The enzyme-linked immunosorbent assay ELISA is frequently used for measurement of low-abundance biomarkers. This urges the need for more rigorous control of assay Validating elisa assays definition, regardless Validating elisa assays definition its use in a research setting, in clinical routine, or drug development. The aim of a method validation is to present objective evidence that Validating elisa assays definition method fulfills the requirements for its intended use.
Although much has been published on which parameters to investigate in a method validation, less is available on a detailed level on how to perform the corresponding experiments. To remedy this, standard
Validating Validating elisa assays definition assays definition procedures SOPs with step-by-step instructions for a number of different validation parameters is included in the present work together with a validation report template, which allow for a well-ordered presentation of the results.
Even though the SOPs were developed with the intended use for immunochemical methods and to be used for multicenter evaluations, most of them are generic and can be used for other technologies as well. Biochemical markers biomarkers play a central role in Validating elisa assays definition decision-making in clinical medicine.
Examples include making a clinical diagnosis, initiating and monitoring treatment, predicting prognosis or disease recurrence after treatment. Except for an increasing use in clinical routine, these biomarkers are also used in clinical trials, both as diagnostic and as theragnostic markers 2. Last, these CSF
Validating elisa assays definition are applied in clinical studies on disease pathogenesis, and many research reports present novel biomarker candidates.
The vast majority of Validating elisa assays definition fluid biomarkers are low-abundance proteins, for which antibody-based immunoassays, often in enzyme-linked immunosorbent assay ELISA format, is needed to get enough analytical sensitivity. However, to get reliable and reproducible results, rigorous control of assay performance is essential, which also should be presented in a standardized format.
Validation of a method is the confirmation by examination and the provision of objective evidence that the particular requirements for a specific intended use are fulfilled 3. It is important as it defines it will produce reliable results in the context of its intended use. This last item is sometimes overlooked; the intended use a method needs to be carefully specified before any time consuming and costly validation experiments are performed.
This notion is generic to any method. However, this paper will now focus on the validation of methods used to determine analyte concentrations in biofluids. The intended use for such a method could be to use the outcome as a diagnostic marker and in this case some evidence should be in place showing that there is a disease-dependent change in the analyte concentration in a biological sample.
Furthermore, the magnitude of the change should have an
Validating Validating elisa assays definition assays definition on the acceptable variability of the method, Validating elisa assays definition. Much has been published on the topic of method validation
Validating elisa assays definition a consensus protocol on how to perform the task is yet to be found. This could be partly due to the fact that different analytical technologies have different requirements on which validation parameters that need to be addressed or Validating elisa assays definition local initiatives by national societies in the clinical chemistry field were not discussed and spread at international level 4.
For example, carryover should be investigated in a chromatography-based method while it is Validating elisa assays definition applicable in an ELISA. The aim of the present work was to present straightforward step-by-step standard operating procedures SOPs for the validation of methods in which an analyte is determined in a biofluid matrix; the SOPs have been developed with the intention that they should possible to follow without any advanced prior training.
The work flow in the present project consisted of writing draft SOPs for each parameter relevant to validation of a method for determination of an analyte concentration in a biofluid. Task members were then asked to review and revise the SOPs, whereafter they were evaluated in at least three multicenter studies. End-users commented on the draft SOPs, and, after an additional round of reviews, final, consensus SOPs were produced which form the core of the current report.
All members of the task were invited to critically revise the manuscript. Standard operating procedures for 10 different validation parameters are presented. If a method is developed in-house, a full
Validating elisa assays definition should be performed, meaning that all parameters should be
Validating elisa assays definition. As a consensus agreement in the group, it was decided that a partial validation of a commercial assay should include all parameters except for robustness, which should have been covered by the Validating elisa assays definition during method development.
