The Cygnus Technologies ELISA Kits for detection of bioprocess impurities are very sensitive methodologies capable of detecting analytes in the pg/mL to ng/mL range. Some of these analytes such as HCPs, BSA, HSA, Immunoglobulins and Transferrin may be found in the laboratory in very concentrated forms such as cell culture media, upstream product purification samples, human and animal sera, or other laboratory buffer reagents. These samples can contain on the order of mg/mL or several million-fold higher than the limit of detection of the impurity assay. These concentrated sources of the analytes can contaminate work surfaces and pipettes and then become airborne. Even parts per million contamination of the air or equipment could result in very significant contamination of the ELISA kit reagents and thus cause a false elevation of the apparent analyte levels by the assay. For example, airborne contamination of the microtiter strips used in our kits will typically manifest itself as poor duplicate precision with the inappropriate value being high. Similarly, contamination of one of the liquid reagents such as the antibody conjugate will result in high background absorbances, and thus reduced assay sensitivity even though precision may appear acceptable. To minimize these problems and insure optimum performance of the assay we recommend the following precautions:
• Do not perform the assay in areas where concentrated forms of cell culture media or sera have been utilized.
• Clean all work surfaces and equipment before performing the assay to reduce dust and other airborne particles.
• Dander or mucosal aerosols from the technician performing the assay can be a significant source of contamination in human cell line-based HCP assays or in assays for human proteins. Do not talk or breathe over an uncovered microtiter plate. Consider pipetting reagents in laminar flow barrier hood.
• Avoid use of pipettes previously used to dispense concentrated forms of the analyte.
• If possible, use disposable pipette tips with aerosol barrier filters like those used in molecular biology procedures such as PCR.
• Do not use automated plate washers that have been subjected to concentrated solutions of the analyte, i.e. many ELISA assays already in use in your laboratory will employ a wash buffer containing BSA or various animal sera to block and wash off non-specific binding. Even after extensive flushing of this equipment with water, significant contamination will remain.
• The PNPP substrate used in some kits can be easily contaminated with environmental sources of phosphatase enzymes such as airborne bacteria or human dander or mucosal aerosols. To minimize this source contamination, only withdraw as much substrate from the bottle as is needed for the particular assay run. Recap the substrate vial and return to the refrigerator. Do not return unused substrate back to the substrate bottle.
• After adding reagents to the wells, place the microtiter strips into a zip-lock plastic bag to protect from airborne contamination during incubation steps. We do not recommend the use of adhesive backed plate sealing tape for covering the wells as these can often introduce assay variability. Tightly recap all reagent bottles immediately after use.
Elevated background or non-specific binding (NSB) in an ELISA as evidenced by high absorbances in the zero standard can be due to a number of problems as discussed below. Keep in mind that different assays may have widely different expected absorbances for the zero standard, due to inherent design features of that assay. For example, HCP assays tend to have much higher background NSB than assays for single analyte. This is because HCP assays typically must use on the order of 20 to 50 times the amount of enzyme:antibody conjugate compared to single analyte assays such as our kits for Protein A or BSA. To determine the expected absorbance for the zero standard in your particular kit lot, please refer to the Certificate of Analysis that comes with each shipment. This COA will show the absorbance for the zero standard obtained by our QC laboratory.
1. Washing Procedure – Incomplete washing of the wells can result in carryover of unbound reagent and thus high and variable background. Review the kit package insert for proper washing technique and review “Washing of Microtiter Wells” to get a more detailed explanation of the recommended washing technique. Use only the diluted wash concentrate solution provided with the kit as other formulations, particularly those with detergent, may increase NSB. Do not wash plates more than 4 times or allow the wash solution to soak in the wells for any period of time as this will reduce specific binding for the assay. Or you can review the “Washing Technique for Microtiter Plate ELISA Video” to view the washing technique we recommend.
2. Contamination of kit reagents by concentrated sources of the analyte in your laboratory can also result in high NSB. Please refer to the article “Avoiding Contamination of Kit Reagents” for a more detailed discussion on how to prevent contamination. Our ELISAs are very sensitive methods, capable of measuring analytes in the pg/mL to ng/mL range. Many laboratories will have sources of the analyte in question at very high concentrations near to where they perform the ELISA. For example, culture media or samples from very upstream in the purification process may have HCPs or growth media additives like BSA in the mg/mL range. Such upstream samples can have more than a million-fold greater concentration than the sensitivity of the assay for that analyte. In such cases it is easy to contaminate some of the kit reagents such as random microtiter plate wells, a standards’ vial, or conjugate bottle.
