Antibody Labelling Made Simple
Blog Nov 26, 2014
Direct antibody labelling can be a difficult and time consuming process. We spoke to Klaudyna Schmidt, Marketing Manager, Innova Biosciences, to find out how the new Lightning-Link® Rapid range can simplify and speed up the process.
AM: What are the differences between indirect and direct antibody labelling?
KS: The main difference between direct and indirect antibody labelling is determined by whether the label used within the assay is attached directly to the primary antibody detecting the antigen or instead conjugated to a secondary antibody which binds to the unlabelled primary antibody.
There are many advantages of directly labelling primary antibodies, for example the requirement for fewer reagents, the elimination of cross-species reactivity and non-specific binding, and a significantly simpler and quicker assay protocol which avoids the need for multiple labelling steps.
AM: Can you describe some of the traditional direct methods of antibody labelling?
KS: Traditionally direct antibody labelling is seen as complex and time consuming, carried out only by those with specialist knowledge of chemical modification techniques.
There are various methods for traditional conjugation, which depend to a large extent on the nature of the label being attached to the antibody. Methods for linking large fluorescent proteins or enzymes are especially complex, often requiring chemical modification of both the label and the antibody. Smaller fluorescent molecules are often linked to antibodies using NHS-Ester chemistry, which although somewhat simpler still requires an understanding of the chemical processes involved and the factors that may impact upon conjugation efficiency.
A common feature of traditional antibody labelling is the requirement to separate labelled antibody from free dye, a step which leads to significant losses of highly valuable antibody.
AM: How does Lightning-Link® Rapid simplify the labelling process?
KS: Lightning-Link technology enables direct labelling of antibodies, proteins and peptides or any biomolecule with an amine group in just 20 minutes with only 30 seconds hands-on time.
The researcher simply has to pipette their biomolecule into a vial of lyophilized Lightning-Link mix and incubate for 15 minutes. Therefore specialist knowledge is not required, literally anyone who can use a pipette can successfully label their own primary antibody.
Additionally, in contrast to other systems that claim rapid labelling, there is no need for the researcher to send their antibody to the kit manufacturer to have it modified in advance.
AM: What other benefits does Lightning-Link® Rapid offer?
KS: One of the main benefits of using Lightning-Link Rapid kits, in addition to the obvious simplicity and efficiency, is the removal of extra wash and separation steps in the actual assay in which the antibody is used, which can be both tedious and time consuming. It is amazing how many times people state how much they hate those wash steps! Many researchers that we have spoken to have also highlighted how eliminating the use of secondary antibodies in their assay is an attractive time-saver.
Furthermore the weaker non-covalent bond formed between the secondary and primary antibody in an indirect assay may lead to loss of low affinity antibody during the additional wash steps required. This is the underlying reason for the fact that the amplification that is generally expected with secondary antibody staining is not always seen in practice.
Direct assays also avoid the problem of cross-species reactivity and non-specific binding associated with indirect labelling which can make data interpretation difficult or even impossible.
AM: How do results compare to traditional methods, and what has feedback been like?
KS: Despite its apparent simplicity, the Lightning-Link® process is sophisticated and generates conjugates with performance characteristics identical to, or better than, those prepared with laborious multistep conjugation procedures.
The original Lightning-Link range was launched in 2006, and since that time has received a huge amount of positive feedback. There are now just under 400 published references quoting the use of the kits in a wide range of applications, and there are numerous testimonials such as the one below.
"We’ve been really impressed with Innova’s products. Their biotin kit is simple to use and proved more durable than many competitors’ kits, and we’ve seen significant improvements in reproducibility since we started to use them regularly. We’re looking forward to continuing to work with the Lightning–Link® technology.”
Dr Jake Micallef, CSO of VolitionRx
For more information visit www.innovabiosciences.com/
Klaudyna Schmidt was speaking to Anna-Marie MacDonald, Editor for Technology Networks.