The ELISA test carried out by Eurogentec is an indirect ELISA. This means that a constant amount of antigen has been coated into the wells of the ELISA plate (100 ng/well), and tested with different dilutions of the serum or antibody in question. The development is done colorimetric, using a secondary HRP-conjugated antibody, and o-phenylenediamine as chromogenic substrate. The optical density of the chromogenic substrate is measured at 492 nm OD(492).

The method has to be considered as semi-quantitative, since the measured reactivity with growing dilutions can be due to:

• The concentration of specific antibodies against the antigen
• The affinity of the evolved antibodies against the antigen

The results are influenced by the following factors:

• The general success of the immunisation against the antigen
• The suitability of the antigen (peptide or protein) to be coated onto ELISA plates
• The exposure of the immunisation relevant epitopes after coating

The measured optical densities at 492 nm are plotted against the dilution of the antibody or serum. The following curve types can be found:

Figure 1 : Different shapes of ELISA curves that can be found after ELISA testing. “No reactivity determined” (light blue), “Low [conc.] or affinity” (black), “Average [conc.] or affinity” (blue), and “High [conc.] or affinity” (yellow) display flat, hyperbolic, or sigmoidal ELISA curve shapes. The optical density OD(492) can be correlated to antibody affinity or concentration in the window between the “Background” (red), and Plateau (turquoise).

Flat ELISA curves

Flat ELISA curves (Figure 1, light blue) are typical for pre-immune sera. Since the immunisation host did not encounter the antigen, no immune response could be determined. A flat curve can also result, if antigens coated with the epitope towards the ELISA plate, or which did not coat at all. Potential program difficulties: a flat ELISA curve can result in rare cases, if the antigen is a serum protein, cell surface expressed, and evolutionary conserved – the generation of an immune response will just be suppressed, in order to protect the host (which is normal).
Given all the variables, testing and establishment of the antibody should be carried out accordingly.

Hyperbolic ELISA curves

Hyperbolic ELISA curves (Figure 1, black) are typical for the immune response generation phase during an immunisation program. This reactivity kinetics can be found very often when testing the small test bleed (PP) of 87-day programs. The curve might be understood as “a low concentration of high affinity antibodies” has been generated, but it also can result from antigen coating difficulties. Given this, testing the antibody in your application might give completely different results.

Sigmoidal ELISA curves

The first case of sigmoidal ELISA curves represents the average of ELISA tests carried out by Eurogentec. In Figure 1 the curve is represented in blue. The curve falls in a sigmoidal shape from the “Plateau” of saturation usually at a dilution of 1:1.000 towards the “Background”, which is reached with a dilution higher than 1:10.000. The curve might be read like “the immunisation provided a mixed population of high and low affine antibodies, which detect the antigen even if highly diluted”. Antibodies or sera producing curves like this can be used in a lot of applications, since the antibody population is rich – the ELISA test only detects antibodies that bind to the epitope immobilised on the ELISA plate, other populations of the antibody are not reflected here.

The second case of sigmoidal ELISA curves is over the average of ELISA tests carried out by Eurogentec. This curve is displayed in yellow in Figure 1. The curve is characterised by keeping the “Plateau” level of saturation very long, and falling in a sigmoidal fashion towards the “Background” level beyond the concentration range considered for ELISA plots. Curves like this result from very immunogenic peptides, large proteins like maybe a provided antigen from customers or the peptide carrier protein KLH used by Eurogentec, or so called “super antigens”. While strong reactivity against your peptide, or protein is desired, and a strong reactivity against KLH is a positive control for general successful immunisation using peptides, super antigens represent epitopes that can be found randomly. This random occurrence of structural instead of sequence-related motifs implies as well that the antibody providing this is non-specific. The cases where immunisations provided antibodies to super antigens are rare. Antibodies or sera producing curves like this can usually be used in a lot of applications, since the antibody population is richly composed.

Purifications

After an immunisation, you might have included affinity purification of the produced antibodies against the peptide or your antigen in the sufficient amount. Eurogentec offers different formats of purifications, 5 ml, 20 ml and 50 ml. The ELISA can be added optionally to 5 and 20 ml purifications, but it is a part of 50 ml affinity purification. The ELISA has to be considered here, as a mean of characterisation for purified antibodies, and not as a tool providing a quality control. As above already mentioned, the results of the ELISA tests are influenced by

• The accessibility of the epitope for the antibody
• The concentration and plating suitability of the antigen
• Simply the fact that the antibody has been purified with the antigen coupled to a matrix, and is now probed with the antigen immobilised on an ELISA plate (structural differences of the antigen in two different applications)

Given these factors, different results might be possible for the ELISA of the purified antibodies:

Purification 3 : a flat ELISA curve might suggest that the purification did not provide any working antibody (Example 3). Please be advised that also in such a case testing is worth the effort, since it might still be possible that the antigen looks different on the ELISA plate compared to its structure during affinity purification, and finally in your application. Try to characterise the antibody by Western or dot blotting – these applications work with far over 70% of custom-made antibodies from Eurogentec. A starting dilution might be 1:500. If it tests positive in Western blot – or dot blot, and you want to use your new antibody in your desired application, you have to establish it accordingly, maybe change existing protocols. Please compare the performance of the purified antibody always to the serum (1:500 to 1:10.000 diluted, depending on your optimisation results) in paralleled tests on identical samples. Should you experience difficulties, please feel free to contact us at info@eurogentec.com for further advice.

