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Secondary Antibodies

antibodies Secondary antibody conjugates are important tools in biochemical and cell-based studies. Enzyme-labeled secondary antibodies, such as HRP secondaries, have high catalytic turnover rates and are routinely used in various assays for their rapid and robust signal generation. HRP secondary antibodies have been used extensively in enzyme-linked immunosorbent assays (ELISA), lateral flow assays (LFA), immunohistochemistry (IHC) and Western blot for the diagnosis of infectious diseases. Secondary antibodies conjugated to fluorescent dyes provide brighter signals and multiplexing capabilities in cell analysis and protein analysis applications including immunofluorescence microscopy and cell imaging.

The most important characteristics of enzyme-labeled and dye-labeled secondary antibodies are high sensitivity and signal amplification. Two desirable qualities when detecting target proteins or antigens of low abundance.

 

 

Using secondary antibodies


Secondary antibodies are key components of the indirect detection method. Used in sequence with primary antibodies, secondary antibodies facilitate in the detection, quantification and purification of target antigens by binding to the primary antibody, which directly binds to the antigen. Reporter molecules (e.g. an enzyme or a fluorophore) conjugated to the secondary antibodies enables visualization of the immune complex. The choice of reporter molecule is contingent upon the application and how the secondary antibody will be detected. While the use of secondary antibodies requires an additional immunolabeling step, it presents numerous advantages over the direct detecion method, namely signal amplification and flexibility. Signal amplification arises from the specificity of the secondary antibody for a designated reigion on the primary antibody (e.g. Fc region). This permits multiple secondary antibodies to bind to a single primary antibody, amplifying the signal and improving sensitivity (Figure 1). The use of secondary antibodies also affords the user flexibility contributed by the diversity of available reporter molecules. For example, iFluor™ 800 dye-labeled and iFluor™ 680 dye-labeled secondary antibodies enable two-color imaging in protein detection applications such Western blot.


Representation of direct and indirect antigen detection using target specific antibodies.

 

Selecting the optimal secondary antibody


Selecting the right antibody will improve sensitivity of an assay by increasing signal detection while minimizing non-specific binding and background interference. When selecting a secondary antibody consider the following key factors:

Match the host species of the primary antibody


Secondary antibodies should be raised against the host and target species of the primary anibody being used. For example, if using a primary monoclonal antibody raised in humans, then an anti-human secondary antibody raised in a species other than human must be used, such as a goat anti-human IgG. Common host-specific secondary antibodies from AAT Bioquest include:

Match the class and subclass of the primary antibody


Monoclonal antibodies are subclass specific and require the use of secondary antibodies directed against that specific subclass. For example, if the primary antibody is mouse IgM, then the secondary conjugate must react with mouse IgM such as a goat anti-mouse IgM. If the primary antibody is mouse IgG1, then an anti-mouse IgG1 secondary antibody can be used, this is especially critical in multiplex assays.

Polyclonal antibodies are usually on the IgG isotype and are recognized with anti-IgG antibody H+L (heavy and light chains).

Cross-adsorption


For improved specificity, greater sensitivity and lower background consider using cross-adsorbed secondary antibodies. Cross-adsorption of secondary antibodies, via affinity purification, eliminates cross-reactivity and improves antibody specificity.

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Select the correct reporter molecule for your application


Secondary antibodies are generally supplied conjugated to a detectable label such as an enzyme, fluorophore, or biotin. The choice of label depends upon the downstream application for which the secondary antibody will be used and detected.

 

Table 1. Common Applications for Secondary Antibody Conjugates

Application
Types of secondary antibodies recommended
ELISA
  • Enzyme (HRP or AP) conjugated secondary antibodies are most commonly used for indirect, sandwich and competitive ELISA
LFA
  • Fluorescently labeled secondary antibodies (e.g. iFluor® dyes or Alexa Fluor®)
  • Enzyme (HRP or AP) conjugated secondary antibodies
Western Blot
  • Enzyme (HRP or AP) conjugated secondary antibodies are most commonly used
  • Fluorescently labeled secondary antibodies (e.g. iFluor® dyes or Alexa Fluor®) are also used for multiplexing and infrared immunoblotting
Immunofluorescence
Immunohistochemistry
  • Enzyme (HRP or AP) conjugated secondary antibodies
  • HRP-mediated Styramide™ signal amplification
Immunocytochemistry
  • Enzyme (HRP or AP) conjugated secondary antibodies
  • HRP-mediated Styramide™ signal amplification
  • Fluorescently labeled secondary antibodies (e.g. iFluor® dyes or Alexa Fluor®)

 

Enzyme-labeled secondary antibodies and other reagents for ELISA


Monoclonal antibodies, polyclonal antibodies or a combination of both are typically used in ELISA assays as either detection or capture antibodies. Monoclonal antibodies are inherently monovalent, with specificity towards a single epitope per antigen. In all ELISA formats, monoclonal antibodies are commonly used as detection antibodies. Because they seldom cross-react with other proteins, they are less likely to generate non-specific signals. In contrast, polyclonal antibodies, which are a complex mixture of antibodies, recognize multiple epitopes found in a single antigen. While they are often used in sandwich ELISA assays as 'capture antibodies' to pull down as much of the antigen as possible, they can also be used as detection antibodies. Polyclonal antibodies are highly susceptible to batch-to-batch variation, and should be thoroughly tested and validated prior to using.

