Troubleshooting & FAQs

The absolute quantitation of HCP assays is exceedingly difficult for several reasons. First of all, there is no recognized reference preparation of HCP against which we can calibrate our assays. While ELISA is inherently a quantitative method when applied to a single analyte, ELISAs that attempt to measure simultaneously all of the hundreds of potential HCP impurities in the same well using a single reporter/detector system are at best semi-quantitative assays. Many arbitrary choices and assumptions are used when preparing anti-HCP antibodies and later the choice of material to use when preparing “standards”.
 
How to obtain the HCP material for immunogen is the first choice that must be made. 

• Do we use a null cell line or a mock transfected cell line or one with the actual product plasmid and what differences in HCP might we see from each?  
• At what point in the purification process do we obtain the HCPs for the immunogen or standards? 

In almost every case the array and relative concentrations of HCPs in final product are different from the HCP in the ELISA standards. 

• How does one assign a total HCP concentration to an indeterminant mixture of many proteins with different molecular weights?  
• What effect will the different affinities of antibodies to various HCPs as well as the different concentrations of those antibodies have on the ability of the assay to be quantitative? 
• How do we know that we have antibody to all the HCPs present in a given sample type?                               

All of these questions and arbitrary choices mean HCP assays will at best be semi-quantitative methods capable of making only relative estimates of HCP concentrations from one sample to the next. We estimate the potential error in reporting an HCP level in a sample that has a different array of HCPs from the standards to be as high as 4-fold even if you have a very good broadly reactive antibody. The commonly expressed theoretical concern is that HCP assays might under-estimate results due to incomplete coverage of all HCPs. In reality, the quantitative errors in HCP assays due to the arbitrary choices and fundamental limitations of the method as discussed above can result in either over-estimation or under-estimation and tend to do so to about the same degree. In our experience, a well generated and affinity purified antibody will react to more than 70% of individual HCPs as demonstrated by traditional 2D Western blot correlated to silver stain. What is more important than the number of individual proteins with antibody reactivity, is the total mass of those proteins that are detected. In our experience and as you might expect from theoretical considerations, the proteins in highest concentration have the best chance of generating an antibody. As such, an antibody with about 70% reactivity to an individual HCP will react with those proteins that account for more than 95% of the total mass of HCP. Given this level of uncertainty in the absolute HCP concentration, the most important criteria by which we can judge any HCP assay is on other objective parameters by which any analytical method should be qualified or validated. Those criteria include specificity and accuracy as demonstrated by sample dilution linearity and spike recovery experiments, precision, robustness, and sensitivity. Provided the assay has sensitivity to detect at least a relative portion of the HCP in your final drug substance and the assay also meets the other objective analytical parameter specifications, such an assay is a valuable analytical method capable of demonstrating process control and reporting relative levels of HCP contamination from lot to lot as a release test.

Due to the impracticality of obtaining real final product HCPs that co-purify with product down to the final step, most of our HCP assays will utilize a source of HCPs from very upstream in the purification process. Those HCPs typically do not come from the actual product cell line but rather a null cell or mock transfected cell line. Once we have decided on the source material for the kit standards we first perform some partial purifications to remove non-HCP materials such as growth media additives, buffer salts and extraction reagents. Our initial approach to calibrate this processed material is to simply perform a BCA total protein assay using bovine serum albumin (BSA) as an arbitrary standard. When standards made with the HCP material calibrated by BCA give reasonable “stoichiometric agreement” with the amounts of antibody used in the ELISA then we feel the calibration by BCA is good enough. What is meant by “stoichiometric agreement” is that we know how much antibody is used in the ELISA. It is the quantity of antibody that actually dictates the analytical range and dose response curve of the ELISA. If we assume an average molecular weight for the total HCPs, then we can reasonably estimate HCP concentrations across the valid analytical range of the assay. When the BCA assay concentrations do not reasonably approximate the ELISA stoichiometry, we process the HCP material further. Such processing involves various purification steps to remove components registering in the BCA assay but that are in fact not HCP or at least not immunoreactive HCPs. This processing might also involve affinity purification against the anti-HCP antibody. The purification stops as soon as the BCA concentration gives a realistic stoichiometric agreement in the ELISA.

