Commercial antibodies and their validation

Specific binding of non-specific antibodies

The term non-specificity is used when an antibody binds to unintended proteins. Each antibody molecule has a certain affinity to one part of the protein called an epitope, and this affinity is determined by the epitope’s amino acid sequence. It is therefore very difficult to find antibodies that react exclusively to one protein when this protein is very similar to other (closely related) proteins. Only antibodies that will bind to a unique epitope will react specifically to its intended target protein. However, most antibodies do not bind to unique epitopes and so they will cross-react.

In the case of shared epitopes between closely related proteins cross-reactivity is inevitable. Then the actual binding of the antibody may be specific, yet the antibody is deemed non-specific in relation to the intended target protein. Further diluting the antibody and optimizing blocking conditions will not work in these cases. In other words, the specificity of an antibody relies on the uniqueness of the protein part it binds to (i.e. the epitope).

An antibody specific to an epitope that is shared between one or two other (closely related) proteins may not be useless. It may still be useful in tissues or cell types where those cross-reacting proteins are not present. Or the scientist can take advantage in relating the intensities of bands representing the different proteins in Western blot.

Proteins unrelated to the intended target protein may have epitopes similar but not identical to the specific epitopes. Then the antibody’s affinity for the similar epitope will be lower than for the specific epitope. This will result in cross-reactivity with a proportionate lower signal, called non-specific background.

Non-specific background

Non-specific background can be reduced by further diluting the antibody. The reason is simple: by diluting the antibody only higher affinity interactions are sustained. The lower affinity interactions (to remotely similar epitopes) will not last at lower antibody concentrations. Proper blocking conditions can also help to prevent low affinity interactions. NaCl will interfere with weak hydrostatic interactions while non-ionic detergent (for example Tween-20) will interfere with weak hydrophobic interactions between the antibodies and the unintended target proteins. Increasing the concentrations of such blocking agents may help to reduce the non-specific background.

Noise

Poor experimental conditions will incur random noise and this is typically not related to the primary antibody. Lacking certain blocking components or the use of dirty containers/contaminated buffers are usually to blame. Especially in fluorescence-based assays, noise can be a big issue. There is a risk of antibodies being dismissed prematurely because non-specific background and noise are not considered and dealt with separately.

Secondary antibody derived signals

In addition to primary antibody-derived issues, the secondary antibody can be a source of problems as well. The quality of the secondary antibody can be tested by side-by-side comparison of a complete experiment with another experiment lacking the primary antibody. Noise and background caused by the secondary antibody will become apparent in this negative control.

Batch-to-batch variations

The vast majority of vendors do not manufacture all the antibodies on the catalogue, and most of their antibodies have been obtained from a wide variety of different manufacturers from all over the world under OEM agreement (Other External Manufacturer). Such an agreement usually has a clause to forbid the supplier from publishing which of their products are sold by their OEM vendor. The vendors keep up the appearance that they themselves are the primary source of all their antibodies. This enables them to keep QC data on the product sheet that were generated many years ago thus keeping the sales going, while the actual antibody that generated these data may have sold out and has been replaced by successive other batches (from different animals) and the current batch on sale may no longer be able to generate such data at all.

Even monoclonal antibodies suffer from batch-to-batch variations, but not to such severe extent as some types (see above) of polyclonal antibodies. Nonetheless, certain hybridoma clone numbers are still being used for decades while, just like with cell lines, hybridomas cannot be the same after so many passages anymore. It is therefore misleading to use QC data that were generated decades ago, unless the current batch has proven to still be capable of generating such data (in which case one might as well show the latest version of the data).

Assay developers are advised to buy antibodies straight from the manufacturer and ask for a free validation sample from a large batch in stock. Once validated for the required platform, the same batch then can be purchased in bulk so to prevent batch-to-batch variations during the entire project. Identifying the manufacturer can be a challenge though, and the only way to find them is to start looking for overlap between the product sheets from the different vendors. This way, a list can be generated at which point one can guess who the manufacturer was based on additional details still present on the manufacturer’s product sheet but not elsewhere.

QC data on the vendor’s product sheet

Vendors accrue data from their own customers or from their own QC department, thus making the OEM product look unique. This way the same antibody can show different QC data on different catalogues. And while batches run out and are being replaced by others, it can happen that a vendor has still some of the old batch in stock, while another vendor will keep the QC data obtained from the former batch on their product sheet. From this moment on customers start to buy products that are no longer necessarily reflected by their product sheet.

