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Why is ELISA lot-to-lot Reproducibility Critical for your Research?

Brian Conrad
Tags: Successful Research Tips

What describes a reliable result produced from experimental measurement? An experimentally derived value is described as a reliable result when it is precise and accurate. Accuracy is the closeness of measured values to the true value. Precision is the closeness of measured values to one another. In experiments, precision of a measurement method is based on the degree to which repeated measurements under unchanged conditions are able to show the same results. Repeatability and reproducibility of measured values describe precision. Repeatability is the degree of agreement between measurements taken under certain conditions by the same observer in the same laboratory. On the other hand, reproducibility is defined as the degree of agreement between measurements using the same method previously described but independently by a different observer. Repeatability and reproducibility are usually reported as the standard deviation (σ) or as the coefficient of variation (CV or %CV) which are both measures of variability that are evaluated to define precision.

How is reproducibility expressed in ELISAs?

The importance of CVs – within a plate (intra), plate to plate (inter)

In enzyme-linked immunosorbent assay (ELISA), reproducibility is significant to reported results because it fundamentally describes the precision of ELISA’s reagents/components working together to facilitate the antibodies binding to the target of interest and producing signal accurately to quantify its presence within a heterogeneous sample matrix from a population. Reproducibility of reported results from immunoassays are evaluated through calculation of the coefficient of variation (CV); CV is used to describe the relative variation that exists within a population independently of the absolute values of the observations. CV is calculated by dividing the standard deviation (σ) of a set of measurements by the mean of the set (µ) which can then be transformed into a percentage. The overall precision of an immunoassay is defined by examining reproducibility of reported results through examining variation between wells within a single run of a plate (intra-assay precision) or between runs/trials or plate-to-plate (inter-assay precision) by evaluating the %CVs, respectively. Typically, intra-assay CV will be evaluated by comparing replicates of a sample with a measurable concentration in the middle of a range of values (advertised by a kit manufacturer or in the literature) with N=10 minimum, and should not exceed 10-15%. On the other hand, inter-assay CV is evaluated by comparing triplicate measurements of each sample per run of independent runs on 3 different days. Samples should span the range of the standard curve (2 in the low, 1 in the middle, 2 in the high), and should not exceed 15-20% for each point. Following these general guidelines helps to establish an assay with high reproducibility which suggests the values have high precision. Reliable assay results are derived from having good precision and accuracy. Additional suggestions on how to reduce your % CVs and improve your reproducibility and assay precision can be found in our Immunoassay E-Book and FAQs section.

How do we evaluate reproducibility from batch-to-batch as the manufacturer?

Lots need to be tested to ensure low background detection (specificity), consistent assay sensitivity and a linear standard curve with a broad dynamic range. Commercially available ELISA kits that are produced by manufacturers on a lot-by-lot (batch-by-batch) basis need to ensure test results produced at different times through independent end-users are comparable. This is accomplished by lot-to-lot comparisons of ELISA kits to be done to verify whether data from new kit batches will correlate with data from old kit batches by evaluating the extent of correlation between the results produced (if a correlation exists). Correlations are made by plotting data from old and new kit lots in a linear curve fit/regression and interpreting the resultant R-squared value. The extent the data from the new lot relates to the old lot in the correlation can provide insights about the extent of variation between the two lots which helps describe the reproducibility.

Lot-to-lot kit comparison of new and old batches of ELISA kits is conducted before old kit lots expire by evaluating positive samples (target analyte present in a matrix being tested within the detectable range) using old and new kits on the same-day. Same day lot-to-lot comparison is considered to be highly accurate because performing runs of different lots on the same day avoids the variation that originates from differences in stability of analyte in the matrix the presence of the analyte is being tested in. From a manufacturer’s standpoint, the standards of old and new lots are compared to a master calibrated standard that is built during development and calibrated against purified material to be used as an internal control to qualify all future lots of standard. Consistency in the control values of master standard made during kit development over time ensures reproducibility from lot. From the standpoint of customers, same-day lot-to-lot kit comparison is done to avoid variations that originate in positive samples (with target analyte known to be present) that have differing rates of sample degradation in response to prolonged periods of storage. General guidelines for lot-to-lot kit comparison require use of at least 37-40 positive samples with a wide range of positive samples with values in the low and high points of the standard curve range. The reported results from old kit and new kit are plotted in a linear curve-fit with r-squared values is between 0.85-1.00 are considered acceptable. The slope of the fitting line should be between 0.85 – 1.15. An ideal correction factor is 1.00.

At Enzo, our newly manufactured lots of kits are Quality Control (QC) tested for inter-assay precision, intra-assay precision and assessed for lot-to-lot reproducibility with testing that includes internal controls that were developed during kit development. The kit’s performance and the controls need to pass our internal QC specifications before a lot is released. Reproducibility and consistency between standard curve and QC control performance is evaluated with each lot of each kit. All standards, controls and background wells are run and we examine expected EC20, 50, and 80 values for each curve in addition to expected return values for the controls and expected ODs for various standards.

Highly reproducible immunoassay kits from Enzo’s Bioprocess catalog

Protein A ELISA kit (ADI-900-057) - Immunometric

One example of Enzo’s highly reproducible ELISA kits is our Protein A ELISA Kit. This is a commercially available, colorimetric-based immunometric ELISA kit used for quantification of Protein A concentration in various sample matrices. Intra and inter-assay precision was determined by assaying 16 replicates of three buffer controls containing Protein A in a single assay (Figure 1) and multiple assays over several days (Figure 2). Both intra and inter-assay CVs recorded demonstrate ranges of values that fall beneath the general guidelines for the % CV intra-assay (10% or lower; ours is on between 5.2-6.4%), and inter-assay (15% or lower; ours is between 8-13.4%) showing high reproducibility and precision.
Intra-assay precision

Figure 1: Intra- assay precision of Protein A ELISA Kit (ADI-900-057).
Inter-assay precision

Figure 2: Inter-assay precision of Protein A ELISA Kit (ADI-900-057).

