For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome

Hello. We're improving abcam.com and we'd welcome your feedback.

Hello. We're improving abcam.com and we'd welcome your feedback.

Infomation icon

We haven't added this to the BETA yet

New BETA website

New BETA website

Hello. We're improving abcam.com and we'd welcome your feedback.

Take a look at our BETA site and see what we’ve done so far.

Switch on our new BETA site

Now available

Search and browse selected products

  • A selection of primary antibodies

Purchase these through your usual distributor

In the coming months

  • Additional product types
  • Supporting content
  • Sign in to your account
  • Purchase online
United States
Your country/region is currently set to:

If incorrect, please enter your country/region into the box below, to view site information related to your country/region.

Call (888) 77-ABCAM (22226) or contact us
Need help? Contact us

  • My account
  • Sign out
Sign in or Register with us

Welcome

Sign in or

Don't have an account?

Register with us
My basket
Quick order
Abcam homepage

  • Research Products
    By product type
    Primary antibodies
    Secondary antibodies
    ELISA and Matched Antibody Pair Kits
    Cell and tissue imaging tools
    Cellular and biochemical assays
    Proteins and Peptides
    By product type
    Proteomics tools
    Agonists, activators, antagonists and inhibitors
    Cell lines and Lysates
    Multiplex miRNA assays
    Multiplex Assays
    By research area
    Cancer
    Cardiovascular
    Cell Biology
    Epigenetics
    Metabolism
    Developmental Biology
    By research area
    Immunology
    Microbiology
    Neuroscience
    Signal Transduction
    Stem Cells
  • Customized Products & Partnerships
    Customized Products & Partnerships

    Customized products and commercial partnerships to accelerate your diagnostic and therapeutic programs.

    Customized products

    Partner with us

  • Support
    Support hub

    Access advice and support for any research roadblock

    View support hub

    Protocols

    Your experiments laid out step by step

    View protocols

  • Events
    • Conference calendar
    • Cancer
    • Cardiovascular
    • Epigenetics & Nuclear signaling
    • Immunology
    • Neuroscience
    • Stem cells
    • Tradeshows
    • Scientific webinars
    Keep up to date with the latest events

    Full event breakdown with abstracts, speakers, registration and more

    View global event calendar

  • Pathways
    Cell signalling pathways

    View all pathways

    View all interactive pathways

Indirect ELISA protocol

Related

  • ELISA resources

    ​General procedure and tips for ELISA assay requiring a secondary conjugated antibody. 

    ​Print this protocol

    Buffers and reagents

    See direct ELISA protocol buffers and reagents.

    For accurate quantitative results, always compare signal of unknown samples against those of a standard curve. Standards (duplicates or triplicates) and blank must be run with each plate to ensure accuracy.

    General procedure

    Coating antigen to microplate

    1. Dilute the antigen to a final concentration of 20 µg/ml in PBS or other carbonate buffer. Coat the wells of a PVC microtiter plate with the antigen by pipetting 50 µl of the antigen dilution in the top wells of the plate. Dilute down the plate as required.



      Test samples containing pure antigen are usually pipetted onto the plate at less than 2 µg/ml. Pure solutions are not essential, but as a guideline, over 3% of the protein in the test sample should be the target protein (antigen). Antigen protein concentration should not be over 20 µg/ml as this will saturate most of the available sites on the microtiter plate.

      Ensure the samples contain the antigen at a concentration that is within the detection range of the antibody.



    2. Cover the plate with an adhesive plastic and incubate for 2 hr at room temperature, or 4°C overnight. The coating incubation time may require some optimization.
    3. Remove the coating solution and wash the plate three times by filling the wells with 200 µl PBS. The solutions or washes are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel.


    Blocking

    1. ​Block the remaining protein-binding sites in the coated wells by adding 200 µl blocking buffer, 5% non-fat dry milk or 5% serum in PBS, per well. Alternative blocking reagents include blockACE or BSA.
    2. Cover the plate with an adhesive plastic and incubate for at least 2 hr at room temperature or, if more convenient, overnight at 4°C.
    3. Wash the plate twice with PBS.


