developing agent contain H2O2.
    Q: Does this ELISA kit contain any product produced in humans or primates?
    Keywords: component, ingredient
    A: None of the components in our ELISA kits are produced in humans or primates.
    Q: What is the volume of the recombinant protein control? What is its shelf life?
    Keywords: expiration, storage, temperature, size
    A: The volume of the control is around 100pg. If unopened, the shelf life of the control is the same as the whole kit - "Store at 4˚C for 6 months, at -20˚C for 12 months." The reconstituted control can be stored at -20˚C for 2 days.
    Q: Will it be okay to run the experiment if I accidentally used the wrong buffer (e.g. sample diluent buffer) for antibody dilution instead of the antibody diluent buffer? Keywords: dilution, replace, substitute, sample buffer
    A: Using the sample diluent buffer for antibody dilution instead of the antibody diluent buffer will negatively affect the test result to some extent. The values of both standard and sample might be lower than the normal values.
    Q: The protocol says to use neutral PBS and provides a recipe. Could I use neutral PBS made with KCl or KH2PO4 (common constituents for most PBS recipes) instead? Keywords: protocol, alternative buffer, applications, reagent recipe
    A: Yes, it is ok to use common PBS recipes. We have tried many types of buffers with common constituents, and no significant difference was observed whatsoever.
    Q: Why are my O.D. values different than your values on the datasheet? Keyword: troubleshooting, reference
    A: We detected the kit in our lab and got our values on the datasheet before delivery, but protein activity will decrease as time goes on, so you may get lower O.D. values than ours. However, it should still be in a reasonable range and the standards can be used to calculate sample values. Good linearity of curve is more important than the actual numerical value.
    Q: On the ELISA kit datasheet it says "HRP substrate TMB was used to visualize HRP enzymatic reaction." What is HRP and which part of the kit contains HRP? Keyword: kit components, reagents, protocol
    A: It means Avidin-Biotin-Peroxidase Complex(AR1103) and you could find it in Kit Components.
    Q: Do you offer any ELISA kits that can work with whole blood samples instead of plasma or urine? Keyword: applications
    A: We test serum and plasma routinely, and there is very little difference between serum, plasma and whole blood. The whole blood also contains proteins we need to test. The kit can be used to test whole blood in theory.
    Q: Does silicic acid (formula SiOH4) affect the results of the ELISA assay? Keyword: protocol, reagents
    A: The acid may affect the binding of antigen to antibody, it is not recommended to use. That being said, it is unlikely to affect the reaction if the solution remains an overall neutral environment.
    Q: Is there lot-to-lot variation of the ELISA kits? What are Boster's general services when I have questions about your kits? Keyword: technical support, help, customer service
    A: The NIBSC/WHO 1st International Standard is evaluated, we always test our kits before delivery, and customers can find the test result on the protocol. If the customer needs technical support from us to analyze their data, please contact us and we ask that you provide us the following information: the lot# and production date, and when did the customer detect the kit.
    Q: The results of my standards do not look correct, what could be the problem? Keyword: troubleshoot, protocol
    A: Double check the protocol for plate washing. It should be examined on three aspects: 1) Did you make the washing buffer correctly? 2) How long did you let the buffer stay in wells? 3) What was the volume of the washing buffer added to each well? In addition, pay attention when adding sample to avoid contamination. And we suggest testing the standards again. Values of standards at low concentration are more affected than that at higher concentration, so customer can still get expected values at high concentration even if there is an error. If problems persist, please contact technical support with the catalog and lot number of your product.
    Q: What is the well depth of the 96 well plates for the ELISA kits? Keyword: well capacity, product size
    A: The well depth is 300ul, and the max capacity is 350ul. The height of the well is 1.1 cm, and the internal size is 1 cm.
    Q: The kit does not include wash solution, what should I do? Keyword: wash buffer
    A: Our Elisa kits do not come with wash solution, but we have included information about how to make washing buffer on the datasheet. Please refer to the "Material Required But Not Provided" section. We also offer washing buffer for sale separately (Phosphate Buffered Saline Powder SKU: AR0030).
    Q: For your ELISA kits, are the capture and detection antibodies polyclonals or monoclonals? Keyword: antibody clonality
    A: This information can be found for each kit under the "Properties" section, and you can find the immunogen sequence information in the "Overview" section.