Protein A is produced as a cell wall component in bacterial species Staphylococcus aureus which has a high binding affinity for the Fc region of IgGs. Industry has taken advantage of the strong binding affinity to IgG as a means to purify monoclonal antibody (mAb) IgGs in harvest cell cultures supernatants and crude cell lysates of host cell lines used to produce mAb protein of interest. Protein A affinity chromatography is primarily the first step of antibody capture/purification in a monoclonal antibody preparation and yields highly pure mAb products up to 95% purity. However, trace amounts of Protein A in samples being harvested for recombinant mAb sometimes isn’t immobilized by the resin. Therefore, ELISAs become useable as a tool to trace residual Protein A contamination for bioprocesses used to produce recombinant mAbs. Enzo’s Protein A ELISA kit is the most sensitive commercial ELISA kit for Protein A on the market with a limit of detection as low as 9.0 pg/ml with a wide detectable range that spans from 15.63 pg/ml to 1000 pg/ml. Sensitivity was calculated as the ratio of the Mean OD plus 2 standard deviations of 16 replicates of the 0pg/ml standard to the mean of 16 replicates of the lowest standard, multiplied by the concentration of that standard (15.62 pg/ml). This kit is able to recognize both native and recombinant forms of Protein A and has been validated for specificity amongst various Protein A constructs tested in the presence of human IgG.

Figure 3: Assay specificity of Protein A ELISA kit (ADI-900-057) for native and recombinant forms of Protein A determined through evaluating % recovery of four Protein A constructs in the presence of human IgG. Percent recovery was found by dividing the observed recovery in presence of human IgG by the observed recovery from assay buffer. Graphical Key- A= Natural Protein from S. Aureus, B= Recombinant Protein A from E.coli, C = Recombinant Cys-Protein A from E.Coli, D= Recombinant alkaline-resistant Protein A variant from E.coli. Graphical information A-B: n=9, C-D: n=12, graphical data represents statistical mean +/- 1 SD).

PEGylated Protein ELISA kit (ADI-900-213) – Competitive

Specificity of ELISA kit

Figure 4: Specificity of PEGylated Protein ELISA kit (ADI-900-213).
Enzo’s PEGylated protein ELISA kit is a competitive ELISA specific to the backbone of polyethylene glycol (PEG) and validated for use with linear and branched PEGs found in free form and bound to a protein (Figure 4). For this ELISA kit, intra-assay precision was determined by assaying 16 replicates of two buffer controls containing PEG-BSA in a single assay. Figure 5 shows that in development, this ELISA reported a range of low intra-assay %CVs between 3.4 – 4.2%. On the other hand, inter-assay precision was determined by back-calculating three standards off 16 standard curves produced in mulitple assays over several days. Figure 6 shows that in product development, this ELISA reported a range of low inter-assay %CVs between 1.3 – 5.7%. The PEGylated ELISA kit is a highly precision kit with a high standard of reproducibility.
Intra-assay precision for PEGylated ELISA kit

Figure 5: Intra-assay precision for PEGylated Protein ELISA kit (ADI-900-213).
Inter-assay precision for PEGylated ELISA kit

Figure 6: Inter-assay precision for PEGylated Protein ELISA kit (ADI-900-213).
Sensitivity of ELISA kit

Figure 7: Sensitivity of PEGylated Protein ELISA kit (ADI-900-213).

PEG is the most used polymer and is utilized as a gold standard for polymer-based drug delivery systems that conjugate PEG to drug candidates. PEGylation of potential chemical leads that are drug candidates have been found to be desirable because it increases the retention time, reduces the immunogenicity and increases the stability of the drug to be metabolized by enzymes. Possible side effects and complications have been found from the accumulation of metabolized PEGylated therapeutics in various tissues so examination of the influence of PEGylated products has been a focus to characterize hypersensitivity, unexpected changes in pharmacokinetic behavior, toxicity and antagonism from degradation by mechanical stress from them. Assaying for concentrations of PEGylated substituents can be a helpful tool for drug development applications such as pharmacokinetics analysis, drug comparison, lead candidate identification, lot release criteria and in-process QC studies. In addition to the high reproducibility afforded by Enzo’s PEGylated ELISA kit, it also offers a highly sensitive immunoassay with a limit of detection less than 1 ng/ml with a dynamic range from 1.75 ng/ml to 225ng/ml. Sensitivity of the assay was dependent on the molecular weight of PEG tested and quantity of PEG molcueles conjugated to target molecule and evaluated prepared conjugates using 10 kDa linear PEG offered at 1,3, 5, 10 and 15-fold molar excess to BSA (Figure 7).

Summary Statement

Enzo Life Sciences offers a comprehensive bioprocess portfolio with thoroughly validated ELISAs for contamination monitoring of Protein A and Host Cell Protein (HCP) residuals for process optimization in your biologics preparations. We also have our widely cited PROTEOSTAT® assays for monitoring protein aggregation and protein stability under systematic thermal stress conditions to enhance your manufacturing workflows. Please check out our Bioprocess Optimization platform for more information or contact our Technical Support Team for further assistance.

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