Jackson Immuno Research Inc.
specializing in secondary antibodies and conjugates

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Whole IgG Affinity-Purified Secondary Antibodies

"I have used a wide variety of secondaries and Jackson ImmunoResearch has consistently been the best. The fluorophores are bright and stable and their selective (x reactivity removed) secondaries have always shown species specificity in multiple labeling."

Janet Duerr, Ohio University

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Secondary Antibodies for ELISA

ELISA with JIR Secondary Antibodies

An enzyme-linked immunosorbent assay (ELISA) is a robust and sensitive technique used to detect and quantify specific proteins in samples that may contain complex mixtures of proteins. Antibodies are used to detect the specific proteins immobilized on the surface of microplate wells. The technique facilitates high volume and fast throughput analysis, ideal for analyzing large numbers of samples.


ELISA Formats

ELISAs can be performed in a number of ways depending on the sample specifics and the sensitivity required.

Direct ELISA

The sample is applied to a protein-binding solid surface, typically a microtiter plate, coating the analyte directly onto the surface, if present (Fig. A). Next, reporter-conjugated primary antibodies specific to the analyte are added. Signal from the directly conjugated reporter molecule is analyzed to provide a quantitative result when used with a standard curve of known concentration. Depending on the sample characteristics this method may have limited sensitivity.

Indirect ELISA

The analyte is coated directly onto a microtiter plate (Fig. B). The sample (biological fluid: serum, saliva, etc.) is added and antigen-specific antibodies, if present, bind to the analyte, forming a complex. Subsequent washing removes non-binding proteins and other components in the sample mixture. A reporter molecule-conjugated secondary antibody is used to detect the bound antigen-specific antibody. Signal generated may be quantified by comparing to a standard curve.

Sandwich ELISA

Sandwich ELISAs can be performed both directly or indirectly depending on the level of sensitivity required. There are a variety of methods that use the specificity of antibodies to target different fragments of the antibody allowing greater specificity and versatility.

Direct Sandwich

A primary antibody specific to the antigen (analyte) of interest is immobilized onto a microtiter plate and subsequently captures the analyte from the test sample. A reporter-molecule conjugated primary antibody specific to the antigen is added to complete the sandwich. The signal from the reporter molecule is observed either by adding an enzyme substrate, which results in a colorimetric product or fluorescence, resulting in a readout proportional to the analyte concentration.

Indirect Sandwich

A primary antibody specific to the antigen (analyte) of interest is immobilized onto a microtiter plate and subsequently captures the analyte from the test sample (Fig. C). A second primary antibody of a different host species-specific to the antigen is then added to complete the sandwich. A reporter molecule-conjugated secondary antibody binds to the second antigen-specific antibody amplifying signal. The signal from the reporter molecule is observed either by adding an enzyme substrate, which results in a colorimetric product or fluorescence, resulting in a readout proportional to the analyte concentration.

ELISA Formats ELISA Formats

Figure 1:

  1. Direct ELISA. A conjugated primary antibody detects plate-bound analyte.
  2. Indirect ELISA. Multiple conjugated secondary antibodies are able to bind the primary antibody, leading to signal amplification.
  3. Indirect sandwich ELISA. A capture antibody bound to the plate, which binds analyte from the sample, which is then visualized using a conjugated secondary antibody.
  4. Labeled Streptavidin-Biotin (LSAB) signal amplification. Biotinylated secondary antibodies bind the primary antibody which has reacted with plate-bound analyte. Conjugated streptavidin then binds to multiple biotin molecules on the secondary antibody, leading to maximal signal amplification.

Lateral Flow

Point of care lateral flow tests for the diagnosis of disease utilize the principles of ELISA.

Read More About Lateral Flow Tests

Comparison of ELISA Methods by Step

Step Direct Indirect Indirect Sandwich Signal Amplification
Coat Analyte Capture antibody
Block We recommend using 5% normal serum of the species of the detection antibody
Wash To remove unbound reagents
Sample Conjugated primary antibody Unconjugated primary Sample Sample
Wash To remove unbound reagents
2nd N/A AP/HRP or fluorophore-conjugated secondary antibody Biotinylated secondary antibody
Wash N/A To remove unbound reagents
3rd N/A Streptavidin
Wash N/A To remove unbound reagents
Signal Detection Colorimetric, chemiluminescent or fluorescent

ELISA for Diagnostics

ELISAs or Enzyme immunoassays (EIA) are frequently used in clinical diagnostic testing; many are validated and available commercially as kits containing all the necessary reagents to perform the test, allowing labs to have access to standardized procedures.

