Optimization, Validation and Standardization of ELISA

Posted by Jack on November 24, 2022
Table of Contents


    ELISA is a widely used technique in science and medicine. ELISA testing has been used to measure the concentration of specific proteins, lipids, hormones, vitamins and other substances in human serum or plasma samples. The term "immunoassay" was coined by M.C. Potter in 1969 as a generic name for any scientific technique that uses an antibody to identify or quantify antigens.[1]

    Definition of ELISA

    ELISA is a technique to detect the presence of an antigen in a sample. It is a highly sensitive and specific assay method. ELISA stands for Enzyme Linked Immunosorbent Assay.

    ELISA can be defined as "a type of sandwich immunoassay." The ELISA assay uses antibodies that are labeled with enzyme or other reporter molecules (for example, fluorescent dyes) to detect the presence of antigens in test samples.

    Need of Optimization, Validation and Standardization of ELISA

    ELISA is a highly sensitive, qualitative, specific and rapid test. It is simple and inexpensive to perform. ELISA involves extraction of the antigens from the sample followed by their reaction with antibodies that are labeled with different enzyme systems. The results obtained from this method can be visualized without any additional equipment unlike other methods like immunohistochemistry where one needs to have access to sophisticated equipment like microscopes for visualization of results.

    Types of ELISA

    ELISA (Enzyme-Linked Immunosorbent Assay) is a test used to detect the presence of a substance in a sample. ELISA can be qualitative or quantitative. If the result is qualitative, it means that the test shows whether there is an antigen present in your sample, but not how much of it there is (this type of test can be used for things other than antibodies). For example, if you have been exposed to a particular disease, an ELISA will tell you whether or not you have been exposed; however, it cannot say how much exposure has occurred. If the results are quantitative and you’ve tested positive for exposure to something like HIV or hepatitis C virus (HCV), then this means that your body has developed antibodies against these pathogens and they were detected by this particular test kit/kit combo.

    Need of optimization, validation and standardization of ELISA

    Here, we present an example of optimizing the sandwich ELISA. The assay was optimized by varying the concentrations of coated plate and biotinylated capture antibody in order to get an optimal signal intensity. The absorbance values were recorded at 450 nm on a spectrophotometer after each step in the method and plotted against the concentration of coating antigen per ml of phosphate buffered saline (PBS).

    The regression curve drawn through these data is represented by a straight line having coefficient of correlation r2=0.984. This means that 98% variation in signal intensity could be predicted based on 1% variation in coating antigen concentration. Thus optimization was carried out successfully by using these two variables as shown below:

    Optimization, Validation and Standardization in quantitative immunoassays

    • Optimization: In the optimization of an immunoassay, one must determine the optimal amount of antibody to be used in the assay. This involves conducting several trials with different amounts of antibodies and comparing their results with those from a control sample. The optimal amount can then be determined according to which trial gave the best result.
    • Validation: Once you have determined your optimal level of antibody, it is important to make sure that this level is specific for what you are trying to detect. To validate your assay, you will need to run another set of assays using similar methods but different sets of samples; one sample containing your target antigen and another without it (your control). If both sets show similar values when compared with each other then they are said to be valid.[10]
    • Standardization: Standardizing an immunoassay refers to developing consistent procedures so that every time a particular test is performed by different labs using different equipment or settings there would still yield similar results.[11]

    General requirements for validation study design for analytical methods used in clinical studies for drugs and biologics

    The validation study should be conducted according to an approved protocol, which contains the following elements:

    • Protocol title and number. The title of the study and its number must be given in accordance with good clinical practice (GCP) regulations.
    • Purpose of the study. This section describes why you are doing this work and how it fits into your organization's overall objectives. It should also state what you hope to learn from this project and how those outcomes will help improve your product or process development efforts in the future. For example, "The purpose of our studies is to validate a newly developed ELISA method for measuring levels of Factor VIII."
    • Background information about relevant scientific literature that supports using immunoassay technology for measuring levels of Factor VIII in human plasma samples.

    Pre-validation study runs

    In a pre-validation study run, the analytical method is assessed by measuring samples spiked with known concentrations of analyte. This step provides an indication of how well your assay will perform in real-world situations, before you validate it and start using it in production.

    Of course, since you already know what the expected concentrations are going to be—and since you’ll be using those same conditions for your validation study—you should also do a pre-validation study run on the instrument that will be used for this purpose as well (e.g., ELISA plate reader).

    Analytical method validation requirements for studies conducted after FDA approval of a drug or biologic product (post-approval)

    Analytical method validation requirements for studies conducted after FDA approval of a drug or biologic product (post-approval)

    The analytical method used in each study must be validated for the specific purpose of the study. The validation study must be designed to meet the requirements of 21 CFR Part 58 and be conducted in accordance with the protocol.

    The protocol should include, but not be limited to:

    • Definition of test system(s), including species, strain and sex; where appropriate, sampling plans for raw materials and reagents; statistical treatment of data; sample preparation; assay procedure including immunoassay formats (e.g., ELISA) methods used (e.g., direct versus indirect); etc.;
    • Testing matrix for each assay with identification of acceptance criteria for each assay;
    • Testing protocol including validation model description(s), equivalence considerations if applicable, data analysis plan including sensitivity analyses based on acceptance criteria as well as other relevant information such as final testing results presented graphically with acceptance criteria plotted against performance characteristics such as limit-of-detection/limit-of-quantitation values at various concentration intervals along with any other relevant information requested by FDA reviewers during review process;

    Validation study design recommendations based on the post-approval study objectives

    • Validation study design recommendations based on the post-approval study objectives
    • Post-approval study objectives

    The purpose of this section is to provide validation study design recommendations based on the post-approval study objectives. The post-approval studies are divided into four groups, which are: confirmatory studies with clinical relevance, nonclinical and analytical studies for subcutaneous injections, pharmacokinetic studies, and pharmacodynamic studies.

    ELISA is not just a mere test but also needs some technical aspects.

    ELISA is not just a mere test but also needs some technical aspects. The whole process of ELISA can be divided into three parts: optimization, validation, and standardization. These three activities are very essential to get accurate results.[edit]Optimization

    Optimization means finding out the optimal conditions (e.g., antibody concentration and incubation time) that give highest sensitivity and specificity. This step is important because it helps in reducing variability from assay to assay and thus enables you to obtain reproducible results.[edit]Validation

    Validation means determining whether these optimized conditions are suitable for all types of samples or different samples require different optimal conditions for an accurate result.[edit]Standardization

    Standardization refers to standardizing your method so that every time you use it for quantifying an analyte, you get same amount of information about its concentration in sample with each assay performed on another day or at another laboratory[edit]


    The ELISA technique is one of the most important methods for detecting an antigen in a sample. ELISA is used for detection of antigens in food, environmental air and water samples. The aim of this study was to develop a simple method for the determination of cellulose acetate content using ELISA with different types (receptor) antibodies against cellulose acetate or its derivatives. This research was performed using dual-wavelength spectrophotometry with three different calibration curves. A sensitive colorimetric assay based on the formation of triphenylmethane dye at 459 nm has been developed as a qualitative probe for measuring cellulose acetate in solution at low concentration levels. For quantitative determination, we used two kinds of antibodies that were raised against two synthetic polymers: one had affinity towards cellulose acetate and another one towards polyethyleneimine-polyacrylamide copolymers (PEI-PA). In addition, five calibrators were prepared by dissolving known amounts of pure standards into blank solutions containing 200 ppm acetic acid (acetate buffer). The calibration curve was linear up to 500 ppm with correlation coefficients greater than 0.99984 for both PEI-PA and cellulose acetate types antibodies respectively

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