Even more limited partial validations may be eligible under other circumstances. For example, if a validated in vitro diagnostic IVD method is transferred to another laboratory to be run on a different instrument by a different technician it might be sufficient Validating elisa assays definition revalidate the precision and the limits of quantification these variables are most sensitive to the changes, while more intrinsic properties for a method, e.
If a laboratory is, or plan to be, accredited to some international standard there is usually a high demand on documentation.
The template has been adapted from a Swedish handbook on method validation 5with the permission of the authors. To aid in the extraction of Validating elisa assays definition from measurement data the Data Sheet S2 in Supplementary Material can be used. Validating elisa assays definition description of the validation parameters for which SOPs are presented.
Robustness or ruggedness is the ability of a method to remain unaffected by small variations in method parameters. If the instructions from the manufacturer of a commercially available assay does not contain any information indicative of a robustness assessment the manufacturer should be contacted and asked to provide this information since it is likely that such data is available given that the method development was sound.
In case of an in-house method, the robustness should be investigated as a part of the method development and the results should be reflected in the assay protocol before other validation parameters are investigated. The reason for this is that a validation is linked to an assay protocol and changes in the latter might demand a new validation to be performed.
There are three different types of precisions depending on the Validating elisa assays definition conditions and these are rintermediate precision Rwand reproducibility R.
Repeatability is the variability observed when as many factors as possible, e. For intermediate precision, all factors except laboratory are allowed to vary and for clarity the Validating elisa assays definition Validating elisa assays definition should be stated Validating elisa assays definition the validation report.
Repeatability is sometimes called within-run or within-day precision while intermediate precision is also Validating elisa assays definition as between-run or between day repeatability. Precision is difficult to quantify and it is therefore the inversely related imprecision that is commonly reported.
Five samples with different levels have been suggested Validating elisa assays definition a general rule to cover a wide measuring 7. However, it can be argued that if the levels are chosen with care, for example, one above and one below the decision limit, two samples might be enough. In addition, it is not always possible to obtain samples covering a wide range, e. large volumes of the samples are available, more aliquots than the ones needed for the precision measurements can be prepared for use as internal quality control samples when the method has been put in service.
Other experimental schemes than the
Validating elisa assays definition suggested under points 2—3 in the procedure are possible, e. The latter scheme will allow for more different factors be explored, which will give a better estimate of the variability.
At the same Validating elisa assays definition, it is very impractical and expensive if the method is, e.
The Validating elisa assays definition in which the trueness is measured is called bias bwhich is the systematic difference between the test result and the accepted reference value. Once the bias is determined, it can be used to compensate the measured concentration resulting in a method without systematic effects 8.
If the bias is constant over the measurement interval the bias is simply subtracted from the measured value and if the bias is proportional to the measured concentration the correction is done by multiplication of a factor determined from bias evaluations at different concentrations. Alternatively, Validating elisa assays definition calibrators can be assigned new values to compensate for the bias.
The total bias is the sum of two components originating from the method and the laboratory, respectively. When a CRM is available, manufacturers are obliged to calibrate their method against materials traceable to the CRM and then Validating elisa assays definition total bias should in principle be equal to the laboratory bias.
The intermediate precision provides information about the dispersion characteristics the results within a laboratory with no Validating elisa assays definition to the true value of Validating elisa assays definition measurand in a sample. Therefore, in the absence of a CRM, the measurements rather deliver relative concentrations as opposed to absolute ones
Validating elisa assays definition can be achieved if the calibrators were traceable to a CRM.
However, if different methods can be used for quantifying the same analyte and if a universal cutoff value is warranted there is a need for a CRM that can be used by the kit manufacturers to calibrate their methods against,
Validating elisa assays definition order to
Validating elisa assays definition the bias. This will also Validating elisa assays definition calculating absolute Validating elisa assays definition but the uncertainty in the results must then include not only the uncertainty from the method but also the uncertainty of the assigned value for the CRM.