ELISA and Cross Purifications from Phospho-, Methyl-, or other modification specific programs

Since protein modifications play a key role for different cellular functions, and scientific interest for the different types of protein editing events is gaining more attention, Eurogentec is proud to make the detection of post-translational modifications also possible to you.

Immunisation programs for their detection can be carried out in rabbits or guinea pig, as Eurogentec’s standard 87-day mode or as 28-day Super Speedy immunisations, but the crucial point is the ELISA testing during, and after the programs.

Identifying the best host for cross affinity purification of modification specific antibodies by ELISA

As in any immunisation program carried out by Eurogentec two hosts (in this case rabbits or guinea pigs) have been immunised with a peptide carrying the modified amino acid that you want to detect in down stream experiments.

To identify the best responding host, the pre-immune serum (PPI), and the final bleeds (SAB) of the hosts are tested against the modified peptide and against the carrier protein by indirect ELISA (Figure 2)

Figure 2 : In this anti-phospho program the immune responses against the phosphorylated peptide (solid lines, rabbit 1, yellow, and rabbit 2, green) and against the carrier protein (dashed lines) are determined by Indirect ELISA (Coated antigen, two hosts, and two bleeds, part of modification specific programs) and compare the reactivity of the serum before immunisation (pre-immune serum, PPI, day 0), and the final bleed (SAB, end of the program). The serum to be chosen for cross purification based on Eurogentec’s expertise would be rabbit 1, since the response here is a little bit better compared to rabbit 2 (SAB, solid green and yellow lines). This impression is also supported by the finding that the response from rabbit 1 against the carrier protein (SAB, dashed yellow line) is a little bit better than the carrier response of rabbit 2 (SAB, dashed green line). In analogy to Fig. 1 (above) spoken, both responses to the peptides are good, and similar, but rabbit 1 (SAB, solid yellow line) produced a response a little bit closer to the ideal sigmoidal shaped curve.

Purification 1 : isolation of antibodies that are specific to the peptide and to the modification from rabbit 1

After the best responding host has been identified by the above-performed indirect ELISA, the final bleed serum (SAB) of this host will be used for cross purification. The first affinity purification will be carried out against the modified peptide. By this step the serum will be depleted from antibodies that are specific to the modification, and the peptide context in general. Following this purification, the serum (S), the flow through 1 (FT1), and the purified (PA) are tested by indirect ELISA against the modified peptide and the carrier protein. In general the following results can be expected (Please see also Figure 5) :

Peak 2 and FT2 will be characterised by indirect ELISA against the modified peptide, and against the non-modified peptide. In general the following results can be expected:

A sample result is reflected in Figure 4 and Figure 5.

Figure 4 : Testing of Peak 2 (P2) and flow through 2 (FT2) against the modified peptide by indirect ELISA. Antibodies separated with the non-modified peptide react also with the modified peptide – these antibodies cannot be considered as a control for the non-modified protein target in your experimentation. The flow through 2 (FT2) antibodies show a weak reactivity against the modified peptide – please consider that the modification in the peptide might directly interact with the ELISA plate surface, and therefore a restricted access of antibodies to the modified epitope might result, causing a hyperbolical curve shape.

Please be advised that the population of antibodies against the modification in the deciding fraction FT2 can be very low, due to narrowing the variety of specific molecules towards a single feature of a small epitope. But nonetheless, the antibodies can be used in the most common application types diluted in the same way like other polyclonal antibodies.

Figure 5 : Testing of Peak 2 (P2) and flow through 2 (FT2) against the non-modified peptide by indirect ELISA. P2 contains the majority of antibodies to the non-modified peptide, which is reflected by the strong reactivity of the antibodies to the target. FT2 might contain a small population also specific for the non-modified peptide. These antibodies can be diluted out or washed off in down stream applications, the soon drop of the curve from FT2 indicates that these antibodies are no good binders.

Please contact us at info@eurogentec.com if you wish to discuss your ELISA results in detail, or if you wish support for the establishment of your antibody.

