ELISA Application Notes

Science Hub

What is ELISA?

An ELISA kit contains all the necessary reagents for performing the Enzyme-Linked Immunosorbent Assay (ELISA). This assay is a plate-based assay used to detect and quantify various molecules, including proteins, antibodies, hormones, peptides, and occasionally small molecules like vitamins and coenzymes. ELISA method utilize antibody specificity together with enzyme activity to facilitate the measurement of the target molecules. ELISA kits come in various formats, each with slightly different workflow and interaction between antigens, antibodies and enzymes.

The ELISA kit offers a highly sensitive, specific, and versatile method for detecting and quantifying proteins in biological samples. Its ease of use, flexibility, and compatibility with various detection methods make it indispensable in a wide range of research and diagnostic applications.

ELISA workflow steps


The first crucial step involves immobilizing the antigen (sample) onto the surface of a microtiter plate. This can be achieved either by directly adsorbing the antigen onto the plate or indirectly using a capture antibody that has been attached to the plate.


Once the antigen is immobilized, it is detected either directly or indirectly using enzyme-labeled antibodies. In the direct method, an enzyme-labeled primary antibody binds directly to the immobilized antigen. In the indirect method, two antibodies are used; primary antibody that binds to the target molecule and an enzyme-labeled secondary antibody, which recognizes the primary antibody. The detection antibodies are typically conjugated with enzymes such as alkaline phosphatase (AP) or horseradish peroxidase (HRP). These enzymes catalyze reactions that produce detectable signals, usually a color change.

Substrate addition

A wide range of substrates are available for performing the ELISA, depending on the enzyme that is used. The choice of substrate depends on the required assay sensitivity and the type of instrumentation available for signal detection, such as spectrophotometer, fluorometer, or luminometer.

Detection and quantification

Detection is achieved by measuring the enzyme activity, which results in a colorimetric or luminescent signal. For example, incubation with TMB substrate leads to a color change that can be quantified by measuring the absorbance of light at 450 nm. Alternatively, chemiluminescent substrates can be used, which generate a luminescent reaction that can be quantified using appropriate instrumentation.

Types of ELISA

Direct ELISA - an enzyme-labeled primary antibody binds directly to the immobilized antigen

Indirect ELISA - an enzyme-labeled secondary antibody, which recognizes the primary antibody, is used

Sandwich ELISA - antibody pairs are used in series, where each antibody detects a specific part of the antigen

Competitive ELISA - assay plate is coated with antibodies that react to a molecule of interest

How to choose the right ELISA kit

1. Pay close attention to the instructions before buying the kit to make sure it fits your needs.

2. Consider these:

  • What protein biomarker are you measuring? Make sure to use the correct name when searching.
  • Check if the kit is developed for the species you are studying, like human, rat, or mouse.
  • Ensure the kit is compatible with your sample type (serum, plasma, etc.).
  • Check the sample volume required per well.
  • Look into the sensitivity of the kit and whether it fits the concentration range of your biomarker.

3. Remember, always double-check the details before making your selection to avoid any issues later on.

4. When choosing an ELISA kit, it is crucial to assess how well it performs. Look for kits that have undergone validation. Here is what to consider:

  • Accuracy - Can it reliably detect the protein biomarker in biological samples?
  • Dilution linearity and parallelism - Does it accurately recover the analyte when samples are diluted?
  • Specificity and cross-reactivity - Does it only detect the intended analyte without interference from others?
  • Precision - Can it consistently produce precise and reproducible results within and across different runs?
  • Calibration - Does it maintain consistent performance across the range of the assay?
  • Sample Stability - Can the analyte remain stable under various conditions, like multiple freeze-thaw cycles or at room temperature?

5. Ensuring these aspects are well-covered helps guarantee reliable results from your ELISA assay.


Our ELISA Suppliers


Cell Signaling Technology

Cell Signaling Technology offers three types of ELISA kits:

FastScan™ ELISAs are a solution-based sandwich assays with a single-wash protocol saving you time. The kits have a sensitivity that is comparable or better than traditional ELISA, and use colorimetric detection of target quantity.

PathScan™ ELISAs are solid-phase sandwich assays and have a sensitivity that is comparable to traditional ELISA. Target quantity is detected by colorimetric reaction, and chemiluminescent  detection kits are also available. PathScan™ RP ELISA kits have a Rapid Protocol that saves you valuable time. Also available are PathScan™ Sandwich ELISA Antibody pairs that are an economical alternative to complete kits.

All CST ELISAs are compatible with several sample types, such as serum, plasma, and other cell extracts, as well as tissue.

Kits are available for post-translational modifications, endogenous cellular proteins, and biomarkers across neurodegenerative disease, cancer, and other diseases. Kits are available for the study of endogenous cellular proteins, post-translational modifications, as well as biomarkers found in cancer, neurogenerative disease, and other diseases.

Acro Biosystems

ClinMax™ ELISA kits

ClinMax™ ELISA kits are fully validated and ready-to-use kits for measuring proteins and cytokines accurately and quickly. They follow strict quality standards outlined by organizations like ICH M10 and C-Path. These kits are manufactured in facilities that meet GMP-like standards and strictly adhere to ISO13485:2016 and ISO9001:2015 quality management systems.