We manufacture our kits for lot-to-lot consistency and restrict and evaluate changes in any components or procedures that could impact accuracy in the customers' laboratory. However, for generic kits, Cygnus performs QC on a limited range of parameters that may or may not be sensitive to your product specific issues. The routine run-to-run quality control of ELISA is best accomplished by assaying control samples across the important analytical range of the assay. We recommend 2 to 3 controls, a low control in the range of about 2 to 4 times the assay Limit of Quantitation (LOQ), a medium control, and a high control. Ideally, these controls should be made using your source of analyte (e.g. HCPs from your process). Furthermore, the controls should be in the same matrix as your critical samples or after dilution to MRD for your final drug substance. By using your source of analyte in your sample matrices, you will be best able to identify any problems within the run or between runs and kit lots. Cygnus offers Control Sets for several ELISA kits including; CHO, HEK, E.coli, PG13, and Protein A. For more information on Control Sets or advice on how to make and establish controls specific to your needs please contact technical support.

Use of laboratory-specific controls is the only way to assure total quality control of the assay for your needs. Controls should be made in bulk, aliquoted for single use, and frozen at -80°C until stability studies indicate some other storage conditions are adequate. Once you have statistically established a range for these samples, they will become your most sensitive and specific tool to assure quality control of the assay. Do not rely on curve fit parameters as quality control specifications in the absence of true analyte controls. Curve fit parameters such as R square, slope, y-intercept, and upper and lower asymptotes are not sensitive or specific enough to reliably detect assay problems. Use of such parameters in the absence of true analyte controls, will frequently yield erroneous results. Contact our Technical Services Department for advice on how to make and establish controls specific to your needs.

Numbers of Replicates - When precision is very good (average replicate %CV on ODs are less than 5%) we feel duplicate analysis is adequate and the most effective approach. In the case of duplicate analysis, we do not allow for editing of any apparent outliers since there is no statistical basis for establishing which of the duplicates is inappropriate. Thus, in duplicate analysis we suggest repeating any sample that yields a %CV greater than 20%.

This study protocol is suggested as an objective method to qualify that the Cygnus Technologies Host Cell Protein (HCP) ELISA kits and other bioprocess impurity kits will yield accurate, specific and reproducible results for a given product and sample type. In addition to the ELISA validation protocol detailed below, it is necessary to utilize orthogonal methods to determine coverage and to assure all significant HCPs are being detected. Despite its many acknowledged limitations, Western blot, both one- and two-dimensional, have historically been used to characterize reactivity of polyclonal antibodies to individual HCPs and for use in early development of downstream purification processes. Unfortunately, the analysis of anti-HCP antibodies by Western blot is of very limited predictive value due to the lack sensitivity and specificity in detecting HCP in final product and other downstream samples. It is for this reason, ELISA is proven to be the method of choice for determination of total HCP in downstream samples. To better answer the question of antibody reactivity/coverage to individual downstream HCPs, we recommend a method termed Antibody Affinity Extraction or AAE.  For a more detailed discussion of the limitations of Western blot and the advantages of AAE please refer to the technical white paper found on our web site.

ELISA Qualification:

In some cases, your product itself or certain components in product formulation buffer may interfere (either positive or negative interference) in the ability of the assay to detect HCPs or other impurities. Similarly, samples from upstream in the purification process may also contain material in their matrices that can interfere in ELISA methods. Factors such as extremes in pH, detergents, organic solvents, high protein concentration, and high buffer salt concentrations are known interference components. For these reasons, it is necessary to qualify by universally recognized experimental procedures (i.e. ICH & FDA guidelines) that the assay will yield accurate results. Should the end user of this kit determine that there is significant product or matrix interference it may be necessary to further process the sample by methods such as sample dilution or buffer exchange to render it into a more assay compatible buffer. The same diluent used to prepare the kit standards is ideally the preferred material for dilution or buffer exchange of your samples. Please refer to the kit product insert for recommended diluents. In other cases, modification of the assay protocol can effectively improve accuracy in some sample types. Users of our kits are encouraged to contact our Technical Services Department for advice on how best to solve sample accuracy issues. The methods suggested below are intended as a general guidance for the most important parameters of accuracy, specificity and precision. We suggest you review ICH and other similar regulatory guidance for detailed protocols on how to qualify analytical methods.

