Antibody Conjugation Illustrated Assay

This How To Guide Will Cover

• Types of Immuno-experiments and Associated Labels
• Direct vs. Indirect Detection Methods
• Antibody Labeling Methods
• Buffers and Additives
• Antibody Concentration and Purity
• Benefits of Lightning-Link Antibody Labeling Kits

Anyone new to this field will find in the literature a bewildering number of antibody labeling protocols. It is difficult for the newcomer to know what the critical parameters are and what procedure should be applied in any given situation. With traditional labeling methods, a basic understanding of the principles of chemical modification is required because the antibody and/or label must be chemically ‘activated’ before the labeled antibody (or ‘conjugate’) can be formed.

This guide is a user friendly tool that allows you to learn the basics of common antibody labeling methods. It also describes Lightning-Link technology, which massively simplifies the production of labeled antibodies. The Lightning-Link approach requires no knowledge of chemistry and the hands-on time is just 30 seconds.

Antibodies and Labels

Antibodies are widely used in immunoassays to detect and quantify antigens. The antibody that recognizes the antigen is referred to as the ‘primary’ antibody and confers specificity to the assay. A ‘label’ is also incorporated into the assay using one of two methods, direct or indirect detection (see below), to provide measurability. Some commonly used immunoassay techniques are given in Table 1 along with examples of the types of labels that may be employed.

Table 1: Types of Immuno-experiments and Associated Labels

Direct vs. Indirect Detection Methods

The label in an immunoassay provides either ‘direct’ or ‘indirect’ detection of the antigen.  With direct detection, the label is attached via a covalent bond to the primary antibody. Alternatively, using indirect detection, the label is covalently attached to a secondary antibody, which is allowed to bind to the primary antibody during the immunoassay.
 
In the case of indirect detection, the assay comprises two distinct parts. First, there is a period of incubation (one hour) with the unlabeled primary antibody, during which a small fraction of the antibody binds to the antigen. Excess unbound primary antibody is then washed away and a labeled secondary reagent is added. After a period of incubation (one hour), excess secondary reagent is washed away and the amount of label associated with the primary antibody is quantified.

With direct detection, the prior covalent attachment of the label to the primary antibody means that only a single incubation step with the antigen is required and only a single round of wash steps, as opposed to two rounds of incubation and wash steps with indirect detection. The assay simplification that is afforded by direct detection tends to decrease assay variability and to improve data quality. Some of the often-stated pros and cons of direct/indirect detection methods are given in Table 2.

Table 2: Pros and Cons of Direct vs. Indirect Labeling

*Find more information on detection methods on our Immunocytochemistry page.

Despite the potential advantages of direct detection, many immunoassays today still employ the principle of indirect detection. Undoubtedly, the main reason for this is that direct labeling of primary antibodies is relatively complicated and, historically, antibody labeling has been carried out only by those with specialist knowledge of chemical-modification techniques.

What about the amplification afforded by the secondary reagent in indirect detection? Is the signal lower with the direct labeling approach? Often the amplification observed in the indirect method may be false.  During incubation, the primary antibody dissociates from the antigen with the secondary reagent and in subsequent wash steps, resulting in amplification due to a diminishing amount of primary antibody. In many cases the same or even a better result can be obtained more easily with direct detection.

Some techniques commonly used in the labeling of antibodies and other biomolecules are outlined in the following section.

Antibody Labeling Methods

Antibodies are composed of amino acids; the lysine side chain (with a -NH2 terminus, a primary amine) is commonly used to covalently attach labels to antibody molecules. There are just four common approaches to labeling, which differ in the creation of the reactive label.

The four main chemical approaches for antibody labeling are summarized below:

1. NHS Esters: In the case of fluorescent dye labels it is usual to purchase an activated form of the label with an inbuilt NHS ester (also called a ‘succinimidyl ester’). The activated dye can be reacted under appropriate conditions with antibodies (all of which have multiple lysine groups). Excess reactive dye is removed by one of several possible methods (often column chromatography) before the labeled antibody can be used in an immunoassay.
2. Heterobifunctional Reagents: If the label is a protein molecule (e.g. HRP, alkaline phosphatase, or phycoerythrin) the antibody labeling procedure is complicated by the fact that the antibody and label have multiple amines. In this situation it is usual to modify some of the lysines on one molecule (e.g. the antibody) to create a new reactive group (X) and lysines on the label to create another reactive group (Y). A ‘heterobifunctional reagent’ is used to introduce the Y groups, which subsequently react with X groups when the antibody and label are mixed, thus creating heterodimeric conjugates. There are many variations on this theme and you will find hundreds of examples in the literature on the use of heterobifunctional reagents to create labeled antibodies and other labeled biomolecules.
3. Carbodiimides: These reagents (EDC is one very common example) are used to create covalent links between amine- and carboxyl-containing molecules. Carbodiimides activate carboxyl groups, and the activated intermediate is then attacked by an amine (e.g. provided by a lysine residue on an antibody). Carbodimides are commonly used to conjugate antibodies to carboxylated particles (e.g. latex particles, magnetic beads), and to other carboxylated surfaces, such as microwell plates or chip surfaces. Carbodiimides are rarely used to attach dyes or protein labels to antibodies, although they are important in the production of NHS-activated dyes (see above).
4. Sodium Periodate: This chemical cannot be employed with the vast majority of labels but is quite an important reagent in that it is applicable to HRP, the most popular diagnostic enzyme. Periodate activates carbohydrate chains on the HRP molecule to create aldehyde groups, which are capable of reacting with lysines on antibody molecules. Since HRP itself has very few lysines it is relatively easy to create antibody-HRP conjugates without significant HRP polymerization.

