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Improving the efficiency of antibody conjugation to latex beads

Latex beads can be used in a wide range of applications, but are especially popular for lateral flow assays and latex agglutination experiments.

Lateral flow assays

Lateral flow assays (LFA) are used to quickly and easily establish whether a target analyte is present in a test sample. In a typical LFA, the sample is added to a sample application pad at one end of a test strip, from which it flows to a conjugate release pad, into which the detection reagent has been dried. The detection reagent is commonly an antibody which has been conjugated to a colored moiety such as a latex bead. The sample and detection reagent then migrate along the membrane, upon which antibodies have been immobilized as a Test line and a Control line; the Test line typically consists of a second antigen-specific antibody, while the Control line is often composed of an anti-species antibody. Accumulation of the detection reagent at the Test line is indicative of a positive result, while accumulation at the Control line demonstrates that the assay has run successfully.

 

A typical lateral flow immunoassay

A typical lateral flow immunoassay. An antibody specific to the target analyte is conjugated to a latex bead, and dried in to the conjugate release pad. This detection reagent will bind any target analyte which is present in the test sample. A second specific antibody, immobilized at the Test line, will capture the analyte, resulting in the appearance of a colored line. An anti-species antibody, against the host species of the detection antibody, will capture the antibody-latex bead conjugate at the Control line, demonstrating the assay to have run successfully.

Agglutination assays

Agglutination assays are another method of identifying the presence of a target analyte in a test sample, and often rely on the use of antibodies which have been conjugated to latex beads. In a typical agglutination assay, the antibody-latex bead conjugate is mixed with a sample which is believed to contain the target analyte; the presence of the analyte results in clumping (agglutination) of the beads, which can be assessed visually, or using a spectrophotometer. 100nm latex beads are most commonly used for agglutination assays.

A typical agglutination assay

A typical agglutination assay. An antibody specific to the target analyte is conjugated to a latex bead, and added to a test sample. Antibody binding to the target analyte results in agglutination of the beads, which can be assessed by eye or spectrophotometrically.

Passive adsorption of antibodies to latex beads

Passive adsorption relies primarily on attractions between the hydrophobic regions of the antibodies and the latex beads. During passive adsorption, the antibody is dissolved in a buffer, mixed with the bead suspension and then incubated for a fixed length of time, often several hours or overnight. The reaction is stopped by centrifugation to precipitate the pellet, followed by washing until no unbound antibody remains in the supernatant. Although this sounds straightforward, passive adsorption can be an extremely time-consuming and laborious process since extensive optimization is necessary to identify the appropriate reaction buffer. Typically, the pH of this buffer should be close to the isoelectric point of the antibody, while other considerations include the antibody: latex bead ratio, and whether to include a surfactant to prevent aggregation.Unfortunately, passively adsorbed antibody-latex bead conjugates are not always able to withstand the various incubation and wash steps during the downstream application, and antibody desorption from the latex beads can result in a significant loss of assay sensitivity.

Covalent conjugation of antibodies to latex beads

Unlike passive adsorption, covalent attachment of antibodies to latex beads is irreversible, providing a stable conjugate and therefore a much greater level of reproducibility within the downstream assay. There are a variety of methods of covalent conjugation, all of which require specialist knowledge of chemical modification techniques.

Expedeon’s Latex Conjugation Kits provide an easy to use alternative to traditional, time-consuming, latex conjugation protocols. The kits utilize a one-step method for covalently conjugating antibodies, proteins and peptides (or any other biomolecule with an amine group) to specially treated 400nm latex beads, and are unlike any other commercially available kit.

Expedeon's latex bead conjugation process

Expedeon’s latex bead conjugation process. The antibody is added to a vial of lyophilized latex beads and incubated for 15 minutes. After quenching the reaction, the latex is pelleted, and the antibody-latex bead conjugate is resuspended. The entire procedure requires 3 minutes hands-on time, and just 35 minutes total time until the conjugates are ready to use.

Our Latex Conjugation Kits offer several advantages:

Features Benefits
Quick and easy to use Save time, no specialist knowledge required
Different colors available Flexible, allows multiplexing
Stringently QC tested Consistent high quality, excellent batch-to-batch reproducibility
Covalent bond Highly stable conjugates
Freeze dried Ships at ambient temperature, long shelf-life
Fully scalable Easy transfer from R&D to manufacturing
No extensive pH optimization required Save precious antibody / protein
Resistant to aggregation Generation of high quality data

Considerations for antibody conjugation to latex beads

Every antibody storage buffer will contain substances other than the antibody itself, and some of these components may be incompatible with the labeling technology. Our Latex Conjugation Kits require your antibody to be in 10-50mM MES, MOPS or HEPES pH 6-7 and free from additives such as sodium azide, BSA and Tris. If your antibody does not meet these criteria, we offer a range of purification kits which enable you to efficiently separate your antibody from an unfavorable starting point. Our easy to use flow chart enables you to select the most appropriate purification kit for your particular antibody.

Conjugate Check&Go! Kits for confirming successful antibody-latex bead conjugation

Although our Latex Conjugation Kits have been designed to guarantee successful conjugation provided the protocol is followed correctly, for extra peace of mind we have developed our Check&Go! product range. Our original Conjugate Check&Go! Kit is a dipstick lateral flow assay for confirming the successful conjugation of an IgG antibody to a colored label such as a latex bead, and is compatible with IgG antibodies from multiple species, provided that they have affinity for either Protein A or G, which are immobilized at the Test line.

Schematic representation of the Conjugate Check&Go! process

Schematic representation of the Conjugate Check&Go! process. The antibody conjugate flows along the nitrocellulose membrane and binds to the Protein A and Protein G which are concentrated at the test line. When the antibody is successfully conjugated to a colored label, a visible line appears on the strip.

We have used Conjugate Check&Go! to test antibody conjugates produced using our Latex Conjugation Kits.

Conjugate Check&Go! evaluation of antibody conjugates generated using Latex Conjugation Kits

Conjugate Check&Go! evaluation of antibody conjugates generated using Latex Conjugation Kits. Conjugate Check&Go! was used to confirm successful conjugation of a rabbit IgG to blue, red or black latex beads. 0.02% w/v conjugate solutions were tested in duplicate.

For more information on our Latex Conjugation Kits, Purification KitsCheck&Go! Kits or any of our products please contact us.