1. Dilution linearity/parallelism experiments – All sample types to be tested that contain levels of impurity greater than the LOQ of the assay, should initially be evaluated for dilution linearity as part of assay validation. This experiment involves performing a series of doubling dilutions using an approved assay sample diluent. These dilutions are then assayed and a dilution corrected impurity concentration is determined at each dilution. This dilution linearity study establishes freedom of sample matrix interference as well as the important condition of antibody excess in the ELISA for the array of impurities in your samples. If you will be routinely testing in-process samples in addition to final product, you should validate dilution linearity of each sample type. This analysis is particularly critical for HCP assays because very high concentrations of certain individual HCPs may approach saturation of the antibody against that particular HCP. When this happens, there is a risk of under-quantitation for that HCP. By performing dilution analysis, one can verify if the antibody is in excess and that the sample matrix itself does not interfere. If the antibody is in a limiting concentration or the sample matrix causes a negative interference, what will be observed is that the apparent HCP concentration for a sample increases with increasing dilution. In most cases a dilution will be reached where the dilution corrected value remains essentially constant. This dilution is what we term the Minimum Required Dilution or MRD. Table 1 below shows example data where an in-process sample did not yield good dilution linearity at high concentration, but with further dilution, an MRD was determined at which acceptable dilution linearity was obtained. In this example we conclude that the MRD for this in-process sample is 1:8 and that the concentration of HCP to be reported is 361ng/m. Once an MRD is established for a particular sample type, your SOP should reflect that such sample needs to be diluted before assay. We suggest defining acceptable dilution linearity as “dilution corrected analyte concentrations that vary no more than 80% to 120% between doubling dilutions”. Due to the statistical limitations in the low end of the assay range you should avoid consideration of dilution data where the assay value before dilution correction falls below two times the LOQ of the assay. Acceptable diluents may vary from assay to assay and you are encouraged to verify with Cygnus that your sample diluent is acceptable. In general, the best diluent is the same one used to prepare the kit standards. See your kit product insert for recommended diluents. Assay specific diluents can be purchased from Cygnus in 100ml, 500ml or 1000mL bottles. Contact Cygnus for information on acceptable diluents. 

Table 1: Example Dilution Linearity Data for an In-Process sample

2. Spike and recovery experiments - For each sample type to be tested, be it final product or in-process samples, you should demonstrate that the assay can recover added HCP or other impurity spiked into that sample matrix. This can be simply performed by spiking some of the highest standard provided with the kit into your sample types and then testing in the assay. Spiking studies are only relevant at or below the MRD as established in Section 1 above. Using our E. coli HCP kit, F410 as an example, we suggest spiking 1 part of the 100ng/mL standard into 4 parts of your sample (e.g. spike 100mL of 100ng/mL standard into 400mL of sample). The spiked concentration into the sample in this case is 20ng/mL. A control dilution of 1 part of assay diluent (zero standard) to 4 parts of sample is also performed to determine the contribution of endogenous HCP in the sample prior to spiking. Both the spiked and diluted unspiked samples are assayed. Percent total recovery is determined by dividing the total HCP measured (from endogenous and spike) by the sum of the spike level + the endogenous contribution of HCP. We suggest acceptable recovery should be within 80% to 120% of the spiked HCP. Table 2 shows example data. If you desire spike and recovery at more than one concentration we recommend that the lowest spike levels should be at least 2 times the Limit of Quantitation (LOQ) of the assay and that the contribution of the endogenous HCP in the sample prior to spiking not exceed two times the spike level to be tested. These two conditions will insure better statistical accuracy.

Table 2: Example Spike and Recovery Data

3. Precision Experiments - We also recommend that each laboratory and perhaps each technician perform a precision study to demonstrate that they can achieve acceptable precision both within and between runs. Such precision would be best accomplished on controls prepared in your laboratory. Those controls should be made in your product matrix and from HCP derived from your cell line and process. Once prepared at the desired levels these controls can be aliquoted and stored frozen to insure stability. Once statistical values have been established on your controls, you will then have an important QC tool to assure that the accuracy is acceptable from kit to kit and assay run to assay run. Beyond the above critical validation studies, you may want to perform other experiments that statistically establish the LOQ and LOD in your laboratories, robustness, stability, etc. as listed in FDA and ICH guidelines. However, you may want to consider using our in-house generic validation study for this less critical data rather than perform it yourself since we view such non-critical data as inherent in the method and not something each laboratory needs to do.

