Secondary Antibodies: their role in serological tests

What are secondary antibodies?

Secondary antibodies are essential components in various immunoassays and immunohistochemistry techniques.

In general antibodies are proteins known as immunoglobulins, produced by plasma cells, tasked with identifying and neutralizing potentially harmful foreign molecules called antigens (‘the non self’). The specific region of an antibody that recognizes the antigen is called the “paratope,” which specifically binds to the antigen’s “epitope.” This binding triggers various immune responses, including the deployment of macrophages to destroy ‘the non self’.

To fully grasp the role and importance of secondary antibodies, it’s essential to distinguish them from primary antibodies.

Primary antibodies, directly produced by the immune system, both as polyclonal and monoclonal varieties, are directly used in diagnosing diseases and detecting specific biomarkers associated with health conditions like diabetes, cancer, and neurological disorders such as Alzheimer’s and Parkinson’s diseases. They bind directly to antigens, but they must be detected to highlight the pathology.

In contrast, secondary antibodies have a different role. They do not bind directly to antigens. Instead, they bind to primary antibodies, creating an indirect linkage to the antigen. This unique interaction is critical as it amplifies the detection signal, enhancing the visualization and quantification of the primary antibody, bound to the target antigens. This amplification is vital in diagnostic assays where sensitivity and specificity are paramount.

Understanding the pivotal interactions between primary and secondary antibodies can significantly enhance the diagnostic accuracy and provide deeper insights into immune system functioning and disease pathology. These secondary antibodies are not just components of the immune response but are powerful tools that enable researchers and clinicians to achieve more precise and reliable results in various immunological assays.

What are Secondary Antibodies Used For?

Secondary antibodies are vital tools in both research and diagnostic settings. They are generated against the host species and isotype of a primary antibody. For example, to detect a human IgG polyclonal primary antibody, a goat anti-human IgG secondary antibody (targeting Fcγ or light and heavy chains) is typically used. These antibodies fulfill several key roles:

Signal Amplification: Secondary antibodies are often conjugated with labels such as enzymes (e.g., peroxidase or alkaline phosphatase for colorimetric detection) or fluorescent dyes (e.g., Alexa Fluor for fluorescence-based assays). This conjugation amplifies the primary antibody-antigen signal , enhancing the detectability of the primary antibody across various assay types.

Increased Selectivity and Sensitivity: Secondary antibodies provide high selectivity when binding to primary antibodies present in very low concentrations in complex biological fluids. This property is crucial for the accurate isolation or quantification of specific antibodies.

Enhancing Specificity: By using isotype-specific secondary antibodies, researchers can minimize non-specific binding and background noise, thereby improving the specificity and clarity of experimental results. This is particularly important in studies requiring high specificity and reliability.

Fields of Application:

• Assays: Secondary antibodies are integral to various laboratory techniques, including Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), immunofluorescence, and flow cytometry. Each technique benefits from the specific properties of the conjugated secondary antibodies.
• Clinical and Diagnostic Applications: They are crucial in diagnosing and studying diseases, particularly in detecting antibodies linked to tissue antigens and autoantibodies in autoimmune disorders. Additionally, they are used to isolate and quantify specific mouse monoclonal immunoglobulin classes such as IgG1, IgG2a, IgG2b, IgG3, and light or heavy chains.

These applications highlight the indispensable role that secondary antibodies play in enhancing the accuracy, sensitivity, and specificity of immunodetection methods across a wide array of biological and medical sciences.

Biomat has developed the Secondary Antibodies Goat anti Rabbit IgG Coated 96 Well Plates and Secondary Antibodies Goat anti Mouse IgG Coated 96 Well Plates in Breakable Strip, Strip and Solid format ready to use for your serological tests.

Examples of application of secondary antibodies

1° Example: Indirect ELISA assay

Let’s start by describing the indirect ELISA assay.

An indirect ELISA is one where the primary antigen-specific antibody is recognized by a secondary conjugated antibody, as shown in figure below:

The figures represent the various steps of an indirect assay.

