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

Technical Service e-mail
tech@jacksonimmuno.com

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 which 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 are performed in a number of ways, some of which are illustrated in Figure 1.

ELISA Formats Figure 1:
A. In direct ELISA a conjugated primary antibody detects plate-bound antigen.
B. In the indirect ELISA method multiple conjugated secondary antibodies are able to bind the primary antibody, leading to signal amplification.
C. Sandwich ELISA uses a capture antibody bound to the plate, which binds antigen from the sample, which is then visualized using a conjugated secondary antibody.
D. Biotin/streptavidin signal amplification. Biotinylated secondary antibodies bind the primary antibody which has reacted with plate-bound antigen. Conjugated streptavidin then binds to multiple biotin molecules on the secondary antibody, leading to maximal signal amplification.

Comparison of ELISA methods by step

Step Direct Indirect Sandwich Signal Amplification
Coat Antigen 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
Wash N/A To remove unbound reagents
3rd N/A Streptavidin
Wash N/A To remove unbound reagents
Signal detection Colorimetric, chemiluminescent or fluorescent.

Blocking

Blocking reagents are especially important in ELISA. To block all unsaturated binding sites on the microplate, use normal serum (5% v/v) derived from the host species of the labeled antibody.

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). 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

Suggested dilution ranges for ELISA are shown in the table below.

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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