3. Contamination of the substrate used in the kit can also result in high NSB. This is most often seen with alkaline phosphatase-based ELISA using PNPP substrate. It is rarely a problem in HRP based assays with TMB as the substrate. Review the package insert for techniques to minimize contamination of PNPP substrate. If your substrate has become contaminated, you should order replacement substrate.
Generally, we do not recommend the use of linear regression to fit ELISA data and particularly HCP assays. HCP ELISAs are rarely perfectly linear in dose response. Even though you may get an R squared value of .99, which looks to be good does not mean the assay is truly linear. To force an inherently non-linear method to fit the best straight line will lead to inaccuracies in reporting values on your samples. Those inaccuracies are most significant at the extremes of the standard curve, most often in the low end but sometimes in the high end as well. For this reason, we strongly urge our clients to use Point to Point, Cubic Spline or 4 Parameter as the curve fitting routines since these will yield the most accurate results.
It may be that curve fit routines other than the three we recommend will work satisfactorily for a given immunoassay method. If you decide to use another method for interpolation of sample values, it will be important for you to perform a careful analytical evaluation of your proposed algorithm. If after considering the points below, your method meets or exceeds the interpolation accuracy and precision of the other 3 methods then by all means use it. A word of caution, that just because your method works well in one immunoassay is no guarantee it will be optimal for another and therefore, each assay should be validated on a case by case basis. We recommend the 3 methods above because they are the most robust and the most accurate for immunoassay and have been shown to work for our assays. We specifically warn against the use of linear regression methods because most immunoassays are not linear nor is linearity a requirement for a good immunoassay. To force data from an assay to fit the best straight line when the inherent dose response is not a straight line is certainly a way to introduce inaccuracy into your results. Similarly, other more sophisticated regression methods can also introduce mathematical assumptions and enforce arbitrary and inappropriate rules that actually reduce the inherent accuracy and precision of the assay.
An easy way to determine the optimal curve fit routine is by “back fitting” the signals of your standards as unknowns. If the standards when “back fit’ as unknowns do not report back their nominal values, there may be artifacts introduced by inappropriate assumptions or restrictions in your curve fit algorithm. Finally, the most direct and objective way to assess the accuracy of an immunoassay is to assay controls with known levels of analyte across the important analytical range of the assay. Do not rely on arbitrary and indirect parameters such as R squared, slope, y-intercept, or asymptotes such as QC specifications. These parameters are often too insensitive to be useful in flagging a bad assay run.
Some samples, particularly those from upstream in your purification process will have impurity analyte concentrations above the analytical range of our very sensitive ELISA kits. Such samples may require very large dilutions in order to overcome “Hook Effect” and to achieve acceptable “sample dilution linearity”. Please refer to “Poor Dilution Linearity” for a more detailed discussion on sample dilution issues. In addition to the “Hook Effect,” the matrix of some samples may interfere non-specifically with the assay and result in under recovery of the true analyte levels. Simple dilution of those samples is often adequate to buffer out such interference provided the dilution does not reduce analyte concentrations below the limit of quantitation of the assay. In cases where dilution of your samples is not an option, contact our Technical Service Department for advice on how best to overcome sample matrix interference.
Cygnus offers assay specific diluents for each of its kits. The catalog numbers for these diluents can be found in the kit product insert or by contacting Customer Service. We strongly recommend use of those diluents because they are the same formulation as the matrix used for the kit standards. Thus, as you dilute your samples in our diluent, your sample matrix begins to approach that of the standards and in this way greatly minimizes any dilutional artifacts that could occur if you were to use another diluent. If you elect to use another diluent you must validate that it provides accurate results. We recommend two critical experiments for validation:
• First, you must assay the diluent alone to determine that it does not yield absorbance values significantly above or below the absorbance for the kit zero standard. OD values above the zero standard indicate that the diluent may have low levels of the analyte or that the diluent causes an increase non-specific binding, relative to the kit standards matrix. OD values less than the kit standard may indicate that the proposed diluent is lowering non-specific binding and may also be inhibiting specific binding. In any event use of a diluent that does not match the standards
risks significant errors when the sample concentration gets multiplied by the dilution factor.