Orthogonal Methods

We find no predictive value in 2D WB of upstream samples as it is too insensitive and non-specific to determine how an antibody when used in an ELISA will quantitatively reactive to the more limited array of downstream HCPs. Instead, we recommend performing AAE on at least 2 samples. The first sample can be very upstream such as harvest material. AAE will provide a more sensitive and accurate assessment of percent coverage than can be achieved by 2D WB. The second sample will come from downstream in your purification process determined by the level of total HCP as measured by ELISA. AAE on this sample will allow you to identify those individual HCPs to which the antibody is reactive. Any individual HCPs spots showing up in a 2D PAGE silver stain or similar method that do not have a corresponding AAE spots can be picked from the 2D PAGE and subjected to Mass Spec analysis to confirm if they are in fact HCPs and if so what is their structure and biological function. Any sample for which dilution linearity cannot be obtained should also be analyzed by Mass Spectrometry to identify major impurities for which there is lack of antibody excess.

Many of our customers have expressed an interest in automating our ELISAs to improve throughput and assay performance. Cygnus Technologies has much experience with various automation platforms. These systems include automated microtiter plate instruments and other liquid handling systems as well as several “biosensor” platforms. Using our proven and well-qualified antibodies and reagents in a biosensor platform still presents your lab with significant challenges in re-qualifying and optimizing the reagents. Before considering adapting our reagents to an automated system, you are encouraged to contact our Technical Service Department for guidance on how to approach assay development, qualification, and validation.

The promises of automation are higher throughput, faster turnaround of results, and improved performance in terms of parameters like sensitivity and precision. The practical ability of the many systems now being marketed to meet these promises is variable. Before embarking on a quest for automation, we urge you to contact our Technical Services Department. After evaluating your testing needs we can guide you to the system that best meets your requirements.

There have been some erroneous reports in the literature of cross-reactivity of anti-HCP antibodies to recombinant drug substances. These mistaken conclusions are usually based on the misuse and misinterpretation of Western blot data. Loading a large amount of product protein in the range of micrograms, in an effort to see trace HCP contaminants in downstream samples, will often result in a "ghost" WB band to the product protein. This is rarely true immunological cross-reactivity, but rather a non-specific protein-to-protein interaction due to overloading of the product. WB is not a good method to assess immunological cross-reactivity, as it is often non-specific and rarely has the sensitivity to detect downstream HCPs. Furthermore, WB is of very little value in predicting how cross-reactivity would manifest itself in the more sensitive ELISA. Without the appropriate negative controls, WB should not be used to conclude immunological cross-reactivity. If you want to use WB to determine cross-reactivity, contact our Technical Services Department for advice on how to design your WB experiments with the proper controls. Also, review our FAQ on What is the difference in Sensitivity and Specificity, Western Blot vs ELISA.

Given that most HCP antibodies are made to null cell strains, cross-reactivity to the product would not be expected unless there is a significant conservation of epitopes on the product with those in the natural proteome of the host organism. Even if there are some cross-reacting epitopes on the product that manifest as a specific band by Western blot, it should be understood that in most cases this cross-reactivity will not result in a false increase in HCP concentration by ELISA or similar sandwich assay method used to quantitate HCP in final drug substance. To understand why this is the case, one must have a comprehensive appreciation for the stoichiometric limitations of ELISA. Any cross-reacting antibody is a small subset of the total anti-HCP activity. Downstream samples contain mostly the product itself, often in the mg/mL range. This high concentration of product will greatly exceed the amount of cross-reacting antibody in the ELISA, often by over a million-fold. With such an excess of antigen, the dose response curve for drug substance will be well past the "high dose hook" in the negatively sloped region, very near to the background signal for the assay. In this region, any ELISA reactivity to your drug substance would be observed either to not change with dilution of drug substance sample, or to actually increase with decreasing drug substance concentration. The dilution linearity experiment we recommend in our FAQs on Dilution Linearity is critical to validation of any HCP method. Dilution linearity can indicate either an excess of certain HCPs relative to the amount of kit antibody against them or it can indicate if you have cross-reactivity to your drug substance. If you have a lack of dilution linearity, a stoichiometric analysis of your dilution data will usually allow for the discrimination of true drug substance cross-reactivity from the more common lack of antibody excess for certain HCPs that persist through your purification process.

