Validated Immunochemical Assay for Comprehensive Determination of the Human Epidermal Growth Factor Receptor 2 Released from and Bound to Cells

We present a validated sandwich, ELISA, using novel monoclonal antibodies, which enables precise quantification of cell bound and released extracellular domain of HER2 protein for in vitro cultured cells and patient samples.

The protocol addresses the need for accurate and validated assay to correlate the concentration of circulating HER2 protein with disease clinical manifestations in diagnostics and research.

Broad working range of the assay, variety of sample types and reliability of results might open new opportunities for a comprehensive analysis of the receptor status in in vitro and in vivo models.

The developed ELISA will be used for quantification of HER2 released from cancer tissues into different human body excretions like blood, peritoneal fluids or mucus.

[Instructor] To begin detach the cells using trypsin after they reach 80% confluency and collect them into a separate 15 milliliter conical tube. After counting the cells using an automatic cell counter and seed three times 10 to the power of five cells per well in a 12 well plate with one milliliter of growth medium. Incubate the culture at 37 degrees Celsius in a humidified atmosphere containing 5% carbon dioxide. From the 24 hour culture, collect the culture medium from one well of each cell line into a 1.5 milliliter tube. Centrifuge the collected samples at 10,000 G for 10 minutes at four degrees Celsius and transfer the supernatant to a new tube for storage at minus 20 degrees Celsius. Wash the remaining cells in each well with one milliliter of cold PBS, aspirate gently and discard the PBS. Then, add 200 microliters of RIPA buffer, supplemented with 1% protease inhibitor to each well and incubate for five minutes. Now, scrape the cells with a cell scraper and mix the lysate by pipetting several times to homogenize. Collect the lysed cells into a new tube and slowly aspirate the sample into a two milliliter syringe fitted with a 21 gauge needle measuring 0.8 by 40 millimeters. After aspirating five to 10 times, centrifuge the collected samples at 10,000 G for 10 minutes at four degrees Celsius and transfer the supernatant to a new tube for storage at minus 20 degrees Celsius. Dilute the Anti-HER2 capturing antibody to one microgram per milliliter in coding buffer. Using a multi-channel pipette, add 100 microliters of the capturing antibody solution to each well of a 96 well ELISA plate. Cover the plate with sealing film. Incubate the plate overnight at four degrees Celsius. After overnight incubation, place the plate at room temperature on a horizontal microplate shaker for one hour. Using a microplate washer, aspirate the coating solution and wash each well three times with 300 microliters of PBST. Then, with a multi-channel pipette, add 100 microliters of blocking buffer containing 5% non-fat dry milk in PBS to each coated well to block non-specific binding sites. Now, prepare the calibration curve and positive control. Add two microliters of one milligram per milliliter, HER2 stock solution to 1,998 microliters of PBS to create a 1000 nanograms per milliliter working standard one. For standard two, add 500 microliters of standard one to 500 microliters of PBS and mix thoroughly to achieve 50 nanograms per milliliter concentration. Prepare serial dilution to generate standards three through seven. Next, prepare matrix samples using PBS cell lysates and culture medium spiked with known HER2 concentrations. Dilute cell lysates one to 2000 with 2.5 microliters of lysate and five milliliters of PBS to obtain HER2 levels below the limit of detection. To prepare a two nanograms per milliliter solution, add eight microliters of standard one to 392 microliters of PBS culture medium or cell lysate and mix thoroughly. Using a single channel pipette, add 100 microliters of standards PBS blank and test samples each in triplicate into their respective wells of the coated and blocked ELISA plate. After one hour at 37 degrees Celsius in a humidity chamber, wash the plate using a microplate washer. To detect antibody binding, dilute the biotinylated detecting antibody in PBS to a working concentration of one microgram per milliliter. Then, using a multi-channel pipette, add 100 microliters of this detecting antibody solution to each well. Next dilute the AVID and HRP conjugate one to 40,000 in 40 milliliters of PBST. Using a multi-channel pipette, add 100 microliters of the diluted conjugate to each well. After incubating and washing the plate, use a multi-channel pipette to add 100 microliters of 3,3 5,5 tetramethylbenzidine substrate to each well to initiate the color metric reaction. Then, cover the plate with a sealing film. Incubate the plate at 37 degrees Celsius for one to five minutes, protecting it from light while monitoring the color development. When the desired color intensity is reached, add 100 microliters of stop solution to each well to terminate the reaction. Finally, measure the absorbance at 450 nanometers using a microplate reader. Using a four parameter calibration curve, calculate the sample concentration based on the curve equation. The developed sandwich, ELISA, demonstrated a working range for HER2 protein from 1.56 to 100 nanograms per milliliter with a coefficient of determination of one indicating excellent matching. The HER2 concentration in the culture medium increased over time for all tested cell lines with the highest values at 72 hours. SK-OV-3 and SK-BR-3 cells showed approximately ten fold and 50 fold higher HER2 concentrations, respectively, compared to MDA-MB-231 cells in the culture medium at 24 hours. Similarly, HER2 levels in lysates were about threefold and fivefold higher for SK-OV-3 and SK-BR-3, respectively. Interestingly, the cell culture medium contained 1000 fold lower concentrations of the tested antigen in comparison to membrane bound protein found in whole cell lysates.