Below is the sequence of the various steps:

1. Coat ELISA plate with testing antigen, seal plate and incubate overnight at 4 °C
2. Remove coating solution and wash plate 3 times with desired buffer
3. Block plate with desired blocking solution and incubate overnight at 4 °C
4. Wash plate 3 times with desired buffer
5. Incubate with unconjugated primary antibody at room temperature 60 minutes
6. Wash plate 3 times with desired buffer
7. Incubate with HRP-labeled secondary antibody at room temperature 30 minutes
8. Wash plate 3 times with desired buffer
9. Incubate plate with TMB substrate Solution at room temperature 15 minutes
10. Stop reaction with sulfuric acid 0.3 N
11. Measure the absorbance signal at 450 nm with an ELISA microplate reader

In the indirect ELISA assay, the concentration of the primary antibody is directly proportional to the intensity of the color developed by the enzymatic reaction.

 

2° Example:  ELISA sandwich assay for the determination of immunoglobulins

This assay is based on the sandwich method in which a Secondary Antibody Goat anti Mouse IgG Fcγ (fragment specific) has been bound to the solid phase and another enzyme-labeled goat anti-mouse IgG antibody reveals sandwiched mouse IgG.

It is important to underline that the sandwich test works only if the two antibodies that form the sandwich come from the same species of animal. Furthermore the two antibodies must be specific for different structures of the sandwiched protein.

To guarantee the best performance if you are measuring mouse IgG, in the specific, it is advisable to have a goat anti mouse IgG Fcγ in the solid phase which binds the IgG through the crystallizable fragment and the second goat specific for the variable portion of the mouse IgG.

Below is the sequence of steps, which describe the test for calculating the binding capacity and sensitivity of the microplate, using the Biomat Goat anti Mouse IgG  Plate, code MT25F4-aM.  

①    goat anti mouse IgG Fcγ (secondary antibody) coated plate

1. Add 100 µl of different concentrations of mouse IgG (from 0.025 to 4 µg/ml) to the wells of goat anti mouse IgG Fcγ coated plate and incubate for 60 minutes at room temperature

②    binding between goat anti mouse IgG Fcγ and mouse IgG (primary antibody under evaluation)

 

2. Empty the wells and wash with 0.1 M PBS pH 7.2,0.05% Tween® 20 four times
3. Add 100 µl /well of Goat anti-mouse IgG (H+ L)-HRP (Jackson ImmunoResearch code 115-035-003, diluted 1: 150,000) and incubate for 30 minutes at room temperature

③  mouse IgG sandwiched between goat anti mouse IgG Fcγ and goat anti mouse IgG( H+L) labeled with peroxidase (labeled secondary antibody)

4. Empty the wells and wash with 0.1 M PBS pH 7.2,0.05% Tween® 20 four times
5. Add 100 µl /well of TMB substrate solution and incubate 15 minutes at room temperature

④ color development due to the action of the peroxidase enzyme; the color is proportional to the concentration mouse IgG

 

6. Stop the substrate reaction by adding 100 µl /well of sulfuric acid 0.3 N ( the blue color changes to yellow after adding the acid ) and read the optical density values at 450 nm

The data show that a plateau has started with an IgG mouse concentration of 1.0µg/ml. This concentration means the well binding capacity we can express as:

– µg/well = 0.1 (100 ng/well)

– pmol/well= 0.625 (this result is calculated considering the IgG M.W. = 160,000)

The microplate sensitivity was calculated as the lowest mouse IgG concentration higher than the mean optical density plus 5 S.D. of 0 µg/ml mouse IgG concentration.

Our experiment gave the following results:

– 0 µg/ml mouse IgG optical density mean (coming from 8 replicates) = 0.108

– standard deviation = 0.014

– mean + 5 S.D. = 0.178

– sensitivity = 0.012µg/well of mouse IgG

 

Related Products

Biomat Secondary Antibodies Plates (offered in Breakable Strip, Strip and Solid format and in clear, white and black color):

Goat anti Rabbit IgG Coated 96 Well Plates

Secondary Antibodies Goat anti Mouse IgG Coated 96 Well Plates