We have developed over 150 anti-HCP antibodies to virtually all the recombinant and transgenic expression systems in use today. The only cases where we have seen true immunological cross-reactivity that interfered in the accurate detection of HCP by ELISA has been with Pichia pastoris and two transgenic plant expression systems. In all three cases, it was determined that the cross-reacting epitopes were due to glycosylation of the drug substances. We have developed various methods to overcome cross-reactivity. If you have confirmed cross-reactivity that interferes with accurate measurement of HCP please contact our Technical Service Department for advice on how to proceed.

We are not aware of limits of HCP impurities established by the FDA or any other international regulatory bodies. You should contact your regulatory body for advice and current points of view.  We do not presume to speak for current regulatory positions on HCP detection and limits, but we can relate our considerable experience with products expressed in a variety of biological expression systems. In addition, we are pleased to offer what we feel is a rational approach to the questions of HCP impurities.

Each product submission should and will likely be considered on its own merits regarding the question of HCP impurities. Issues such as the route of administration, quantity of drug given, and frequency of dosage are all factors that could influence safety problems from HCPs. The approach should be to get HCP levels as low as reasonably possible. With advances in antibody generation and immunoassay development such as those implemented in our kits, the sensitivity and specificity of HCP analysis is significantly improved over Western blot and PAGE analysis. When the purification process is developed with feedback from a sensitive HCP ELISA, the levels of HCPs are typically less than 10 ppm. When a sensitive ELISA is not used during process development, and instead methods like PAGE and Western Blot are employed, it is common to see HCP levels greater than 100 ppm and often in the ppt range. We believe the most rational and cost-effective strategy is to employ a sensitive generic ELISA methodology early in product development rather than waiting until Phase 3. Such an assay will provide very valuable feedback to purification process developers and may help insure a more positive outcome to early clinical trials.

For more information on HCP guidance, please refer to the USP Chapter 1132 (i.e. United States Pharmacopeia's chapter on Residual Host Cell Protein Measurement in Biopharmaceuticals)

https://www.usp.org/sites/default/files/usp/document/our-work/biologics/USPNF810G-GC-1132-2017-01.pdf

Please review our technical article in the Publications section for our website for a very detailed discussion on the definitions, limitations, misunderstandings and myths of Process-Specific versus Multi-Use assays.

Most so-called Process-Specific Assays are not purification process specific but are rather ambiguously termed Process-Specific because they use a very upstream, null cell line preparation of antigen to generate the antibody. This arbitrary selection of an upstream source of null cell HCP such as conditioned media or lysate does not guarantee that the antibody or assay development from this antibody will detect the different and more limited array of HCPs that persist through a purification process. The vast majority of HCPs are conserved among all the strains of a given expression system. For example, of the hundreds to few thousand HCPs in the CHO proteome, there are only a very small number that significantly differ antigenically from strain to strain or growth process to growth process. There are four key factors in making a broadly reactive antibody that can have a much greater effect on the relevant activity of the antibody to downstream HCP than the minor antigenic differences that might be seen from strain to strain:
     1. Selection of an antigen that is truly representative of HCPs found at that step in the purification process.
     2. The processing of the antigen to make a potent immunogen.
     3. Methods for immunizing animals that insure broad reactivity and high antibody titer.
     4. Subsequent affinity purification of that antibody.

If one fails to understand how to make a good HCP antibody, then simply calling it Process Specific, based on the use of upstream, null cell antigen material does not insure it is a good antibody. The proof of the validity of an antibody is found in the validation of the assay for detection of HCP in downstream and final drug substance. All assays, whether they are arbitrarily termed Process-Specific, Platform, Generic, Commercial or Multi-Use must be validated the same way using real downstream and final drug substance samples while following conventional, objective parameters for validation of analytical methods. 

 One- and two-dimensional Western blot have traditionally been used as orthogonal methods in an attempt to characterize antibody reactivity to individual upstream HCPs.  Unfortunately, Western blot is too insensitive and non-specific to be of any practical value in demonstrating antibody coverage to individual downstream HCPs. While Western blot can detect the more abundant upstream HCPs it has very little predictive value for ELISA performance. The absence of a western blot band or spot to an individual HCP does not necessarily mean the ELISA will fail to detect that HCP. Cygnus developed and now offers a service much superior to WB in both sensitivity and specificity. This method is termed Antibody Affinity Extraction (AAE). AAE is >100 times more sensitive the 2D WB and more specific. As such, AAE can identify and determine antibody coverage to the most important HCPs, those that co-purify with drug substance. Along with qualification of the ELISA, AAE can better answer the question of reactivity and help objectively answer the question if a generic or presumed process specific assay is fit for purpose. See our AAE white paper on our web site. Contact our expert scientists for how to use state of the art orthogonal methods like AAE and Mass Spectrometry to determine if a generic kit is acceptable for lot release testing of if a process specific assay is needed.

