The Enzyme-Linked Immunosorbent Assay (ELISA) is a widely utilized analytical biochemistry assay, predominantly employed for the detection and quantification of proteins, peptides, antibodies, and hormones. The development of a custom-made ELISA kit allows for the precise measurement of specific biomarkers tailored to research and diagnostic needs. This study outlines the technical process involved in the design, development, and validation of a custom ELISA kit for the quantification of a target biomarker.
Custom ELISA kits are essential in research and clinical diagnostics due to their high specificity, sensitivity, and versatility. This study describes the step-by-step process involved in creating a custom ELISA kit, focusing on the selection of antibodies, optimization of assay conditions, and validation of the assay performance.
Materials and Methods
Antigen and Antibody Selection: The target biomarker was identified, and specific monoclonal antibodies were selected. The antigen was synthesized and purified using affinity chromatography.
Coating of Microplates: High-binding ELISA plates were coated with the capture antibody at a concentration of 2 µg/mL in a carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. Unbound antibodies were removed by washing with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBST).
Blocking: Non-specific binding sites were blocked with 1% bovine serum albumin (BSA) in PBST for 1 hour at room temperature.
Sample and Standard Preparation: Samples and standards were prepared in duplicate, with serial dilutions of the target biomarker ranging from 0.1 to 100 ng/mL.
Assay Procedure:
- Addition of Samples/Standards: 100 µL of samples or standards were added to the wells and incubated for 2 hours at room temperature.
- Washing: Plates were washed three times with PBST.
- Detection Antibody: 100 µL of biotinylated detection antibody (1 µg/mL) was added and incubated for 1 hour at room temperature.
- Washing: Plates were washed three times with PBST.
- Streptavidin-HRP Conjugate: 100 µL of streptavidin-HRP (diluted 1:1000) was added and incubated for 30 minutes at room temperature.
- Washing: Plates were washed three times with PBST.
- Substrate Reaction: 100 µL of TMB substrate was added and incubated in the dark for 15 minutes.
- Stopping Reaction: The reaction was stopped by adding 50 µL of 2N sulfuric acid.
- Measurement: Absorbance was measured at 450 nm using a microplate reader.
Validation: The assay was validated for specificity, sensitivity, precision, and accuracy. The lower limit of detection (LOD) and lower limit of quantification (LOQ) were determined. Inter- and intra-assay variations were assessed by running multiple replicates of samples across different days and within the same day.
The custom ELISA kit demonstrated high specificity with negligible cross-reactivity with other proteins. The LOD and LOQ were determined to be 0.05 ng/mL and 0.1 ng/mL, respectively. The assay showed excellent precision with intra-assay CV < 5% and inter-assay CV < 10%. The recovery rates ranged from 95% to 105%.
The developed custom ELISA kit provides a reliable and robust method for the quantitative analysis of the target biomarker. The high specificity and sensitivity make it suitable for both research and clinical diagnostics. The validation parameters confirm the assay's reproducibility and accuracy, ensuring its utility in various applications.
The comprehensive methodology for the development of a custom ELISA kit, from antigen and antibody selection to assay validation. The optimized and validated assay offers a powerful tool for the precise measurement of specific biomarkers, facilitating advancements in biomedical research and clinical diagnostics.