Our range of enzyme-antibody conjugates and conjugate stabilizers helps you get the best performance from your ELISA assay.
ImmunoChemistry Technologies offers a range of enzyme-antibody conjugates and conjugate stabilizers to help you get the best performance from your ELISA assay. We take a look at the key features of these products to help you choose the right ones to meet the specific requirements of your ELISA.
The Enzyme-Antibody Conjugate
The detection step of any ELISA assay - be it antigen-down or antibody-sandwich format - requires an enzyme-antibody conjugate to provide a readout signal, as shown below. The conjugate consists of a signal-generating enzyme covalently linked to the detection antibody. In antibody-sandwich ELISAs, the "up" antibody can be directly conjugated to a signal generating enzyme (direct conjugate format) or a conjugated secondary antibody can be used to detect the "up" antibody (indirect conjugate format).
The Signal-Generating Enzyme
The two most commonly used signal-generating enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP).
While it is possible to configure these enzymes for colorimetric, fluorescent or chemiluminescent readouts, we will focus on colorimetric assays as they can be read using absorbance plate readers that are standard equipment in most laboratories.
In colorimetric readout systems, both HRP and AP work by converting a colorless substrate to a colored product.
HRP does this in a redox reaction involving H2O2 as a co-substrate (Our HRP Redox Reaction Driven TMB Color Development gives a detailed explanation of HRP chemistry).
AP works by hydrolyzing the phosphate group from a colorless substrate to generate a colored product.
Which enzyme is best for enzyme-antibody conjugates?
HRP is often the signal generating enzyme of choice for colorimetric readouts (See our webinar "Features of HRP which provide the Chemical Basis for its routine inclusion within Colorimetric ELISA assays"). This is due to its high catalytic efficiency and relatively small size. The high catalytic efficiency enables good sensitivity and shorter assay times, while the small size of the enzyme reduces the likelihood of steric hindrance impeding the performance of the HRP-conjugated detection antibody.
AP is preferred in certain situations, despite its slower reaction kinetics, as the time-course of the AP reaction tends to be linear for extended periods of time – this can be advantageous if you want to follow the time course of the reaction.
- Off the shelf enzyme-antibody conjugates
We offer a number of HRP and AP conjugated secondary antibodies from ImmunoChemistry Technologies, Antibodies Inc, and Aves Labs. This gives you various off-the-shelf options for building ELISA assays with either signal generating enzyme.
| Conjugated Enzyme | Conjugated Secondary Antibody | Brand |
| HRP | Goat Anti-Chicken IgY (H&L) | Aves Labs |
| HRP | Goat Anti-Mouse IgG (H&L) | Antibodies Inc |
| HRP | Goat Anti-Mouse IgG Fc | ImmunoChemistry |
| HRP | Goat Anti-Rabbit IgG (H&L) | Antibodies Inc |
| HRP | Goat Anti-Rabbit IgG Fc | ImmunoChemistry |
| HRP | Goat Anti-Guinea Pig IgG (H&L) | Antibodies Inc |
| HRP | Donkey Anti-Sheep IgG (H&L) | Antibodies Inc |
| HRP | Goat Anti-Human IgG Fc | ImmunoChemistry |
| HRP |
Goat Anti-Human IgG (Monkey adsorbed) |
Antibodies Inc |
| HRP | Mouse Anti-Human IgG Fc | Antibodies Inc |
| AP | Goat Anti-Chicken IgY (H&L) | Aves Labs |
| AP | Goat Anti-Mouse IgG (H&L) | Antibodies Inc |
| AP | Goat Anti-Rabbit IgG (H&L) | Antibodies Inc |
| AP | Donkey Anti-Sheep IgG (H&L) | Antibodies Inc |
- Making your own enzyme antibody conjugates
Of course, you may want to make your own custom-made enzyme-antibody conjugate – perhaps the secondary antibody host/target species combination you need isn’t commercially available as an enzyme conjugate, or maybe you want to use your favorite anti-target antibody in a direct conjugate format antibody-sandwich ELISA.
If this is the case, our Conjugation Ready HRP Maleimide can be used to conjugate HRP to your antibody of choice. The maleimide-activated HRP forms a stable bond with free SH groups on the antibody in a two step thiolation-conjugation approach. The HRP:protein ratio of your conjugate can be controlled while avoiding the HRP inactivation that accompanies periodate oxidation protocols and the formation of multimeric protein aggregates than occurs with glutaraldehyde-based methods.
What is a conjugate stabilizer and what does it do?
ICT's Conjugate Stabilizers perform two functions.
First, they extend the shelf life of diluted enzyme-antibody conjugates by stabilizing and preserving the catalytic activity of the signal-generating enzyme and the functional integrity of the antigen binding sites on the antibody. Due to their stabilizing effects, they can be used to preserve concentrated stocks of IgG-HRP conjugates, reconstitute lyophilized conjugates, and dilute conjugates to their final working titre.
Second, Conjugate Stabilizers help minimize non-specific interactions during the assay process, thus reducing background noise.
Which conjugate stabilizer should I use for my ELISA?
If you are using an HRP-conjugate, you have the choice of four different ICT Conjugate Stabilizers – each with its own unique properties. We also offer a specially formulated AP-conjugate stabilizer.
Need a general purpose HRP-antibody conjugate stabilizer?
HRP Conjugate Stock Stabilizer, 5X is designed as an all-purpose reagent for HRP conjugate dilution and stabilization. The proprietary formulation contains BSA. It can be used as a base formulation to which further specialized components can be added to deal with unique ELISA sample situations/issues.
Running an Antigen-Down ELISA with HRP detection?
Antigen-Down HRP Conjugate Stabilizer, 5X is specifically formulated to stabilize anti-IgG HRP complexes. It used proprietary hydrolyzed, non mammalian proteins - the lower molecular weight protein matrix can lead to vastly improved stabilizing performance. In addition to its stabilizing effects, it inhibits the non-specific binding of the HRP-IgG complex to serum proteins that may be bound to the plate.
This stabilizer may be particularly useful in antigen-down ELISAs designed to detect low-positive, humoral antibody responses.
Need a stabilizer for HRP configured antigen-down and antibody-sandwich ELISAs that doesn't contain mammalian proteins?
HRP Conjugate Stabilizer, Non-Mammalian, 1X (formerly known as Neptune HRP Conjugate Stabilizer) uses a proprietary formulation of hydrolyzed non-mammalian proteins with enhanced ability to stabilize IgG-HRP complexes. It is suitable for both antigen-down and antibody-sandwich ELISA formats. It has been demonstrated to maintain 100% activity of working dilution HRP conjugates for at least 12 months.
Want an HRP conjugate stabilizer containing mammalian proteins?
HRP Conjugate Stabilizer, Mammalian, 1X uses a proprietary formulation that is suitable for use in both antigen-down and antibody-sandwich ELISAs.
Using an AP antibody conjugate?
Alkaline Phosphatase Conjugate Stabilizer, 1X is a ready to use reagent containing mammalian proteins. It stabilizes AP-antibody conjugates and reduces non-specific binding. It is suitalbe for both antigen-down and antibody-sandwich ELISA formats.
All ICT Conjugates and Stabilizers are available in 100 mL, 500 mL, and 1L sizes. Bulk volumes are also available. Complete the form on the product page for a quote on bulk quantities!
Learn more about our Conjugates and Stabilizers
View our ELISA & Assay Reagents Catalog
View our ELISA & Assay Reagents Advantages Catalog
View our Guide to Developing Optimized ELISAs