Antibody Evolution (meaningful for 87-day immunisation)

Eurogentec offers you the possibility to have ELISA testing done – this can be chosen as option for any immunisation program. Simply ask for a complete program including ELISA right at the start (Please note, ELISA testing represents for 28-day Super Speedy immunisation a set of supplementary characterisation data – the test can for technical reasons not be performed to decide on programme extension or stop).
In case ELISA testing with your program is ordered, we carry out the same standard program however without sending the peptide and sera directly to the customer. We keep the free peptide, the preimmune and small test sample (obtained after 3 injections) until the large bleed sample is available (day 66, after the 4th injection). At this moment, we carry out the ELISA test using one 96-well micro titre plate per animal. We test in parallel per ELISA dilutions from:

• Preimmune (PPI),
• Small (PP) and
• Large (GP) test bleed

Against

• The free peptide and
• Against the carrier protein,
• Including positive and negative controls.

The test report is sent to you per e-mail, and this is as well announced in the program related immunisation schedule.
This paralleled testing not only allows to have a good view of the antibody evolution with the last boost, but it also gives a relatively good decision facilitation for program prolongation with additional boosts and bleeds, termination as scheduled, or immediate termination (Example 1).

Figure 6 : In this anti-peptide program the immune responses against the peptide (solid lines) and against the carrier protein (dashed lines) are determined by Indirect ELISA (Coated antigen, one host, and three bleeds, referenced as AS-ELIS-01) and compare the reactivity of the serum before immunisation (pre-immune serum, PPI, day 0), the small test bleed (PP, day 38), and the large bleed (GP, day 66). The success of the program is reflected by the sigmoidal curve shape of the plot, which evolved from “no response” (PPI), already strong responses after day 38 (PP), and the strongest responses after day 66 (GP). The program in this host can be finished, if desired, since the response evolution reached already at day 66 a maximum being characteristic for day 87 (the end point of a conventional 87-day program).

If a good increase is observed between the first and the second test bleed, the chances to get still higher titres with additional injections are quite good (Example 2). If however both bleeds (first and second test bleed) show more or less the same titre, an additional injection will most of the time result in only slight or no titre increase of specific antibodies.

Figure 7 : In this anti-peptide program the immune responses against the peptide (solid lines) and against the carrier protein (dashed lines) are determined by Indirect ELISA (Coated antigen, one host, and three bleeds, referenced as AS-ELIS-01) and compare the reactivity of the serum before immunisation (pre-immune serum, PPI, day 0), the small test bleed (PP, day 38), and the large bleed (GP, day 66). The antibody evolution of the program is slow, and reflected by the hyperbolic curve shape for the PP and GP for the peptide (blue) of the plots. The carrier (yellow, compare also to Fig. 1) produced already strong responses, which evolved from “no response” (PPI), to almost maximum at day 38 (PP), and the strongest responses after day 66 (GP). The program in this host should be extended by additional boosts in order to receive a sigmoidal curve shape (compare Fig.: 1, PP and GP, blue) reflecting the desired antibody reactivity against the antigen (Compare to blue curves PP and GP in Fig.: 1).

Please be advised that ELISA results are telling the customers and us if the immunisation worked. Nonetheless, this information can also bear weaknesses. Usually proteins bind very good to ELISA plates but especially small antigens like peptides or other haptens might

• Not bind at all (a false negative result)
• Bind insufficiently (the result might partially reflect the immune response which has been evolved in the animal)
• Bind to the plate with by the epitope (a false negative result)

Please note the general efficiency in case of a peptide immunisation program is reflected by the strong immune response to the carrier protein (a global positive control, Fig.: 1 and 2, yellow curves). If the response against the peptide appears to be strikingly weaker (Fig.: 2 for example; Fig.: 1 reflects rather the ideal case), you should in each case test the serum in your intended application, or by a different method, like dot or Western blotting.

Please note, it is hardly possible to extrapolate results from working ELISA tests to other applications, or to conclude from an antibody’s efficiency in one application to its potential capabilities in other applications: this means that an antibody providing bad results in ELISA testing, might be excellent in your down stream application, where the antigen looks different – but also vice versa is possible.

Please contact us at info@eurogentec.com if you further wish to discuss your ELISA results.

General considerations:

Please be advised that the antigen binding characteristics of an antibody after affinity purification might be shifted from polyclonal to a monoclonal-like specificity. A protein as antigen has much more epitopes for antibodies than a peptide. It is therefore in a few instances likely that especially in the case of peptides that key epitopes get hidden on ELISA plates, and ELISA results do not reflect properly the detection capabilities of the tested antibody, like they might be revealed in your intended application.

Eurogentec works with standardised protocols for purification of antibodies. Though these protocols are optimised, it might still be possible in rare cases that antibodies are not completely eluted from the purification column, nor can it be excluded that all partially denatured antibodies fold back to functional molecules during neutralisation after elution. In these cases affinity purification might not be the right way to concentrate your antibody.

Please note that immunisation hosts are not transparent, and very rare occurring failure of immunisation programs are not foreseeable to us, Eurogentec cannot guaranty for any antibody efficiency, nor warrant any quantities or capabilities of the produced molecules.