The overall goal of the following experiment is to detect neuronal anti D two R or N-M-D-A-R antibodies in patient samples using a high-throughput flow cytometry cell-based assay. This is achieved by sub cling, the full CDNA sequence of human D two R or N-M-D-A-R within a plasmid. As a second step, a human cell line is transfected, which leads to the cell surface expression of neuronal antigens.
Next transfected cells are incubated with patient serum and an appropriate secondary antibody before flow cytometry acquisition. In order to detect antibody binding to cell surface antigens, results are obtained based on flow cytometry analysis. This Our high three port flow cytometry cell-based assay is fast, quantitative and efficient for large cohorts of patient samples.
This is a significant advantage over using a cell-based assay with immunochemistry and confocal analysis. Generally, individuals new to this method will struggle with the loss of cells over the course of the experiment in order to acquire the appropriate number of cells at the flow cytometer. Take care not to aspirate cells between washes.
Select an expression vector that is suitable for expression of transmembrane proteins and enables the cellular co-expression of the antigens and the green fluorescent protein separately. For example, this vector has an enhanced GFP reporter under the control of an internal ribosome entry site sub clone the full length human CD NA into the vector using the appropriate restriction enzymes after verifying the clones by sequencing, make plasmid stocks that are suitable for use in transfect culture. The HEK 2 93 cells in complete DMEM with 10%fetal bovine serum.
Harvest the cells by trypsin after one wash centrifuge the cells and resus suspend the cell pellet in fresh complete dmm. Count the cells, then seed two milliliter cultures in six well plates at around 70%Co fluency assign the wells of HEK 2 93 cells for transfection with the expression constructs as well as the control vector. Keep some on transfected HEK 2 93 cells for compensation later on.
Prepare the transfection mixes comprising of 200 microliters of 0.9%sodium chloride, 2.5 micrograms of DNA and four micrograms per milliliter of polyethaline. Immediately Vortex for 10 seconds and incubate at room temperature for 10 minutes before adding the transfection mix to each well of two milliliter fresh complete dmm. Cover the plate, wrap the sides with perfil and centrifuge at 280 GS for five minutes to aid cell transfection.
Then remove the para film and then place the plate under culture conditions for 18 hours. Replace the culture media with fresh complete DM EM and maintain in culture for another 72 hours. To detach the cells, add 500 microliters of varice to each well and incubate for approximately five minutes at 37 degrees Celsius.
Then resuspend the cells of each well in two milliliters of 2%FBS in PBS and transfer the cells to a conical tube after three washes. Resuspend the cell pallets at 1 million cells per milliliter in 2%FBS solution. Now design the 96 well template for flow cytometry acquisition such that the different cell lines are treated with each patient sample or primary antibody.
Then seed 50, 000 cells per well in a V bottom 96 well plate pellet the cells by centrifuging at 450 Gs for five minutes. Tilt a plate on an angle and gently remove the supra natin taking care not to aspirate the cell pellet. Next, add serial dilution of primary antibody in order to assess the antigen surface expression.
And then patient samples in order to detect antibody, incubate the cells for one hour at room temperature in the dark after three washes in 170 microliters of 2%FBS solution, add the appropriate AF 6 47 conjugated secondary antibody anti-human IgG for the patient and anti-US. IgG for primary antibodies. Hybridize for one hour at room temperature in the dark after three washes.
Resus suspend the cells in 35 microliters of 2%FBS solution for the flow cytometry cell acquisition at the flow cytometer. First set up the high throughput sampler. Acquire 10, 000 events per well according to the flow cytometry acquisition template.
Export and then analyze the data using a flow cytometry software package gate the live HEK 2 93 cells based on forward inside scatters. Also gate on high GFP positivity to analyze only the transfected cells. Determine the mean fluorescence intensity of the AF 6 47 channel within the live GFP positive cells.
Then export the data into Excel or prism for analysis. Plot the concentrations of primary antibody versus the MFI obtained from the corresponding samples for each patient and control sample. Determine the delta MFI calculate the threshold of the antibody positivity by adding three standard deviations above the mean delta MFI of the control cohort.
Then plot the individual samples grouped according to the patient group on a graph. Represent the threshold as a line here. Cells expressing either the n methyl de aspartate receptor or dopamine two receptor were acquired at the flow cytometer using a high throughput sampler.
During the analysis cells were gated based on forward scatter for size and on side scatter for granularity. TRANSFECTED HK 2 9 3 cells expressed the GFP reporter molecule in the cytoplasm and UNT transfected cells were excluded from analysis within the GFP positive gait. The MFI associated with AF 6 47 conjugated anti-human IgG secondary antibody binding was measured in order to eliminate the background fluorescence when analyzing human sera.
The delta MFI was calculated by subtracting the background MFI of the control cells. This flow cytometry cell based assay is based on antibody detection of the cell surface antigen of interest. For example, the a GK 2 93 cells transfected with the expression plasmid for N-M-D-A-R showed high expression of the surface and methyl de receptor.
Similarly, the D two R transfected cells expressed the dopamine two receptor, whereas the controlled cells expressed neither of these antigens. This clinical study evaluated a patient cohort with N-M-D-A-R encephalitis, a patient cohort with D two R antibody associated encephalitis and a control cohort with other non-inflammatory neurological diseases. Note that none of the positive patients were double positive for antibodies.
Targeting N-M-D-A-R-M-D two R as expected. The N-M-D-A-R and D two R antibodies were not detected in any of the controls analyzed. Once mastered, this technique can be performed properly in five hours with one hour of flow acquisition for about 80 samples After its development.
This technique paved the way for researchers in the field of neuroimmunology to discover novel antibody targets in immune mediated disorders.