ELISA tests detect immunoglobulins produced as part of an immune or allergic response allowing the diagnosis of infections and allergic diseases, such as food allergy. Alternatively, ELISA can be used to identify causative agents through the detection of the antigen, such as allergens, virus particles, or bacteria, allowing identification of infectious disease.

Typically performed using a polystyrene microtiter plate, the analyte may be coated on the plate, or coated with a capture antibody in the case of a sandwich ELISA. A blocking step using an appropriate serum such as Bovine Serum Albumin (BSA) minimizes the potential for background signal from non-specific interactions between the patient sample and the plate. The patient sample, which may be blood, saliva, or another biological fluid, is introduced to the plate allowing either immunoglobulins or antigens to complex with the capture material. Depending on the format of the assay, signal may be confirmed and quantified by, a reporter molecule-conjugated-primary or secondary antibody, or a biotinylated antigen-specific antibody followed by labeled streptavidin to amplify signal.

Learn About Reagents for Diagnostic Testing

Blocking

Blocking reagents are especially important in ELISA. We recommend using 5% (v/v) normal serum derived from the host species of the labeled antibody to block all unsaturated binding sites on the microplate, although BSA may also be appropriate.

Browse Normal Serum

Secondary Antibody Conjugates for ELISA

Jackson ImmunoResearch alkaline phosphatase (AP) and horseradish peroxidase (HRP) conjugates can be used for colorimetric assays using a chromogenic substrate. For chemiluminescent detection, a luminol based substrate is commonly used with peroxidase conjugates for highly sensitive detection.

Read More About Reporter Enzyme Conjugates

ELISAs can also be performed using fluorescent conjugates to allow simultaneous detection of multiple primary antibodies derived from different species. By using labeled secondary antibodies each antigen can be distinguished specifically by the individual fluorescent signal. The detection limit for fluorescent ELISA is typically lower than colorimetric or chemiluminescent detection using a reporter enzyme.

Read More About Fluorophore Selection

Labeled Streptavidin with Biotinylated Antibodies for Enhanced Sensitivity

Signal enhancement can be achieved using labeled streptavidin to detect a biotinylated antibody (primary or secondary antibody) (Fig. D). Each antibody can present multiple biotin molecules, which are then able to bind to multiple streptavidin molecules. These combined factors mean that multiple probe molecules are available to either catalyze the detection substrate to its end product or generate fluorescent emission, achieving a brighter signal and greater sensitivity.

Read More About Signal Enhancement

ELISA with JIR Secondary Antibodies

We recommend determining appropriate dilution ranges for your experiment, the ranges suggested in the table below are only a guide. We find that higher dilutions may be more appropriate particularly when using the HRP conjugates.

Product Conjugate ELISA
Whole IgG and F(ab')2 secondary antibodies Unconjugated 10-20 µg / ml
Whole IgG, F(ab')2 and Fab secondary antibodies Alexa Fluor® 488, 594, 647, DyLight™ 405, and Cy™3 1:100 - 1:800
Whole IgG, F(ab')2 and Fab secondary antibodies AMCA, BV421™, BV480™, Cy2, FITC, TRITC, RRX, and Texas Red 1:50 - 1:200
Whole IgG secondary antibodies Cy™5 1:100 - 1:400
Whole IgG and F(ab')2 secondary antibodies Horseradish Peroxidase 1:5,000 - 1:100,000
Whole IgG and F(ab')2 secondary antibodies Alkaline Phosphatase 1:5,000 - 1:50,000
Whole IgG and F(ab')2 secondary antibodies Biotin-SP (using enzyme-Conjugated Streptavidin) 1:20,000 - 1:400,000
Streptavidin Horseradish Peroxidase 1-2 µg / ml
Streptavidin Alkaline Phosphatase 1-2 µg / ml
Normal Serum 5% (v/v) for blocking
ChromPure™ 10 µg/ml
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