You may find more detailed instruction on these four approaches from any specialist book on antibody labeling.

Buffers and Additives - Key Considerations for Antibody labeling

It is important to remember that your antibody will contain substances other than antibody; minimally a buffer and/or salts, and possibly other proteins and additives. Compatibility of the mixture with labeling methods may not have been a key consideration when the antibody was initially purified and formulated; occasionally it will be necessary to re-purify the antibody prior to carrying out the labeling reaction. Purification may involve the removal of stabilizing proteins (e.g. BSA) or the removal of low molecular weight substances, such as sodium azide, tris buffer or glycine. The different labeling methods may be negatively impacted to different extents by the various additives but, as mentioned earlier, the majority of labeling methods exploit lysine residues on antibodies, thus substances with primary amines should generally be avoided in antibody labeling procedures. For example, glycine is often used to elute antibodies from antigen affinity columns and antibodies that have been purified in this way should be dialysed before use in labeling reactions. One should be particularly aware with dialysis that the removal of unwanted low molecular weight substances is more efficient if the buffer is changed two or three times. This does not mean that you have to make three times as much buffer, quite the contrary, though the total time required for the dialysis procedure will be greater. For example, calculation shows that dialysis against 3 x 1L volumes is far more effective than dialysis against a single 5L volume. PBS, MES or bicarbonate are often recommended as suitable buffers in which to carry out conjugation reactions; the best buffer is determined from the pH requirements of the labeling reaction.

Antibody Concentration and Purity

For most labeling reactions, the antibody will need to be reasonably pure (e.g. >90%, preferably >95%) and at a concentration of >0.5mg/ml. Many commercially available antibodies are provided in a form suitable for labeling, but you should be aware that this is not always so. For example, antibodies may be sold in the form of hybridoma tissue culture supernatant (TCS), ascites fluid or crude serum. TCS often contains many other proteins and culture nutrients (e.g. amino acids) which are particularly problematic. Purification (e.g. on protein A columns) is more or less obligatory if TCS is your starting point. Ascites fluids and crude serum have higher concentrations of antibody than TCS, but these materials are impure and contain high concentrations of amino acids; further purification of the antibody will generally be required.

At this point it is probably useful to provide a summary of the above. You now know about:

• (i) the key differences between direct and indirect detection;
• (ii) the four main approaches to antibody labeling;
• (iii) the importance of buffer formulations; and
• (iv) antibody concentration.

In reality, today, because of advances in antibody labeling technology, anyone who can use a hand-held pipette can make labeled antibodies with ease. The Lightning-Link one-step antibody labeling method is discussed below.

Lightning-Link - The World's Easiest Antibody Labeling Kits

The simplification of the antibody labeling process (most notably the elimination of column separation steps) circumvents many issues that have beset traditional procedures for years -loss of material, sample dilution during column chromatography, batch-to-batch variation and difficulties in scaling up.

The Lightning-Link process is summarized in Figure 1. You simply pipette the antibody to be labeled into a vial of lyophilized mixture containing the label of interest. Dissolution of the vial contents activates the chemicals that mediate the antibody labeling reaction. As there are no purification or separation steps (by-products of the reaction are completely benign), antibody recovery is close to 100%. Furthermore the labeling reaction can be set up using this simple method in less than thirty seconds. 

Although the antibody labeling procedure is very simple, the chemical approach is sophisticated, allowing the formation only of antibody-label conjugates in a gentle and controlled process.  Antibodies labeled with Lightning-Link exhibit performance characteristics that are identical with, or better than, those prepared with laborious multistep traditional procedures  (see figure 2).

The simplicity of the Lightning-Link approach means that it is very easy to scale up (or scale down) the amount of antibody to be labeled. Whether you are labeling small or bulk amounts of antibody, the hands on time remains the same – just 30 seconds. As there are no new technical issues created by scaling up, the performance of trial conjugate prepared at small scale is essentially identical to that prepared at bulk scale (see Figure 3 below). For any new antibody, or in situations where the antibody is extremely valuable and in limited supply, Lightning-Link kits allow labeling reactions to be carried out with as little as 10 µg of antibody. Other regular pack sizes include 100 μg, 1 mg and 5 mg.

Although the hands-on aspect of doing antibody labeling reactions is now incredibly simple, as mentioned above it is still necessary to think about what else is present in your preparation of antibody before undertaking a labeling reaction. However, Lightning-Link is remarkably tolerant of many additives found in antibody preparations.

Sodium azide, which is widely employed as an antimicrobial agent in commercially available antibodies, typically at levels of 0.02% or 0.1%, causes serious interference with some labeling methods. Fortunately, the Lightning-Link approach is minimally impacted by sodium azide (figure 4) at concentrations of 0.1% or less.

Another common additive in commercially available antibodies is BSA, which is used as a stabilizer. While one would anticipate serious interference from a molecule such as BSA in labeling reactions, BSA has only a very modest impact on Lightning-Link. Indeed, the possible benefit of removing BSA from very small amounts of antibody is likely to be offset by losses of antibody during any attempted purification. For example, with 1% BSA, which equates to a 20-fold molar excess over 1mg/mL antibody, the normal ELISA absorbance could be achieved simply by diluting the antibody labeled in the presence of BSA by 1/3000, instead of 1/10,000 for the BSA-free reaction (see figure 5).