Same antibody on many catalogues seemingly different by their QC data

Vendors do not only obtain their antibodies from the original manufacturers. There is a network of vendors obtaining each other’s catalogue items. Consequently, the same antibody starts to occur several times in one catalogue: one time with the current QC data provided by the original manufacturer, and one or more times with QC data obtained from the other vendor’s direct customers or QC department. Potentially, assay developers buy several antibodies from several vendors thinking they are buying different antibodies, yet a number of them originate from the same manufacturer’s catalogue number.

Storage

Antibodies deteriorate by repeated freeze/thaw cycles. Such cycles should be kept to an absolute minimum. One way is to keep aliquots frozen at all times and have one aliquot in the fridge for daily use until finished. Primary antibodies usually come with preservatives to keep them good at 4°C for many months. Please be aware that some freezers are opened extremely often during a working day and then antibodies stored at the front may endure freeze/thaw cycles because of the frequent and long browsing and possibly in combination with the summer sun shining straight in. When this applies, antibodies are best kept in a different freezer that is less often opened (eg. -80°C).

Western blot

Western blot is seen by many as the easiest and most straightforward type of immune assay. This systematic underestimation is thought to be holding back science at a great scale. With so many pitfalls unrecognized, many good antibodies are dismissed after one or two poor experiments. The Western blot is possibly the most complicated immune assay because of the many layers where the assay can fail. The most trivial problems and their remedies are highlighted in Table 1.

Potential problems may have already occurred during preparations of the biological material to be analyzed, well before the assay takes place. Proteolysis and oxidation are two notorious factors that will introduce low reproducibility of results when comparing different lysates from the exact same cell type with each other. The conditions (including temperature) of protein separation in SDS PAGE will influence the banding patterns. And finally, different tissue types and cell types do not necessarily give mutually identical patterns in WB. It depends on the type of protein of interest and whether any posttranslational modifications (PTM) and/or degradation may take place in a different fashion in each cell type. The PTMs may be prone to (partial) removal during the lysate preparation by endogenous enzymes. Proper inhibitors (making sure they did not get inactivated during preparation) should be added to the lysates to minimize such impairments. Hence, a commercial antibody shown to work properly in one tissue type or species should still be validated in other tissue types or species. And it is best advised to generate several lysates of the same cell type or tissue type at different days before starting analysis of all of them, side by side.

Immunohistochemistry and immunocytochemistry (IHC/ICC)

When cells, tissue blocks or their sections have been stored after dehydration, the structure of the proteins in the cells will have changed thus affecting the binding of antibodies. Antigen/epitope retrieval will restore this, but its success is very dependent on the method of retrieval and the method may have to be adjusted from antibody to antibody (or from target protein to target protein). Heat induced epitope retrieval (HIER) can be done at pH6 and at pH9 by either high pressure steaming or by microwave. The quality of results can heavily depend on the conditions (pH value, temp, the heating duration, and the method of heating). When all attempts through HIER fail, one could opt for protease induced epitope retrieval (PIER), although some scientists prefer PIER over HIER as their sole approach.

When fresh tissues have been fixed in alcohols or acetone, one usually does not store the samples, and cryosectioning and subsequent probing with the antibodies go ahead in one flow. In this case epitope retrieval may not be required. I would however recommend epitope retrieval if the tissues have been stored long-term in the alcohol or acetone before cryosectioning. The dehydration of the tissues may mimic the paraffin embedding procedure, particularly in the case of storage in the hydrophobic acetone. Hence, rehydration of the tissue during the removal of the alcohol/acetone may still not be enough to expose the epitopes fully (again) after long term dehydrated storage.

Immunofluorescence (IHC, ICC, FC)

Fluorescent labels are commonly used in Flow Cytometry (FC), ImmunoCytoChemistry (ICC) and in IHC as well. The major issue with fluorescence is the noise. Because of the high sensitivity of this detection type, noise is much more prevalent compared to other less sensitive detection types.

As with all assays, different dilutions of the primary (including one without primary) will have to be compared in order to appreciate the level of constant noise. One has to be careful in choosing the right blocking agents, but more importantly, one has to take into account the presence of endogenous molecules (even at low abundance) that bind to either the fluorophore itself or to its conjugated carrier (for example avoid using streptavidin-fluorophore when there is endogenous biotin, and avoid using anti-IgG-fluorophore when there are endogenous Ig-receptors).

Immunoprecipitation (IP/ChIP)

Some antibodies will not pull their target proteins down under certain conditions. Particularly when working with linear epitope antibodies (e.g. peptide antibodies) one is advised to compare native conditions with denatured conditions (reduced and in presence of 0.1% SDS) as a positive control.

