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CaptSure™ DIY ELISA

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Immunoassays are the gold-standard method for the detection of proteins and biomarkers in discovery processes. However, ELISA development usually involves costly plate coating, antibody pair screening, long development and optimization times, and variable success rates. These limitations are now solved by CaptSure DIY ELISA.

CaptSure DIY ELISA is the latest cutting-edge ELISA kit with a built-in system for antibody labeling, enabling the rapid and easy development of in-house ELISAs using any antibody pair while eliminating the need to perform plate coating procedures.

Furthermore, the CaptSure DIY ELISA enables you to reduce the amount of capture antibody required, the ability to measure different targets on the same plate with different assay antibody pairs, and streamline your ELISA to a single-wash format if desired.

The CaptSure DIY ELISA optimized protocol results in faster ELISA development, reduced overall costs, and a greater assay flexibility to increase the research output with a reduced chance of error.

How does CaptSure DIY ELISA work?

CaptSure DIY ELISA is powered by the combination of Expedeon’s proprietary CaptSure and Lightning-Link technologies.

Unlike standard ELISA systems, the CaptSure DIY ELISA kit has a ready-to-go CaptSure assay plate, pre-coated with our CaptSure antibody, which is specific for the CaptSure peptide. The capture antibody is quickly and easily conjugated with the Lightning-Link CaptSure peptide supplied within the kit.  Once added to the plate, the interaction between the CaptSure antibody and the CaptSure peptide immobilizes the immuno-complex to the plate.

The detection antibody is similarly conjugated with HRP using the Lightning-Link HRP conjugation reagent, also supplied in the kit. Thus, with minimal hands-on time, you can set up both of your antibodies to ensure the best performance of your ELISA.

 

CaptSure DIY ELISA how it works image

Lightning-Link is the world’s fastest conjugation technology to label antibodies and proteins by targeting free amine groups. As amine groups are ubiquitous and numerous in antibodies, any antibody can be labeled with the Lightning-Link CaptSure peptide and Lightning-Link HRP kits.

This labeling protocol is comprised of just a few simple steps:

  1. Resuspend the lyophilized Lightning-Link CaptSure peptide and Lightning-Link HRP with the solution of capture and detection antibodies, respectively;
  2. Incubate each at room temperature for the specified times;
  3. Quench the reactions, and your labeled capture and detection antibodies are ready to use for the ELISA.

CaptSure DIY ELISA labeling protocol image

CaptSure DIY ELISA workflow

 

The workflow of an ELISA utilizing the CaptSure system can be either multi-wash or single-wash, depending on your laboratory setup and desired level of optimization and automation.

In a multi-wash format, the assay sequence is very similar to that of a standard ELISA (bottom, left panel). The assay can, however, be simplified to a single-wash format, where the assay antibody concentrations are first optimized for the target concentration (bottom, right panel).

 

A more detailed diagram of a single-wash CaptSure ELISA is shown below. To set up your ELISA for a single-wash protocol, the concentration of CaptSure– and HRP- conjugated antibodies must first be optimized to be at the correct concentration for your analyte. Typically, this will be approximately 100-200 ng/mL final concentration in well of each antibody to a final volume of 100 μL in the well.

 

CaptSure DIY ELISA single-wash workflow

 

CaptSure DIY ELISA single-wash workflow image

  1. Label your capture and detection antibodies of choice to Lightning-Link CaptSure peptide and Lightning-Link HRP, respectively
  2. Add the labeled antibody pair and the sample into the plate
  3. Incubate for one hour
  4. Wash the plate and detect

Work with us

Expedeon can provide assay development services, including CaptSure™ assay development services to partners, as well as aspects of product validation, up to full product manufacture.

For further information about collaboration, partnership, and licensing please get in contact with us.

Ask the expert >

CaptSure DIY ELISA and conventional passive coating approach were compared. With CaptSure DIY ELISA, 10x less capture antibody can be used without affecting the assay performance.CaptSure DIY ELISA and conventional passive coating approach were compared graph

Other solution-phase binding ELISA formats typically use biotin to immobilise immunocomplexes. These can constrain the sample types that can be used with the assay. In contrast, CaptSure DIY ELISA has been specifically designed to be used with most sample matrices without interference.

 

CaptSure DIY ELISA phase binding graph

 

1. Can I use a different ELISA plate to the one that is supplied?

CaptSure Assay Plates are specific to CaptSure DIY ELISA and cannot be substituted with any other type of 96 well microplate. CaptSure Assay Plates, which rely on our CaptSure technology, are optimized to provide an active surface for binding the immunocomplex by means of the interaction between the CaptSure antibody precoated onto the plate and the CaptSure peptide conjugated to the capture antibody.

2. How are CaptSure Assay Plates supplied?

CaptSure Assay Plates are supplied in a 12×8 strip well plate. The plate format allows the user to use only the number of the strip wells needed, without incurring additional waste when performing small scale experiments.

3. How are CaptSure Assay Plates stored?

CaptSure Assay Plates can be stored at +4°C or room temperature. If stored at +4°C, the strip wells should be allowed to equilibrate to room temperature prior to opening the pouch to minimize condensation.

4. I did not use all the strip wells supplied with my CaptSure DIY ELISA kit. How should I store the remaining strip wells?

