ELISA Development: Developing Antibody Pair to Optimizing LLOQ

ELISA or Enzyme-linked Immunoassay is defined as a powerful technique both for the detection and quantification of the specific proteins. This method typically requires an antigen of interest to be immobilized or captured onto a solid surface. This is subsequently complexed with an enzyme-linked antibody. The detection of this complex is accomplished by conjugating enzyme activity. 

Although the market today contains various types of ELISA, each of them is accomplished with the same essential assay elements: 

Coating or capture – 

This is a type of direct or indirect antigenic immobilization technique where you immobilize your antigen to the solid surface of polystyrene microplate well. 

Plate blocking – 

In this stage of ELISA, you have an addition of irrelevant proteins or other related molecules to the unsaturated surface binding sites of your polystyrene microplate wells. 

Probing or detection – 

This type of ELISA Method technique involves incubation of antigen and antigen-specific antibodies 

Signal measurement – 

The last and the final stage of your ELISA development is the detection and analysis of the signal generated through direct or secondary tags attached to your specific antibody. 

ELISA assay development – 

ELISA assay development involves various steps for achieving success. While beginning with your ELISA assay development, the first and foremost step is to determine an immobilization strategy and optimization of the pre-coating conditions. 

Immobilization is a process wherein your antigen of interest is captured on to a solid surface. This results in reducing or loss of mobility. The process through which and how your proteins immobilize helps in affixing the properties of ELISA. In some cases, immobilization results in loss of protein activity, either wholly or partially. Both structural deformation and random orientation influence the loss of protein activity. For complete retaining the biological activity of your proteins of interest, protein attachment to the solid surface should be accomplished without disturbing both its conformation and function. The physicochemical and chemical properties of both the immobilized proteins and the surface facilitate the selection of suitable immobilization activity. 

Antigen and antibody – 

Nature and type of both antigen and antibody are influencing factors for the development of your ELISA assays. The purity and stability of your antigen are the key parameters affecting your ELISA assay’s performance. High antigen purity directly influences the capability of capturing antibody, thereby increasing assay sensitivity. 

Conjugate strategy – 

Enzyme labeled antibody functions as the key to your ELISA signal output. Conjugation of the enzyme with the antibody involves forming a stable covalent linkage between your antigen-specific monoclonal antibody and your enzyme. In this conjugate formation, you do not notice any alteration in the antigen combining site of your antibody or your enzyme’s active site. 

Enzyme and Chromogen – 

The last and the final stage of your ELISA assay is dictation. In this stage, the intensity of your final signal produced stands directly proportional to the concentration of antigen present on your plate and the bounding of your detection reagents. 

Besides revolutionizing the field of immunology, ELISA finds its way into commercial applications such as the detection of allergens and detection markers across the food and diagnostic industries.

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