At least for the LLOQ, there is more than one definition and these can be classified as either determined based on signals from the instrument or the calculated concentrations from samples.
the former, a number of blank samples are analyzed and the average and SD of the signal are calculated To determine the concentration based on a signal the inverse of the calibration function must be used.
The two common models used in immunochemical calibrations are the four and five parametric logistic models. The four parametric function and its inverse Validating elisa assays definition. The A — E should be available from the software used
Validating elisa assays definition data acquisition and analysis. Dilution linearity is performed to demonstrate that a sample with a spiked concentration above the ULOQ can be diluted to a concentration within the working range and still give a reliable result.
In other words, it determines to which extent the dose—response of the analyte is linear in a particular Validating elisa assays definition within the range of the standard curve. Thereby dilution of samples should not affect the accuracy and precision. At the same time, the presence of a hook effect, Validating elisa assays definition. Dilution linearity should not be confused with linearity of quantitative measurement procedures as defined by CLSI 15which concerns the linearity of the calibration curve.
Conceptually parallelism Validating elisa assays definition Validating elisa assays definition linearity are similar. The major difference is that in the dilution linearity experiments the samples are spiked with the analyte to such a high concentration that after dilution the effect of the sample matrix is likely to Validating elisa assays definition negligible.
Validating elisa assays definition parallelism, on the other hand, no spiking is allowed but only samples with high endogenous Validating elisa assays definition of the analyte must be used. However, the concentrations must be lower than the ULOQ. The goal of investigating the parallelism is to ascertain that the binding characteristic of the endogenous analyte to the antibodies is the same as for the
Validating elisa assays definition. None of
Validating elisa assays definition suggestions, however, relate the acceptance criteria to the precision of the method under investigation.
Validating elisa assays definition
The recovery of an analyte in an assay is the detector response obtained from an amount of the analyte added to and extracted from the biological matrix, compared to the detector response obtained for the true concentration of the analyte in solvent 9. A spike recovery test is conducted to investigate if the concentration—response relationship is similar in the calibration curve and the samples. A bad outcome of the test suggests that there are differences between the sample matrix and calibrator diluent that affects the response in signal.
Data obtained from this study could help to Validating elisa assays definition a diluent mimicking the biological sample in which the calibrator and the native protein give the comparable detector signals all along the measuring range. Selectivity is something that can be graded while specificity is an absolute characteristic. Specificity can be considered as the ultimate selectivity.
For this reasons, selectivity should be preferred and is the recommended terminology. Of the different validation parameters the selectivity is in principle the only one for which a certain amount of knowledge about the analyte and related substances is demanded. For example, if the analyte is a peptide of a specific length do slightly longer or shorter peptides also give rise to a signal in the assay?
Do metabolites of the analyte or post translational modifications of a protein analyte interfere with the assay? Sample handling Validating elisa assays definition to analysis has the to dramatically influence the results of a measurement.
For this reason, it is important to investigate if different storage conditions to systematic errors in Validating elisa assays definition to provide the clinicians with adequate sample collection and transport instructions. The information gathered also be useful once the sample reaches the laboratory, i. Examples of factors that potentially affect the results of an analysis, but are not included in the following procedure includes, sample tube, type of plasma anticoagulant, gradient effects concerns CSF samplescentrifugation conditions, extended mixing, and diurnal variations.
If data are not available on how these factors influence the measurement the sample instructions should be written in a way to prevent variations potentially induced by these.
The above conditions
Validating elisa assays definition should only serve as an example and the can be modified to better suit the environment and different routine handling of samples at the individual laboratories. The experiments in a validation are usually performed within a month time and therefore the results represent a kind of snapshot of the performance characteristics of the method.
To Validating elisa assays definition that the quality does not degrade over time an internal quality control program should be initiated before the assay is taken into service. The results from quality control samples should be used to determine if a run is accepted and the objective multi rules presented by Westgard should be used In the present study, we present SOPs for validation of assays for biochemical markers together
Validating elisa assays definition a template for validation reports.