• Second, you must perform a spike & recovery experiment into your proposed diluent at several levels across the analytical range of the assay. For a diluent to be deemed acceptable we suggest a recovery specification of 95% to 105%. In general, the formulation of diluents should be a neutral pH with some carrier protein added to block non-specific adsorptive losses of the analyte. Use of just PBS or TBS without a carrier protein can be problematic because the analyte diluted in the range of the assay (ng/mL) can very significantly adsorb to the dilution tube resulting low recovery! Your diluents should not have sodium azide as a preservative or significant detergent concentrations as these will reduce assay accuracy.
Low ODs or absorbance values can be due to a number of problems as discussed below. To determine the expected absorbances for your particular kit lot please refer to the Certificate of Analysis included with each shipment. The COA will show the absorbances obtained by our QC laboratory for the zero standard and the highest kit standard. Some variation in absorbance values is expected from lab-to-lab and technician-to technician. Provided you are obtaining acceptable precision and sensitivity, ODs lower than what is given on our C of A are acceptable.
1. Washing technique or equipment – Use of automated plate washers or hand-held vacuum aspiration devices could significantly dissociate specific bound analyte. Washing more than 4 times or soaking the wells in wash solution for any period of time can likewise lower expected ODs. Use of wash solutions other than the one provided with the kit may reduce your ODs. Review the kit package insert for proper washing technique. Visit our web site www.cygnustechnologies.com to view a video of how we perform plate washing. (Troubleshooting Video: Washing Technique for Microtiter Plate - ELISA) Do not be overly aggressive in banging the plates to remove all residual liquid. It is not necessary to remove all liquid. The 4 washes will sufficiently dilute unbound material such that less than 3µL of residual liquid at each wash step will not significantly add to background ODs after the 4th wash.
2. Shaking of the plate during immunological incubations – Placing the plate on a microtiter plate shaker can significantly accelerate the rate of binding. As various shaker instruments can have somewhat variable speeds and range of motion, the magnitude of shaking effect can vary. We have determined that the optimal rate of shaking is in the range of 400 to 600 rpms for most assays and most plate shakers. If ODs are lower than expected you may consider increasing the speed of shaking. Be advised that speeds greater than 600 rpm risk spilling liquid from well-to-well or causing it to make contact with the material used to cover/seal the plate resulting in very poor precision. If you do not shake the plate but instead perform the immunological incubations without agitation it may be necessary to increase the incubation time by up to 2-fold to achieve the same absorbances that are seen with shaking.
3. Laboratory or reagent temperature can have a significant effect on assay ODs. The QC of our kits takes place at 25°C. If your laboratory is colder than this, it may be necessary to extend incubation times in order to achieve higher ODs. Alternatively, you may use an incubator set at 25°C. Make certain that the kit reagents are up to room temperature prior to pipetting.
4. For HRP assays with TMB substrate, the plate should be read within 30 minutes after addition of stop solution since color will fade over time.
5. Check plate reader for proper performance. The instrument filters for both the test and reference wavelengths can deteriorate over time.
6. Carefully review the kit directions insert for proper technique. When possible have another technician in another laboratory and with different equipment perform the assay. This is often the best way to identify the cause of low ODs.
7. If you would like higher ODs than those shown in the C of A it is often possible to simply extend the incubation time of one or all of the various assay steps. Contact our Technical Service Department ([email protected]) for advice on how best to modify the assay protocol.
A common and unfortunate artifact of western blot is that some proteins particularly those at very high loads will non-specifically bind to a western blot antibody. Attempts to increase the sensitivity of western blot for HCP contaminants in the presence of a very large excess of product often involve use of higher concentrations of HCP antibody and loading of more sample. Increasing the sensitivity by these approaches will also tend to compromise specificity. Almost all non-immunoreactive proteins if present in very high concentration (e.g. your drug substance) will 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. There are a number of approaches to confirm the specificity of your western blot as discussed below.
1. You can often demonstrate that binding to your product protein at its chosen load is non-specific by performing a negative control blot at the same time you blot with the HCP antibody. The control blot uses a non-immune or normal immunoglobulin conjugate at the same concentration and from the same animal species as the anti-HCP antibody. If the intensity of the drug substance band is the same with both the normal IgG and the HCP antibody, you can conclude the band is nonspecific.