    Q: Do your ELISA kits come with sealants or plate covers? Keyword: storage, sequential use
    A: The kit can be used within a month sequentially if it's opened and stored at 4 degree. There is no need to use sealants, the plate can be packaged with aluminum foil bag, and for other reagents keep bottle tightly closed.
    Q: What is the concentration of Sodium Azide in your Elisa kits? Keyword: preservative
    A: Our Elisa kits contain 0.02% Sodium Azide. All of the components except TMB colour developing reagent and stop solution contains 0.02% Sodium azide as the preservative.
    Q: Are there positive and negative controls available for my ELISA kit? Keyword: positive control, negative control
    A: We can provide a recombinant protein as a control. It costs $50 per control and takes 2 weeks to manufacture. We cannot provide a positive or negative control in serum.
    Q: Why do I get positive results for my knockout (KO) model when used as a control?
    A: The knockout (KO) model may contain truncated forms of the target protein which can be detected by the ELISA assay.
    Q: How long do I soak my plate in the wash buffer?
    A: The plate should soak in 0.3mL PBS or TBS wash buffer for at least 1-2 minutes in an automated wash station.
    Q: Can I use Tween in my wash buffer?
    A: While it is not recommended to use Tween in your wash buffer, small amounts (<0.1% concentration) may decrease background due to insufficient blocking.
    Q: What plate type do I use to set up the microplate reader?
    A: Our plates are made with the Corning costar plates similar to the DNA-BIND 96 -well plates.
    Q: What should I use for negative control?
    A: Please contact us for negative control suggestions. You can also check expression databases such as genecards, uniprot etc. Due to logistic reasons, we do not sell serum or lysates that we use internally for positive or negative control.
    Q: Where can I find troubleshooting information? What should I do if I have unexpected bands, high background, no signal, weak signal
    A: You can find Boster's troubleshoot guides under tech support tab. Please contact us for further assistance on troubleshooting your experiment.
    Q: What is the normal level of this protein in my sample of interest?
    A: We have reviewed literature and have gathered this information for most of our ELISA kits. You can find this information on the product page or contact us if you cannot find it. However this information is only suggestive and cannot replace pilot studies in determining the optimal sample dilution ratio.
    Q: Is the plate separable? Can I use only part of the kit and save the rest for later? How many samples can I run with one kit?
    A: Yes the plate is separable if the kit says that there are removable strips. There are 12 strips of 8 wells each, all removable from the plate. The amount of samples you can run depend on a few factors. In the most common ELISA set up, you will use two strips for standards, and 10 strips for samples using duplicates, which let you run up to 40 samples per kit. Contact us if you have questions regarding other situations.
    Q: Does the kit contain sample preparation reagents? How do I prepare my samples?
    A: Since different sample types require different reagents, we do not include them in the ELISA kit. However we do have each reagent mentioned in the file below available at very reasonable prices. Be sure to check them out. For sample preparation protocols please download the file below:
    Q: Can this ELISA kit work on brain tissue homogenate, cell culture supernatant, saliva, urine, serum, whole blood or any other sample type?
    A: In theory the ELISA kit will work for all sample types if the target protein is present at a level that falls within the linear range of the ELISA kit detection range. We guarantee the kit to work on the sample types that we have tested. If you want dilution ratio suggestions on these sample types please contact our technical support. For sample types that we have not tested for, we suggest you run pilot experiments to decide the optimal sample dilution.
    Q: Can this ELISA kit react with the pro-form of the target protein? Can this ELISA kit react with an isoform of the protein?
    A: In general, unless otherwise specified, the ELISA kit is pan-specific, meaning that it will react with all different forms of the target protein if they share the majority of the target protein's sequence. The capture and detection antibodies are reactive to the entire sequence of the standard protein. You can find the sequence information of the standard protein in the "immunogen" section. For more information about the specificity of the kit for your particular experiment, please contact our technical support.
    Q: Can this ELISA kit react with human, mouse, rat or other species?
    A: If the kit is reactive to another commonly used species (human, mouse, and/or rat), we would have listed it as a separate product. If you want to check cross-reactivity to a species that is not included in the 3 species listed above, please contact our technical support for more information. As a rule of thumb, if the sequences are 90%+ identical, there is a high chance of cross-reactivity for your species of interest.