Krishgen ELISA are always fully validated using a stringent, thorough protocol that guarantees quality and performance. They are designed and developed to meet high standards set by the best manufacturers around the world. 

In this blog, learn more about our validation process.

Optimization

Optimization of an ELISA is essential to its success. Since ELISA is a multistep procedure, each component can be individually tested prior to the start of an experiment.

ELISA procedure consists of antigen or antibody coating, saturation, analyte application, detection with appropriate antibodies, primary or secondary and signal detection. Between each step the plate is washed. A variety of samples can be tested with ELISA, and the choice of assay conditions will depend upon the complexity of the sample and the expected amount of analyte present. Optimization is the establishment of ideal concentrations of each assay reagent and ideal conditions for each step and that must be done empirically.

The cornerstone of any ELISA is the selection of the protocol type: direct, indirect or sandwich, which is dependent on the type of sample, available reagents, and the concentration of the analyte, keeping in mind that the procedure should be as straight forward as possible. Numerous factors are tested, such as the concentration of antigen, or antibody used for coating, temperature, the duration of individual steps the type of coating buffer, such as phosphate-buffered saline (PBS) or carbonate buffer, sample preparation methods (with or without EDTA, decomplementation, serum or plasma or whole samples). Plate saturation is also a step which requires optimization such as different concentration of bovine serum albumin (BSA), non-fat-dried milk, or whole serum from different animals.

Standardization

The second step is standardization. Today, leading regulatory agencies for specific guidance on immunogenicity assessment of biotherapeutic products are part of EMA and WHO, and there are other agencies. The National Institute for Biological Standards and Control (NIBSC), for example, part of UK Medicines and Healthcare products Regulatory Agency (MHRA), is of great importance to the field of biological standardization. It produces over 90% of the biological international standards in use around the world. The WHO Biological Reference Materials are established through a standard procedure, in which representative materials are tested by participating laboratories using their own methodologies and coordinated by a responsible WHO Collaborating Center. Upon establishment of the reference preparation by the Expert Committee on Biological Standardization (ECBS), the material is assigned a unitage and serves as the comparator against which results from laboratories can be standardized and compared, irrespective of the location or the methods employed. This enables the results of bioanalytical methods, including ELISA, to be comparable. Based on international standards „ working standard” (i.e. in-house or secondary standards) are evaluated and compared, and subsequently adequately used.

Validation

Validated analytical methods such as ELISA for quantification of biomarkers, drugs, biological products, and their metabolites in a given biological matrix (e.g. blood, plasma, serum, or urine) are critical for the successful conduct of nonclinical and clinical studies. Validating the analytical method ensures that the data are reliable. Validated methods provide critical data to support the safety and effectiveness of drugs and biological products.

Although there is abundant literature relating to immunochemical methods, EMEA and US FDA have clearly defined the characteristics of the validation procedure for bioanalytical methods. These also applies to the validation of ELISAs, which are intended for use in diagnostics, toxicology, basic or applied research or production control. Methodology for the validation of bioanalytical methods must follow clear recommendations from reference institutions such as the EMEA or the WHO because that provides important measurements to be of satisfactory quality all over the world. The latest such guideline, released July 2022, is the ICH M10 guideline for bioassay validation.

In accordance with it, Krishgen follows a seven parameter GOLD standard quality testing that each batch of ELISA kits is subjected to before release, amongst others:

1. Inter-Assay Precision

2. Intra-Assay Precision

3. Recovery

4. Sensitivity

5. Specificity

6. Linearity

7. Stability

Inter-Assay / Intra Assay Precision

Reproducibility and precision in different lots of ELISA are the key to good results in any experiment. Intra-assay validation shows the reproducibility between wells within an assay plate. Inter-assay precision shows the reproducibility between different assays done on different days. Both play an equally important role in the robustness of the ELISA.

Results are defined as the percent coefficient of variation (%CV) i.e. standard deviation divided by the mean and multiplied by 100. Krishgen focuses on ensuring that precision for any assay is less than 10% CV.

Typically, assay precision is determined by both intra (n=5 assays) and inter assay (n=5 assays) reproducibility on two or three sample pools with low standard, medium standard and high standard.

Recovery / Matrix Effect

The spiking recovery study is a method to evaluate the accuracy of the assay when different kinds of sample matrices are used. Serum and plasma, the typical matrix for ELISA was may have interfering factors or components that could affect the ability of the assay to accurately quantify an analyte.

Known amount of analyte is “spiked” into sample diluent (1X), diluted normal human serum at different concentrations (for example, 1:10, 1:100, 1:500, 1:1000, 1:2000 and 1:5000) and diluted normal human plasma at different concentrations (for example, 1:10, 1:100, 1:500, 1:1000, 1:2000 and 1:5000) and run in the ELISA. The resulting concentration, or “recovery” of the spiked material, demonstrates if the expected value can be measured accurately.

The results are expressed as a percentage of analyte recovered. Our acceptance range for the recovery is 80–120%.

Sensitivity

Limit of detection is defined as the lowest detectable concentration corresponding to a signal of Mean of the zero standard plus 2* SD. 10 replicates of the ‘0’ (zero) standards are typically evaluated, and the sensitivity is accordingly calculated.

Specificity

Both antibodies used in the kit are monoclonal antibodies, anti-idiotypic and specific for the target analyte. The calibrators / standards used are calibrated against commercially sourced drugs / injections. This allows for highly specific testing.

Krishgen also offers custom evaluation of the kit at no additional charge for customers looking to study cross-reactivity to specific molecules that the user feels may cause interference.

Dilutional Linearity:

All dilutions performed should always get the same final analyte concentration for a sample. This is known as assay or dilutional linearity. This data provides insights on the precision of the results and robustness of the ELISA for the sample at various levels of dilution across the quantitative range of the assay.

The results are observed as % deviation from known / expected concentration. To ensure that matrix effect is not observed, both serum and plasma spiked samples are run for dilutional linearity studies.

Typical data from the validation of Krishgen’s Atezolizumab ELISA kit (KBI1017).

Standards provided in the kit were used for measuring the dilutional linearity range of Atezolizumab present in serum and plasma matrix. 

Normal human serum (n=3) was spiked with Atezolizumab (Tecentriq™) and diluted with the Sample Diluent provided with the kit in the ratio of 1:100, 1:1000 and 1:2000. The recoveries obtained were observed.

Normal human plasma (n=3) was spiked with Atezolizumab (Tecentriq™) and diluted with the Sample Diluent provided with the kit in the ratio of 1:100, 1:1000 and 1:2000. The recoveries obtained were observed.

Human Serum-Spiked Data

Dilutional Linearity:

All Krishgen ELISA lots are subject to a three-variable accelerated stability study. The three main components – pre-coated plate, conjugated detection antibody and standard / calibrator are kept at 37^C for 14 days in the below format and known samples are run with for each component every alternate day. A 20% +/- in the results is accepted.

Have questions about our validation process? Contact us at info@krishgen.com.