For lack of better methods, Western blot has been accepted by regulators as a method orthogonal to ELISA. Some companies have used WB to drive early process development and to qualify anti-HCP antibodies for ELISA based on coverage analysis. Due to its lack of sensitivity and specificity, WB is of no predictive value in determining how a given antibody will quantitatively react to the more limited array and concentration of individual HCPs that co-purify with drug substance. For that reason, Cygnus no longer recommends use of 2D WB. We have instead developed a method termed Antibody Affinity Extraction (AAE). AAE is >100 times more sensitive and more specific than 2D WB and for that reason will give a much more accurate indication of coverage to individual HCPs. More importantly, AAE can be used to determine coverage to the most important HCPs, those that persist through the purification process. See our AAE white paper on our web site.

For those who still wish to perform WB considering its limitations and despite the availability of AAE, we offer the following information. Samples downstream in your purification process typically contain HCPs below the sensitivity of Western blot. For Western blot, you are limited in the amount of total protein you can load and still get good PAGE resolution. When you load final product or samples from downstream in the purification process, the vast majority of protein will be the product itself. For example, the maximal load of protein for a PAGE run on a mini gel is on the order of 10µg/lane. If HCP contamination is 100ppm, a level typical of many final drug products, then the amount of total HCP in that 10µg of drug would be 1ng. With the sensitivity of Western blot on the order of 1ng/band it could, in theory, detect HCP contamination down to 100ppm if the 100ppm were a single HCP and not a mixture of several different HCPs. For most purification processes, there are several HCPs that contaminate final product. Because each HCP is very low in concentration relative to the product, Western blot is almost always negative on downstream and final product samples. ELISA demonstrates less interference from drug product and shows sensitivity more than 100-fold lower than Western blot. As such, ELISA will typically allow for the detection of total HCP contamination to less than 1ppm. There are many other fundamental reasons why the sensitivity of Western blot is inferior to ELISA. For example, Western blot often requires that the PAGE step be carried out under reducing conditions (DTT or BME followed by boiling) and in the presence of high concentrations of SDS detergent. These procedural components may actually denature or block some of the native HCP epitopes that would be detectable in an ELISA. Incomplete transfer of the proteins out of the PAGE and onto the membrane and adsorption on the membrane at or near antigenic sites will also limit the amount of binding seen by Western blot.

As you try to increase the sensitivity of Western blot, it is very common that the specificity of the method is also compromised. What is typically seen is that a non-immunoreactive protein present in very high concentration (e.g. your drug substance) will invariably adsorb some of the excess anti-HCP antibody non-specifically leading to the erroneous conclusion that the anti-HCP antibody seems to "cross-react" with your product. The way to confirm this non-specific binding to your product is to use a non-immune immunoglobulin of the same species and at the same concentration as the anti-HCP antibody. If the intensity of the drug substance band is the same with both the normal goat IgG and the anti-HCP antibody, you can conclude the band is non-specific. Specificity can also be confirmed by loading much smaller quantities of your drug substance in the range of 1-4ng/lane. If there is true cross-reacting antibody that might manifest as false HCP levels, you should still see a strong product band. Beyond these experiments, it should be understood that the specificity of the ELISA method is typically orders of magnitude better than Western blot owing in large part to the fact that any protein must be bound simultaneously by both the capture antibody and the detection antibody. For this reason, most artifactual product bands in the Western blot will not yield apparent HCP activity in the ELISA method.

While Western blot is of little value for detecting HCP in all but very upstream samples, it has continually been used for demonstrating that the antibodies in the ELISA kit react with the majority of the HCPs from your cell line. This is typically a one-time experiment where you lyse some cells or use conditioned media from your cell line in a Western blot and compare the blot to a protein stain from PAGE such as colloidal gold or silver stain. If the homology is adequate one can conclude that the antibody has adequate reactivity in the ELISA kit, although it is only through validation of the ELISA that one can conclude that the antibody is adequate.