2. Loading of much lower quantities of your product protein will also help to confirm specificity. The specific sensitivity of western blot is on the order of 1ng/band. If you fail to detect a product band at product loads in this range it is reasonable to suspect that bands seen at much greater loads are nonspecific in nature.
3. Specificity can also be confirmed by ELISA analysis. Our ELISA kits use the same antibody as the respective western blot kit. The specificity of ELISA is in orders of magnitude, better than western blot, owing in largest part to the fact that any protein must be bound simultaneously by both the capture antibody and the enzyme labeled antibody. For this reason, artifactual product bands in the western blot will not yield apparent HCP activity in the ELISA method. The ELISA experiment is performed by diluting your product protein to concentrations within the analytical range of the ELISA (typically from 1 to 200ng/mL). Assuming that your product protein is not heavily contaminated with HCP (has less than parts per hundred HCP), you should not see any apparent HCP activity unless your product cross-reacts with both the capture and enzyme-labeled antibody. Some products, in particular therapeutic antibodies, can non-specifically bridge the anti-HCP antibodies in the kit, but in most cases we have been able to design our kits to overcome this problem. Contact our Technical Service Department if you are having difficulty demonstrating specificity of the HCP antibodies.
Demonstration of dilution linearity (also termed dilution parallelism or dilution recovery) of samples containing the analyte of interest is a critical experiment to validate the specificity and accuracy of a given method. Assays for multiple analytes such as our HCP ELISA will often show a lack of dilution linearity for certain samples. In the case of HCP assays this lack of dilution linearity is usually due to insufficient excess of antibody for some of the HCPs found in your sample. Other causes for poor dilution linearity are due the product protein itself or certain components in product formulation buffer that may interfere (either positive or negative interference) in the ability of the assay to detect HCPs or other contaminants. Similarly, samples from various points 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 validate 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 there is significant product or matrix interference it may be necessary to further process the sample by methods such as 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. In other cases, modification of the assay protocol can improve accuracy in some sample types. Users of our kits are encouraged to contact Technical Services Department for advice on how best to solve sample accuracy issues.
Sample types with levels of contaminants greater than the LOQ of the assay should, as part of assay validation, initially be evaluated for dilution linearity. This experiment involves performing a number of serial dilutions using an approved assay diluent. These dilutions are then assayed, and a dilution corrected HCP concentration is determined at each dilution. This dilution linearity study is particularly important for a HCP assay because it establishes the important condition of antibody excess for the array of HCPs in your samples. If you will be routinely testing in-process samples in addition to final product, each sample type should have a dilution linearity validation. This analysis is necessary because very high concentrations of certain 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, you will observe 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 a sample did not yield good dilution linearity at a high concentration, but with further dilution an MRD was determined at which acceptable dilutional 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/mL. Once an MRD is established for a particular sample type, your SOP should reflect that this sample needs to be diluted before assay. Your samples from a given point in the purification process could vary somewhat from lot to lot. For this reason, we suggest assaying samples at 3 doubling dilutions; one at the MRD, one above, and one below, until your process reproducibility and control can established. 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 Technologies that your sample diluent is acceptable. In general, the best diluent is the same one used to prepare the kit standards. Assay specific diluents can be purchased from Cygnus in 100ml, 500ml or 1000mL bottles. Contact Cygnus for information on acceptable diluents.
Table 1. Example of Dilution Linearity Data for an In-Process Sample
Please consider the following points when experiencing poor assay precision or reproducibility. Poor assay precision is almost always due to procedural or equipment issues within your laboratory. The kit reagents are well validated and tested for stability and the coating of antibody on the plates is very uniform. Given that the other reagents are homogeneous liquids, poor CVs are normally a result of one of the following problems:
1. Washing technique and equipment – Many automated plate washers as well as hand held vacuum aspiration devices can significantly affect assay imprecision. Overly aggressive washing is a common cause of poor precision. Many technicians fail to appreciate that in their efforts to remove unbound reactants they can dissociate antibody bound reactants, and that this dissociation can be highly variable. Automated vacuum aspiration devices or overly hard banging of the plate when using a manual method will cause worse CVs and lower than expected antibody activity. For these reasons we recommend the use of our manual washing procedure for optimal reproducibility. Our web site offers a video of the proper manual wash technique. You may also review “Washing of Microtiter Wells” article to get a more detailed explanation of the recommended washing technique.