    Incubation with primary and secondary antibody

    1. Add 100 µl of diluted primary antibody to each well.
    2. Cover the plate with an adhesive plastic and incubate for 2 hr at room temperature.



      This incubation time may require optimization. Although 2 hr is usually enough to obtain a strong signal, if a weak signal is obtained, stronger staining will often be observed when incubated overnight at 4°C.


    3. Wash the plate four times with PBS.
    4. Add 100 µl of conjugated secondary antibody, diluted at the optimal concentration (according to the manufacturer) in blocking buffer immediately before use.
    5. Cover the plate with an adhesive plastic and incubate for 1-2 hr at room temperature.
    6. Wash the plate four times with PBS.


    Detection

    Although many different types of enzymes have been used for detection, horse radish peroxidase (HRP) and alkaline phosphatase (ALP) are the two widely used enzymes employed in ELISA assay. It is important to consider the fact that some biological materials have high levels of endogenous enzyme activity (such as high ALP in alveolar cells, high peroxidase in red blood cells) and this may result in non-specific signal. If necessary, perform an additional blocking treatment with levamisol (for ALP) or with 0.3% solution of H2O2 in methanol (for peroxidase).

    ALP substrate
    For most applications pNPP (p-Nitrophenyl-phosphate) is the most widely used substrate. The yellow color of nitrophenol can be measured at 405 nm after 15-30 min incubation at room temperature. (This reaction can be stopped by adding equal volume of 0.75 M NaOH). 

    HRP chromogens
    The substrate for HRP is hydrogen peroxide. Cleavage of hydrogen peroxide is coupled to oxidation of a hydrogen donor which changes color during reaction.

    TMB (3,3',5,5'-tetramethylbenzidine)
    Add TMB solution to each well, incubate for 15-30 min, add equal volume of stopping solution (2 M H2SO4) and read the optical density at 450 nm.

    OPD (o-phenylenediamine dihydrochloride)
    ​The end product is measured at 492 nm. Be aware that the substrate is light sensitive so keep and store it in the dark. 

    ABTS (2,2'-azino-di-[3-ethyl]-bensothiazoline-6 sulfonic acid) diammonium salt.
    The end product is green and the optical density can be measured at 416 nm. 



    Some enzyme substrates are considered hazardous (potential carcinogens), therefore always handle with care and wear gloves.


    1. ​Dispense 100 µl (or 50 µl) of the substrate solution per well with a multichannel pipette or a multipipette. 
    2. After sufficient color development (if it is necessary) add 100 µl of stop solution to the wells. 
    3. Read the absorbance (optical density) of each well with a plate reader.


    Analysis of data

    Prepare a standard curve from the data produced from the serial dilutions with concentration on the x axis (log scale) vs. absorbance on the Y axis (linear). Interpolate the concentration of the sample from this standard curve.

    View more of our ELISA resources.

    Get resources and offers direct to your inbox Sign up
    A-Z by research area
    • Cancer
    • Cardiovascular
    • Cell biology
    • Developmental biology
    • Epigenetics & Nuclear signaling
    • Immunology
    • Metabolism
    • Microbiology
    • Neuroscience
    • Signal transduction
    • Stem cells
    A-Z by product type
    • Primary antibodies
    • Secondary antibodies
    • Biochemicals
    • Isotype controls
    • Flow cytometry multi-color selector
    • Kits
    • Loading controls
    • Lysates
    • Peptides
    • Proteins
    • Slides
    • Tags and cell markers
    • Tools & Reagents
    Help & support
    • Support
    • Make an Inquiry
    • Protocols & troubleshooting
    • Placing an order
    • RabMAb products
    • Biochemical product FAQs
    • Training
    • Browse by Target
    Company
    • Corporate site
    • Investor relations
    • Company news
    • Careers
    • About us
    • Blog
    Events
    • Tradeshows
    • Conferences
    International websites
    • abcam.cn
    • abcam.co.jp

    Join with us

    • LinkedIn
    • facebook
    • Twitter
    • YouTube
    • Terms of sale
    • Website terms of use
    • Cookie policy
    • Privacy policy
    • Legal
    • Modern slavery statement
    © 1998-2022 Abcam plc. All rights reserved.