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 VHH Discovery

Anti-Alpaca IgG, Subclass and VHH domain secondaries
  • Ideally time PBMC harvest
  • Enhance detection of VHH antibodies
  • Test for VHH expression and binding activity

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Secondary Antibodies for Enzyme Linked Immunosorbent Assay - ELISA

Introduction to ELISA

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.

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ELISA formats and methods

ELISAs are performed a number of ways, the most common is the sandwich or capture assay or with labeled streptavidin to amplify analyte signal from a biotinylated antibody.

ELISA Methods Common ELISA formats. A. Direct. B. Indirect, C. “Sandwich” capture assay ELISA and D. Streptavidin/ biotin signal amplification using a biotinylated primary antibody.


Comparison of ELISA methods by step

Step

Direct

Indirect

Sandwich

Signal amp


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 for ELISA

Blocking reagents are especially important in ELISA, ideally use serum of the species of the labeled antibody to block all unsaturated binding sites on the microplate to avoid background from nonspecific binding, however BSA may be appropriate. See Normal Serums and Bovine serum albumin (BSA) pages. The addition of a mild detergent such as Tween ® 20 can help to reduce background from hydrophobic interactions.

Secondary antibody conjugates for ELISA

Jackson ImmunoResearch alkaline phosphatase (AP) and horseradish peroxidase (HRP) conjugates can be used for both colorimetric assays, using a chromogenic substrate. For chemiluminescent detection, a luminol based substrate is commonly used with peroxidase conjugates for highly sensitive detection. ELISAs can be performed using fluorescent conjugates to allow simultaneous detection of multiple antigens using primary antibodies derived from different species. Using labeled secondary antibodies each antigen can be distinguished specifically by the individual fluorescent signal. The detection limit for fluorescent ELISA is however typically lower than colorimetric or chemiluminescent detection using a reporter enzyme.

Labeled streptavidin with biotinylated primary antibodies for enhanced sensitivity

Signal enhancement can be achieved using a labeled streptavidin to detect a biotinylated antibody (primary or secondary antibody for enhanced amplification). Each antibody can itself present multiple biotin molecules, which are then, in turn, able to bind with 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

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

Product ELISA
Horseradish Peroxidase Conjugates [IgG, F(ab') 2] 1:5,000 - 1:100,000
Alkaline Phosphatase Conjugates [IgG, F(ab') 2] 1:5,000 - 1:50,000
Biotin-SP Conjugates [IgG, F(ab') 2] (using enzyme-Conjugated Streptavidin) 1:20,000 - 1:400,000
Horseradish Peroxidase-Conjugated Streptavidin 1-2 µg / ml
Alkaline Phosphatase-Conjugated Streptavidin 1-2 µg / ml
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