2. Plate Reader – Malfunctioning plate readers are more common than appreciated. Poor precision, particularly in the low absorbance region at less than 0.1 OD units is rarely detected by routine calibration, maintenance or by instrument self-diagnostics. The best description of this problem is instrument “noise” due most often to a failing light source, bad monochromator or bad filters. It usually manifests as variability in the form of standard deviations of 0.020 OD units or less, from well-to-well or read-to-read in empty wells or wells containing non-adsorbing liquid. This variability may be intermittent with the instrument stable at times and then noisy at others. Obviously, when such variability is seen in the low absorbance end of an assay this can have a very significant impact in assay sensitivity (LOQ or LOD) as these assay performance parameters are direct functions of low end assay precision. A good way to determine if you have instrument noise is to look at absorbance data from your assay using dual wavelength analysis. For example, our HRP-TMB based ELISAs recommend reading at 2 wavelengths; the test wavelength of 450nm where the yellow substrate color absorbs maximally and then at an “offpeak” reference wavelength of 650nm where there is no absorbance due to the yellow substrate. In dual wavelength analysis, the OD reading at 650nm is automatically subtracted from the reading at 450nm. In theory this should provide a measure of improved precision and accuracy. If there are any wellto-well factors that result in a non-wavelength specific absorbance such as smudges or plastic imperfections, then subtraction of that absorbance from that well should improve the results. In practice non-wavelength absorbance is very minimal in our ELISAs. The optical quality of our microtiter strips is such that the absorbance at 650nm is on the order of 0.032 ODs in our well monitored instruments with a standard deviation across 96 wells of only about 0.001 OD units. The mean 650nm OD can vary slightly from instrument to instrument and manufacturer to manufacture but the standard deviation across 96 wells should not be more than 0.002 ODs. If you have a run where the low-end precision is questionable it is a simple matter to look at the raw OD data at 650nm across your plate. If you see a mean value much greater than 0.032 with a standard deviation greater than 0.004 ODs then this may indicate problems with your plate reader that may have a significant impact on the low end precision and sensitivity for the assay.
3. Contamination of kit reagents by concentrated sources of the analyte are a source of poor reproducibility. Our ELISA kits are very sensitive and capable of measuring analytes in the pg/mL to ng/mL range. Many laboratories may have sources of high concentrations of the analyte in close proximity to the area where ELISA is performed. For example, culture media or samples from very upstream in the purification process may have HCPs or growth media additives like BSA in the mg/mL range. Such upstream samples will have on the order of a million-fold greater concentration than the sensitivity of the assay for that analyte. In such cases, it is easy to contaminate some of the kit reagents such as random microtiter plate wells, a standards vial, or the conjugate bottle. Click on “Avoidance of contamination of kit reagents” to get advice on how to prevent contamination.
4. Operator inexperience, poor technique, and laboratory equipment such as pipets, or poor-quality pipet tips can have a significant contribution to poor precision. A good method to isolate and identify the source of the imprecision is to have another technician perform the assay in another lab using different equipment.
Spike and recovery of known amounts of analyte into your various sample types is a critical experiment to validate the accuracy of a given method. In some cases, the product protein itself or certain components in the product formulation buffer may interfere in the ability of the assay to detect HCPs or other contaminants. Factors such as extremes in pH, detergents, organic solvents, high protein concentration, and high buffer salt concentrations are known to interfere. This interference is normally negative in nature and manifests itself as under recovery of the spiked analyte. It is necessary to validate 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 there is significant product or matrix interference, it may be necessary to further process the sample by methods such as 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. In other cases, modification of the assay protocol can affect improved accuracy in some sample types. For each sample type to be tested, be it final product or in-process samples, you should demonstrate that the assay can recover added analyte spiked into that sample matrix. Proper dilution protocol can be found in “How to Perform Spike and Recovery Studies” article in the FAQ section. You are encouraged to contact our Technical Services Department for advice on how best to solve poor spike and recovery problems in our kits.
