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Enzyme labels for immunodetection

One of the most popular methods of immunodetection is to pair an enzyme-labeled antibody with an appropriate substrate. Enzymatic conversion of the substrate produces a colored, fluorescent or chemiluminescent readout, which can be used to determine the quantity of antigen present in the test sample. The most commonly used immunodetection enzymes are Horseradish Peroxidase (HRP), Alkaline Phosphatase and Glucose Oxidase.

Horseradish Peroxidase (HRP)

HRP is an enzyme of approximately 40kDa which is isolated from the roots of the horseradish plant, Armoracia rusticana. It has many different isoforms, and utilizes hydrogen peroxide to oxidize a wide variety of organic and inorganic compounds. The activity of HRP is inhibited by sodium azide.

Alkaline Phosphatase

Alkaline Phosphatase, as the name suggests, is a phosphatase enzyme which functions optimally at alkaline pH. It exists as several different isoforms, named according to the tissue in which they are expressed. The most commonly used isoform is isolated from bovine intestine, and exists as a homodimer with an approximate molecular weight of 150kDa.

Glucose Oxidase

Glucose oxidase is an enzyme which is produced by the fungus Aspergillus niger. It catalyzes the oxidation of β-D-glucose to form D-glucono-1,5-lactone and hydrogen peroxide, and exists as a homodimer with an approximate molecular weight of 160kDa.

Lightning-Link® for direct antibody labeling

Our Lightning-Link® kits enable direct labeling of antibodies, proteins, peptides or any other biomolecule with free amine groups to enzymes such as HRP, Alkaline Phosphatase or Glucose Oxidase. To produce the conjugate, the biomolecule is simply pipetted into a vial of lyophilized mixture containing the label of interest, incubated, and is then ready for use in the downstream application. Since no separation or washing steps are necessary, antibody recovery is 100%. Furthermore, the kits are fully scalable, allowing easy transfer from R&D to manufacturing.

The Lightning-Link® conjugation process.

The Lightning-Link® antibody labeling process

 

Our Lightning-Link® kits have been widely literature cited, with over 300 references to date. Approximately one third of these articles describe the use of Lightning-Link® for direct conjugation with enzyme labels.

LifeXtend HRP Conjugate Stabilizer for protection of antibody-HRP conjugates

The performance of antibody-HRP conjugates is, unfortunately, known to diminish over time due to inactivation of the HRP, microbial attack, or denaturation of the antibody. This loss of performance accelerates with increasing temperature and with increasing dilution of the conjugates, and as such HRP conjugates are often kept in a concentrated form at 4oC. To protect antibody-HRP conjugates, we have developed LifeXtend™ HRP Conjugate Stabilizer, a multi-component reagent system that contains proprietary enzyme stabilizers and antimicrobial agents, as well as blockers to enhance signal to background in downstream applications.

Comparison of different HRP conjugate stabilizer solutions by ELISA.

Comparison of different HRP conjugate stabilizer solutions by ELISA. HRP-conjugated goat anti-rabbit IgG (1mg/ml) was diluted 1:10,000 in each of four stabilizers and incubated for 12 days at 37oC. An undiluted control sample was incubated at 4oC for the same period and diluted 1:10,000 prior to ELISA testing. Conjugates were incubated on a rabbit IgG-coated plate for 1 hour at room temperature, followed by washing and determination of HRP activity using ABTS substrate. LifeXtend HRP Conjugate Stabilizer affords the greatest protection, and can substantially extend the shelf life of antibody-HRP conjugates.

HRP Check&Go! for confirming successful antibody conjugation to HRP

All our Lightning-Link® antibody labeling kits have been carefully designed to guarantee successful conjugation provided the protocol is followed correctly, but for extra peace of mind we have developed our Conjugate Check&Go! product range. This includes HRP Check&Go!, a dipstick lateral flow assay for confirming the successful conjugation of HRP to an IgG antibody.

The HRP Check&Go! process

The HRP Check&Go! process. The HRP-antibody conjugate is run on the Protein A/G Strip. The conjugate binds the Protein A and Protein G which are concentrated at the Test line; following the addition of the HRP detection solution, a visible line on the strip indicates successful conjugation.

The HRP Check&Go! Kit is compatible with IgG antibodies from multiple species, provided they have affinity for either Protein A or G. The entire process takes just 25 minutes, and requires only small volumes of diluted conjugate.

FlexLISA® – quick and easy ELISA development and optimization

To significantly drive down the costs of ELISA development, we have designed FlexLISA®, an assay which reduces the required amount of capture antibody by up to 40-fold. The FlexLISA® Kit contains a Lightning-Link® Streptavidin kit for conjugating your choice of capture antibody to streptavidin, and either a Lightning-Link® HRP kit or a Lightning-Link® Alkaline Phosphatase kit for conjugating your chosen detection antibody to either HRP or alkaline phosphatase. Each of the Lightning-Link® kits provided within FlexLISA® is suitable for the conjugation of 3 x 10µg antibody, allowing you to directly label a range of capture and detection antibodies. Also included is a 96-well microplate (in 12 x 8 strip well format) which has been pre-coated with biotin; this can be supplied as clear or black, depending on the enzyme substrate. FlexLISA® employs a rapid one-step protocol, and is ideal for ELISA assay development, antibody pair screening and ELISA assay optimization.

Schematic representation of the FlexLISA® process

Schematic representation of the FlexLISA® process. 50µl sample is added to a strip well microplate which has been pre-coated with biotin. 50µl antibody mix, consisting of a capture antibody-streptavidin conjugate and a detection antibody-enzyme conjugate is then added, and the plate is incubated for 1 hour at room temperature with shaking. After washing, an assay readout is produced using an appropriate enzyme substrate.

After using FlexLISA® to optimize your assay, it is possible to make a bulk purchase of the individual kit components. Please contact us for pricing or further information.

For more information about any of our products, please get in touch.