During the classic method of IP, a network of antigen and polyclonal antibodies is precipitated and analyzed. A polyclonal antibody generated to the entire protein is essential for this method as many epitopes are involved in the network formation that is precipitated. However, when the target protein is bound to one or more other proteins, the variety of epitopes for the antibody to bind to become limited and the network may not be stable enough for this classic approach. It is therefore recommended to have a control of denatured protein IP side-by-side with the actual experiment.

When using epitope-specific antibodies (monoclonal or peptide-polyclonal) the target protein (complex) is brought down by beads. This approach makes the scientist independent on an antibody-antigen network to form. When the right epitope-specific antibodies are used, extra information can be generated about the binding sites for the interacting other proteins.

Microwell platforms

This assay type represents any micro-well formatted immunoassay. They all have in common that one reagent is coated to a stationary phase (usually the bottom of the well), and other reagents react with the coated reagent proportionally to the content of the analysed material within a natural matrix. Matrix could be a body fluid (plasma, serum, urine, etc), a culture supernatant, or a buffered solution spiked with biological material from which one constituent needs quantifying.

The biggest hurdle in such assays is the notorious matrix effect. To put it simply: the matrix contains molecules that interfere with the reaction between the antibodies and the antigen to be quantified. This interference can be visualized by making serial dilutions of the matrix. When the matrix is diluted with a factor two and the measured antigen (present at sub-saturating levels) therein does not read a reduction by a factor two along the way, you have established matrix effect. Also when readings do not correlate to the levels of antigen spiked into the matrix of interest, there will be matrix effects. Matrix effects are best dealt with by diluting the matrix in assay buffer to such extent that the antigen can be quantified without interference from the matrix. It is essential to establish matrix effect every time a new assay is set up. When a new antibody is introduced in an already existing assay, the matrix effect may respond differently from the previous antibody. A calibration curve needs to be made in the same matrix and run in parallel with each assay.

Polyclonal antibodies may perform better after being pre-adsorbed to abundant serum proteins as a blocking step. This should be a compulsory step when using the antibody in matrix containing high levels of serum protein, especially when all proteins in the matrix were labelled before the antibody was added. One needs to be aware that most commercial primary and secondary antibodies have not been pre-adsorbed to serum proteins. Some detergents may also minimize matrix effect.

Reproducibility

One should not jump to conclusions after a single experiment. Even positive data may sometimes be false positive and lack of signals may be due to trivial factors that can be solved by systematic trouble shooting. Therefore conclusions can only be drawn once identical results have been obtained by the same experiment carried out at different times.

Transportation-related impairments

Anyone working in a subtropical climate (eg. southern state of the USA) knows how hot it becomes on a sun baked parking space during summer and how temperatures can soar inside a delivery van parked out there while taking care of a delivery. Antibodies are inherently made to work at 37–42°C, but it remains a protein and it can cook until inactive when temperatures go well over 50°C. Every antibody is different from the next and the majority will survive the above mentioned conditions. When antibodies in solution arrive on frozen icepack, one can assume that the integrity of the antibody has been maintained during transport. Lyophilized antibodies are more resistant. It is the user’s responsibility to ensure that the ordered antibody is received in proper packaging (particularly during hot weather conditions) and complain to the vendor when this is not the case. When an antibody does not perform as expected based on the product sheet and the antibody was delivered under hot conditions, it is time to ask for replacement.

Endorsement by publication

Today’s advanced internet facilities enable vendors to search for publications describing successful use of their antibodies. However, some publications mistakenly attribute an antibody to the wrong vendor. And sometimes an antibody is correctly reported but no data generated by it are to be seen or presented in the paper, nor in the supporting documentations. In such cases the internet search delivers false-positives, and examples can be found in each vendor’s catalogue. Many such papers are inaccessible to the vendors and therefore they cannot always double check themselves. Although this is a relatively rare phenomenon, it is real all the same and therefore the customer is advised to double check the contents of the referenced paper when such endorsement is a requirement for the scientist to purchase this antibody. Each vendor will appreciate the feedback when a customer identifies a false-positive reference, and we urge customers to contact the vendor when such reference has been identified so they can remove it from the product data sheet.

Although there is a recent change in trend, the vast majority of publishers still do not demand to specify the used antibodies by their catalogue number in scientific papers. Since most catalogues have more than one antibody to one protein, a mere description of an antibody from vendor X to protein Y is not sufficient and prevents peers from replicating the described experiments.