Inhibitor Screening Kits

In drug development, inhibitor screening is vital, especially in early stages, involving screening numerous candidate molecules to identify those with notable pharmacological effects. Rapid and sensitive tools are crucial for efficient screening. ACROBiosystems kits utilize the competitive ELISA method. Kits are high-sensitivity screening kits with unique biotinylated proteins, ideal for preliminary screening and quality control in drug development. Kits provide reliable, efficient, and accurate tools for inhibitor screening. Kits are 4-step ELISAs, have high batch-to-batch consistency ensuring stable and reliable experimental results. The amount of interfering substances, like DMSO, are optimized for small  and macro-molecule inhibitors.

Antibody titer kits

Pharmacokinetics (PK) is the study of how drugs move through the body of a  living organism. This involves studying how the drug is absorbed, distributed, metabolized, and excreted. In both animal and human studies, researchers measure the concentration of the drug in the bloodstream at various times after it is given. This helps them understand how the drug behaves in the body and guides dosing recommendations.

With the rise of biologic drugs, like monoclonal antibodies, there is a need for efficient ways to measure these drugs in serum samples. Traditionally, this was done using indirect ELISA or anti-drug antibodies (ADA), each with its own strengths and weaknesses. ACROBiosystems has developed a new method based on competitive ELISA. In this method, a labeled monoclonal antibody competes with the drug in the sample, and the interaction between biotin and streptavidin is used to measure the results. This method avoids some of the issues seen with traditional assays, like background interference.

ACROBiosystems provides kits specifically for studying CTLA-4, PD-1, and HER-2 in both humans and common experimental animals. These kits make it easier for researchers to study the pharmacokinetics of these important drug targets.

Nuclease detection assays

As biologic treatments gain momentum in medical care, they are revolutionizing how we approach previously untreatable conditions like cancer. Therapies involving antibodies, cell treatments, and gene therapies are opening new doors for the possibility to cure perviously untreatable conditions. However, because these treatments come from biological sources, ensuring their safety is crucial. There is always a risk of contamination or the introduction of harmful substances during production.

ACROBiosystems is committed to advancing the safety and quality of biologic therapies. That is why they offer residue detection kits that align with industry guidelines from organizations like the WHO and FDA. Kits are specifically designed to detect various impurities in therapeutic antibodies, cell therapies, vaccines, and other biologics. By adhering to these stringent standards, the goal is to enhance the safety and effectiveness of biologic treatments for patients worldwide.

Featured products include:

  • resDNA detection kits
  • DNAse/RNase activity detection kits
  • nuclease detection kits

Assay Genie

AssayGenie has several different types of ELISA kits, such as sandwich, competitive, cell based, phosphorylation specific, chemiluminescence immunoassay (CLIA), transcription factor assays, trapping ELISA, biosimilar ELISA, indirect, quickstep ELISA, qualitative ELISA, capture, and enzyme catalysis. Kits are available for a range of species.

Popular products include:

  • Enzymes Recombinant Proteins
  • Bovine Signalling ELISA Kits
  • Growth Factors and Cytokines Recombinant Proteins
  • Cytokines Recombinant Proteins
  • Human Cell Biology ELISA Kits
  • Developmental Biology
  • Porcine ELISA Kits
  • Mouse Neuroscience ELISA Kits
  • Human Immunology ELISA Kits


Europe based Biomedica specializes in developing and manufacturing high-quality ELISA kits for clinical research, focusing on cardiorenal diseases, bone, and mineral disorders. They serve well-known scientific institutions, CROs, and pharmaceutical laboratories globally. Backed by an experienced team, they offer technical and scientific support, adhering to ISO 9001:2015 and GMP/GLP guidelines in manufacturing.Their analytical testing service covers proprietary ELISAs and assays from other brands, including Luminex testing.

Examples of BioMedica’s popular ELISA kits:

  • Anti-oxidized LDL autoantibodies
  • NT-proBNP (1-76).
  • Big Endothelin-1 (Big ET-1 (1-38)).
  • Osteroprotegerin
  • Solube Semaphorin 4D
  • Periostin (all known isoforms)
  • Recombinant and endogenous mouse Periostin
  • Sclerostin
  • Intact FGF23
  • Borrelia IgG (B. burgdorferi ss, B. afzelii, B. garnii, B. bavariensis)
  • Borrelia IgM (B. burgdoreri ss, B. afzelii, B. garnii, B. bavariensis)
  • Human Angiopoietin-2
  • Mouse/Rat Angiopoietin-2
  • Human IL-6
  • LRG (leucine-rich alpha-2-glycoprotein)

Boster Bio

Boster Biological Technology, with over 25 years of experience, specializes in manufacturing high-quality ELISA kits and antibodies. As a reputable provider, they offer a diverse range of over 1000 ELISA test kits, in both sandwich and competitive formats, catering to various mammalian species including human, mouse, rat, and more.

Boster Bio´s enzyme-linked immunosorbent assays undergo rigorous validation to guarantee optimal specificity, sensitivity, and stability. CiteAb.com, a prominent research company, has recognized Boster Bio as the fastest-growing antibody company globally based on publication citations. As of 2019, Boster Bio ELISA kits have been cited in over 6,000 publications worldwide.

BosterBio’s popular ELISA kits include Insulin, Corticol, IL-6, IL-1beta, IL-10, IL-8, CRP, TNF alpha, VEGF, BDNF, IFN Gamma, and Adiponectin.

Need more information? Talk to Katja-Riikka!

Katja-Riikka Louhi

Product Specialist

+358 20 7410 276


Katja-Riikka Louhi