Unused strip wells should be removed from the frame, placed back in their foil pouch, sealed with tape, and stored at +4°C. Unused strip wells should be used within one month from the date of opening.

5. Does the CaptSure Assay Plate need to be blocked before use?

No, the CaptSure Assay Plate does not need to be blocked. CaptSure Assay Plates are pre-coated and ready to use.

6. Which cell types or other samples are compatible with CaptSure DIY ELISA?

CaptSure DIY ELISA can be used for many adherent and nonadherent cell types, including transfected cell lines and primary cells. The concentration of cell lysate should be optimized to ensure that the signal is within the working range of the assay.

Similarly, CaptSure DIY ELISA is also applicable to measurement of secreted proteins in plasma and serum. Should this type of analyte be measured, a sample dilution buffer is provided. It may be necessary to optimize dilutions of the sample, and potentially also use commercially-available matrix blockers (e.g. HAMA blockers).

7. Is cell culture supernatant compatible with CaptSure DIY ELISA?

CaptSure DIY ELISA is compatible with cell culture supernatants, and with many other media. However, factors affecting antibody binding generally can alter assay outputs. Compatibility should be assessed on a case by case basis.

8. What’s the difference between Sample Dilution Buffer (Secreted Proteins) and Lysis Extraction Buffer (5x)?

The Sample Dilution Buffer (Secreted proteins) is the suggested buffer for diluting secreted protein samples or preparing recombinant protein standard curves. The buffer is ready to use.

The Lysis Extraction Buffer (5x) is suggested for use with cellular proteins and lysing cells and it must be diluted before use according to the type of cells being analysed.
For adherent cells, dilute the Lysis Extraction Buffer (5x) with milliQ water to 1X final concentration. Remove medium from cells prior to addition of 1X Lysis Extraction Buffer.
For suspension cells, add 1/5th volume of Lysis Extraction Buffer (5X) to the cells in medium.
This Lysis Extraction Buffer (5x) has been especially validated for use on CaptSure Assay Plate.

9. Are other lysis buffers (e.g. RIPA buffer) compatible with CaptSure DIY ELISA?

Optimal performance is ensured with the Lysis Buffer (5X) provided with CaptSure DIY ELISA. Compatibility with other cell lysis buffers should be assessed on a case by case basis.

10. What can I label using Lightning-Link?

Lightning-Link technology works by targeting free amine groups on your target. It can be used to label antibodies, peptides, proteins and any other molecules with free amine groups.

For advice on labeling molecules other than antibodies please contact our technical support team.

11. How pure does my antibody need to be?

Your antibody must be purified because other molecules with free amine groups will interfere with the reaction resulting in a poor quality conjugate. If your antibody solution contains any of the following substances, you might need to purify your antibody:

12. How do I purify my antibody?

A suitable antibody purification method can be selected among the following ones:

  • Affinity purification – this is the preferred method as it will always result in a purified sample containing only the antibody of interest.
  • Protein A or G purification of serum – this will result in a purified sample of all IgG’s contained in the serum. You will have a purified IgG fraction but only a small percentage of this fraction will be the IgG of interest. This IgG fraction will label with our kits, although adjustments in conjugate dilutions will be required.
  • Protein A or G purification of tissue culture supernatant or ascites fluid – this method will generate purified antibodies equivalent to affinity purification.

Other purification methods, such as ion exchange chromatography and ammonium sulphate precipitation, are low quality purification methods and should be avoided. Filtration using a 0.2/0.22 µm filter is a method of sterilization not purification, and is therefore not appropriate.

13. Is my antibody in a suitable buffer?

For recommended buffer conditions please see the below table:

14. What can I do if my antibody formulation does not fit the requirements?

If your antibody requires purification or the buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link kits.

The appropriate kit to use depends on your particular sample (species, buffer, contaminants, volume, etc.). We have designed a handy flow chart to help you select a kit (please see below) but feel free to contact us if you require further guidance. Please consult the individual kit protocols to see the antibody amount/volume suitable for each kit.

If your antibody is already purified but its concentration is significantly lower than 1mg/ml, you can concentrate it using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link.

If your antibody contains BSA, you can now use our BSA removal kit to purify your antibody in a few simple steps. Please note this kit will also enable you to concentrate your antibody.

NB: All the AbSelect kits will ONLY work with antibodies. They will not purify other molecules. The only exception is the concentration and clean up kit (861-0010), which will work with other molecules greater than 10kD.

15. Do I need a wash or desalt step?

One of the advantages of the Lightning-Link technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. This remaining free label would have its reactive ‘Lightning-Link’ groups blocked by the Quencher provided in the kit, and is washed away during the wash step of your application.

16. How stable will my new conjugate be?

Upon dissolution of the Lightning-Link mixture with a solution of the antibody, proprietary chemicals in the mixture become activated. This results in the directional covalent bonding of the antibody to the label in a gentle and controlled process at near-neutral pH. The stability of your conjugate is dependent on your antibody and label of choice.

Most Lightning-Link conjugates can be stored for up to 18 months, undiluted at 4°C. The exception to this is tandem conjugates which are only stable for up to 3 months.

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CaptSure DIY ELISA is the latest cutting-edge ELISA kit with a built-in system for antibody labeling, enabling the rapid and easy development of in-house ELISAs using any antibody pair while eliminating…

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