It is not uncommon to encounter sample matrix interference when testing for bioprocess contaminants in your various sample types. Such interference can manifest itself as either falsely depressed or falsely elevated levels of analyte. While the product protein itself can be responsible for this interference, other components of the sample matrix such as buffer salts, pH, or other additives like detergent or solvents are often problematic. Experiments to test for sample matrix interference can be found in the sections on Spike & Recovery Studies and Dilutional Linearity. There are several ways to solve sample matrix interference some of which are described below. Should you still encounter problems after trying the following options, we suggest you contact our Technical Service Department for assistance.
1. One of the easiest ways to overcome sample matrix interference is by diluting the sample into a more assay compatible buffer. The ideal diluent is the same material used to prepare the kit standards. As the sample is diluted its matrix begins to approach that of the kit standards and as such tends to improve the specificity and accuracy of the assay. Cygnus sells assay specific diluents in volumes of 100mL, 500mL and 1L bottles. Sample dilution is only an option if your samples either: a) have levels of analyte that remain within the analytical range of the assay after dilution or b) if the allowable limits of the analyte contaminating your product do not fall below the LOQ of the assay after the necessary dilution. Provided a Minimum Required Dilution (MRD) can be experimentally established for your sample types, dilution is the preferred method for solving sample matrix problems.
2. When dilution is not an option because it does not solve the problem or because of assay sensitivity issues, consider sample processing to remove the interfering components. A very effective technique is to perform a buffer exchange of your samples into a more assay compatible matrix. A very easy way to do this is to use small pre-calibrated Sephadex G25 columns sold by GE Healthcare called NAP 10 Columns. The procedure is to first equilibrate the column in assay diluent (the same material used to prepare the kit standards). Apply 1mL of your sample to the column and let it flow into the column; at which point the flow will automatically stop due to surface tension. Next apply 1.5mL of diluent buffer and collect the 1.5mL that elutes from the column. This 1.5mL will contain greater than 98% of the starting proteins in your sample including product and contaminants like HCPs but without greater than 98% of the low molecular weight components such as buffer salts etc. Your sample has only been diluted by a factor of 1.5 but it is now in the ideal assay matrix and thus should allow for good analytical accuracy.
3. Consider simple neutralization of your samples to overcome pH problems. In general, ELISA may not work well if the sample pH is outside of the neutral range, <6.0 or >8.5. Addition of a buffering concentrate can neutralize your sample to the ideal pH of 7.0-7.5 without much dilution of the sample.
4. Modification of the ELISA protocol can also minimize sample matrix interference. Less sample size, longer incubation times, or use of a simultaneous incubation protocol in which the sample is incubated in the coated capture well simultaneously with the enzyme conjugated antibody can also be effective. Please refer to Modification of ELISA Protocol for more information.
When customers obtain unexpected or failed results it is common to speculate that it is either a manufacturing or stability problem. We have many years of experience in manufacture and quality control of these kits and we have invested much effort to enhance stability of ELISA reagents. The claims we make for shelf life in the form of expiration dates and the recommended customer storage conditions at 2-8°C are very conservative given that we have real time and elevated temperature data supporting even longer shelf life. Furthermore, the formulations of our reagents allow for prolonged storage at elevated temperatures and thus assure that problems such as summertime temperatures or delays in shipping or refrigeration problems at the customer location will not cause significant deterioration in the kits. For these reasons, when you are experiencing questionable results we suggest that you examine other potential causes first and not consume your limited kit reagents in trying to prove that instability is causing the problem. Assisting a customer with troubleshooting their kit at our facility to substantiate their problem, almost invariably gives the same excellent performance as when the kit was first released by our QC department. Thus, the problem is typically at the customers’ facility. We have found that a good way to pin point the source of laboratory problems is to have another technician experienced in ELISA from your facility or an associate laboratory run the assay using different equipment. This will identify the problem in most cases.
When customers obtain unexpected or failed results it is common speculation that there is either a manufacturing or stability problem. We have many years of experience in the manufacture and quality control of these kits and we have invested much effort to enhance stability of ELISA reagents. The claims we make for shelf life in the form of expiration dates and the recommended customer storage conditions at 2-8°C are very conservative given that we typically have real time and elevated temperature data supporting even longer shelf life. Furthermore, the formulations of our reagents allow for prolonged storage at elevated temperatures and thus assure that problems such as summertime temperatures or delays in shipping or refrigeration problems at the customer location will not cause significant deterioration in the kits.