HRP and poly-HRP secondary antibodies for ELISA


The enzyme horseradish perxoidase ((HRP, Cat No. 11025) is commonly conjugated to secondary antibodies, and used in either indirect or sandwich ELISA assays. The widespread adoption of HRP as a reporter in ELISA is primarly due to three factors. First, HRP has the capacity to amplify weak signal and enhance the detectability of poorly-expressed antigens. Second, the relatively small size of HRP (?44 kDa) compared to other reporter enzymes (e.g. alkaline phosphatase (~140 kDa)) further improves intracellular penetration into samples, reduces steric hindrance and minimizes immunoreactivity loss. Third, is the high turnover rate and good stability of HRP that enables rapid and strong signal generation. AAT Bioquest offers highly purified and cross-adsorbed secondary antibodies conjugated to HRP and poly-HRP. MegaWox™ poly-HRP secondary conjugates are designed to deliver high levels of sensitivity and low background in ELISA assays. Use MegaWox™ poly-HRP conjugates in immunoassays where sample volume is limited or when the target molecule is poorly-expressed.

 

Table 2. Available HRP-Secondary Antibody Conjugates for ELISA

 

Substrates and detection strategies for enzyme-labeled secondary antibodies


The final step in any using an enzyme-labeled secondary antibody is the detection step by addition of an enzyme substrate. The enzyme (e.g. HRP or AP) catalyzes the substrate into a measurable byproduct, and the magnitude of the signal produced is proportional to the amount of antigen in the sample. Enzyme substrates vary in ease-of-use, sensitivity and compatability with imaging equipment such as spectrophotometers, fluorometers and luminometers.

Colorimetric substrates


Colorimetric (or chromogenic) substrates react with enzymes to generate an observable colored byproduct that can be measured using an absorbance plate reader. AAT Bioquest provides colorimetric substrates for horseradish peroxidase (HRP) and alkaline phosphatase (AP) ranging in sensitivity and optical densities.

 

Table 3. Available Colorimetric Enzyme Substrates for ELISA

Enzyme
Substrate
Absorbance (nm)
Color
Detection Limit
Unit Size
Cat No.
APpNPP405Yellow∼10 ng/well (100 ng/mL)25 mg11619
HRPABTS420Blue-Green∼250 pg/well (2.5 ng/mL)1 L11001
HRPTMB650, 450Blue, Yellow4∼12.5 pg/well (40-120 pg/mL)100 mL11012
HRPTMB650, 450Blue, Yellow4∼12.5 pg/well (40-120 pg/mL)1 L11003



Fluorimetric substrates


Fluorimetric (or fluorogenic) substrates react with enzymes to generate highly fluorescent byproducts that can be measured using a fluorescence plate reader. The degree of photon emission following light excitation is proportional to the amount of antigen in the sample. AAT Bioquest provides fluorimetric substrates for horseradish peroxidase (HRP) and alkaline phosphatase (AP) ranging in sensitivity and fluorescence properties.

 

Table 4. Available Fluorimetric Enzyme Substrates for ELISA

Enzyme
Substrate
Ex (nm)
Em (nm)
Unit Size
Cat No.
APMUP, disodium salt360 nm448 nm25 mg11610
APMUP, disodium salt360 nm448 nm10 g11612
APMUP, free acid360 nm448 nm25 mg11614
APMUP, free acid360 nm448 nm5 g11617
APDiFMUP360 nm450 nm5 mg11627
APFDP497 nm516 nm5 mg11600
APPhosLite™ Green345 nm520 nm1 mg11630
APSunRed™ Phosphate652 nm660 nm5 mg11629
HRPAmplite® Blue324 nm409 nm25 mg11005
HRPAmplite® ADHP570 nm583 nm25 mg11000


Chemiluminescent substrates


Chemiluminescent substrates react with enzymes to generate luminescent byproducts that can be measured using a luminometer. Since the emission of a photon is a result of a chemical reaction and not light excitation, there is minimal background interference. AAT Bioquest provides chemiluminescent substrates for horseradish peroxidase (HRP) and alkaline phosphatase (AP) ranging in sensitivity and chemiluminescence properties.

 

Table 5. Chemiluminescent substrates For Substrates and detection strategies for enzyme-labeled secondary antibodies

Enzyme
Substrate
Em (nm)
Unit Size
Cat No.
APD-Luciferin phosphate 1 mg12512
HRPLuminol410 nm1 mg11050