For these reasons, when you are experiencing problems with your results we suggest that you examine other potential causes and not consume your limited kit reagents in trying to prove that instability is the source of the problem.
We have found that an effective way to pin point the source of laboratory problems is to have another scientist experienced in ELISA from your facility or an associate laboratory run the assay using different equipment. In most cases this will identify the problem. In other instances, please contact our Technical Support Team ([email protected]) for assistance. Prior to contacting us, please be sure to have the following information available:
• Catalog and lot number of the kit.
• OD readings of the standard curve.
• Type of curve fit routine used to calculate standard curve.
• Type of diluents used.
• Control results.
• Assay protocol used, either standard or high sensitivity.
This information will help us troubleshoot your data in a logical manner by defining the exact nature of the problem.
Our experience has shown that the most frequent cause of customer problems in the performance of ELISA is due to plate washing equipment and technique. While the goal of washing is to remove unbound reactants, many technicians fail to understand that the washing step can also dissociate antibody bound reactants and can do so in a highly variable manner. Our experience has shown that automated plate washers and related multi-channel hand-held vacuum aspiration devices give inferior assay performance relative to the manual procedure described below. Some plate washers may leave behind very small but still significant liquid causing variable and higher assay background than can be achieved manually. In an effort to reduce carryover, many washers can be too aggressive in the physics of washing and aspirating and will actually variably dissociate antibody bound analyte resulting in both lower absorbance and higher %CVs. For these reasons we strongly recommend that users use the manual procedure detailed below for optimal assay performance.
Equipment Required: All that is required for equipment is a wash/squirt bottle and low lint absorbent paper. We suggest that the narrow portion of the tip of the squirt bottle be cut off to give the largest possible orifice so that the flow will be generous and gentle. This procedure is best done over a large sink since it can be a little messy.
Step 1. Dumping the liquid from the plate:
Grab the plate from the bottom with the thumb in the middle of one side and the fingers on the other side. If the thumb and fingers slightly overlap the tabs on the ends of the middle strip or strips you will usually be able to avoid having any strips fall out of the strip holder. Holding the plate over the sink, turn the plate upside down just as you rapidly accelerate your arm and hand downward. Abruptly stop your arm causing the liquid to be forced from the strips into the sink. When done properly you should not get any liquid on your fingers or on the outside of the strip wells or plate holder. Repeat the dumping motion a second time.
Step 2. Blotting and tapping the plate:
Immediately blot plate upside down plate onto the blotting paper. Move the plate to an unused section of the blotting paper. Firmly tap the plate 3 times over unused areas of the paper. Do not bang the plate too hard in a misguided effort to remove all the residual liquid, as this may cause variable dissociation due to transferring of unequal amounts of shock energy across the plate.
Step 3. Washing:
Use the squirt bottle to fill all wells staring at the front of the plate and working to the back. Fill to overflowing with the 20-fold diluted wash solution provided with the kit.
o Do not use any other wash buffer formulation as it may negatively impact the performance of the kit.
o Do not worry about overflowing the wells as you will be wiping off the bottom of the wells before adding substrate.
o Do not allow the wash solution to soak in the wells.
Immediately dump & tap the plate as described in Steps 1 & 2 as soon as the last well is filled.
Repeat the washing procedure 3 more times for a total of 4 washes. With the 2nd & 4th washes start by adding the wash solution from the back to the front of the plate. This insures that the total dwell time of wash solution in the wells will be essentially the same for all wells. Additional wash steps should not be necessary and in fact may dissociate specific bound analyte and actually reduce assay sensitivity.
After the last wash, blot and tap as described in Steps 1 & 2. Let the plate rest upside down for about 20 seconds to drain. Firmly tap the plate again 4 times rotating the plate 180° in your hand between each tap. This rotation ensures that the ends of the plate receive on average the same energy and impact from the banging.
Step 4. Wiping the plate:
Wipe the bottom outside of all wells with clean absorbent paper to remove any wash liquid from the overfilling and tapping. Wells are now ready to have the substrate added to them. Add substrate immediately after washing.
o Do not let the wells dry out or enzymatic activity will be lost.
o Do not add substrate near the sink location where your dumping and banging have taken place since the washing procedure can generate aerosols that could recontaminate the wells or your substrate.
Watch the troubleshooting video: Washing Technique for Microtiter Plate - ELISA to get the exact technique.