The overall goal of this method is to quantify allergen loaded ambient particulate matter particles using flow cytometry. This method can help answer key questions regarding the allergen load of PM10 particles. These particles may act as carriers of absorbed allergens, possibly transporting them into the lower respiratory tract and the main advantage of this technique is that it is a fast way to visualize and quantify allergen loaded PM10 particles.
We established this method for the Bet v 1 antigen, but it may also be applied for other antigens. Demonstrating the procedure will be Sabine Bach, a technician from our laboratory. To begin, use a low volume sampler to collect particulate matter that is 10 micrometers or less in diameter from ambient air on polytetrafluoroethylene filters.
Run the collector for one to 10 days depending on the amount of particulate matter in the air and the desired temporal resolution. At the end of the collection, remove the filter from the sampler. And freeze it at minus 20 degrees Celsius until it'll be analyzed.
When ready to continue with sample analysis, remove the polytetrafluoroethylene filter from the freezer. And let it thaw for about five minutes. Then, put the filter in a clean polystyrol petri dish and overlay the filter with enough PBS to obtain a concentration of eight times 10 to the sixth particles per milliliter.
Hold the filter with a pair of tweezers and brush it using an electrical toothbrush with a sensitive brush head for one minute. Then, transfer the particle PBS suspension to a clean reaction tube. Dilute 50 microliters of the PM10 suspension in 10 milliliters of isotonic measurement buffer.
Use a particle counter to measure the concentration of the collected PM10 particles. Calculate the mean particle concentration from three separate measurements. Use the particle PBS suspension from one sample to collect particles from another filter if the particulate matter concentration is found to be too low, and pooling from multiple samples is appropriate.
First, determine the amount of reaction tubes required. For each PM10 suspension to be analyzed, three types of reaction tubes are needed. One tube with the native control sample, one tube with the negative control sample, and one tube with the specifically stained sample.
Next, calculate the volume of PM10 suspension required. Each reaction tube requires 50 microliters of suspension. Then, adjust the particle concentration measured in the specimen to a final concentration of eight times 10 to the sixth particles per milliliter by adding an appropriate amount of PBS.
Next, block non-specific binding by supplementing the PM10 suspension with 0.02%BSA, diluting it from a 1%BSA stock solution prepared with PBS. Briefly vortex the PM10 BSA suspension and incubate for 20 minutes at room temperature. For each sample to be analyzed by flow cytometry, transfer 50 microliters of the suspension to a clean reaction tube.
Add the monoclonal mouse antibody against Bet v 1 at a final concentration of 0.02 micrograms per microliter to the reaction tubes designated for specific staining. Vortex them briefly, and then incubate all the reaction tubes for 60 minutes at room temperature. Next, wash all of the samples by adding 500 microliters of PBS supplemented with 0.02%BSA to each reaction tube and vortexing the samples briefly.
Then, pellet the samples and discard the supernatant carefully using a vacuum pump. Determine the total amount of APC labeled secondary anti-mouse IgG1 antibody needed and dilute it with the respective volume of PBS supplemented with 0.02%BSA. Add 50 microliters of diluted secondary antibody to all of the reaction tubes except for the native control.
To the native control, add 50 microliters of PBS supplemented with 0.02%BSA. Vortex all samples for a few seconds and incubate them for 30 minutes in the dark. Following the incubation, wash all samples twice as previously shown.
After the last wash, add 50 microliters of plain PBS to each reaction tube. Then, vortex the samples and analyze them on a flow cytometer. Using the native control, adjust the cytometer parameters to optimize data analysis.
First, set the forward scatter threshold at the lowest value of 200 and start the analysis. Next, use the scatter voltage controller to adjust the forward scatter, and subsequently the sideward scatter so that all of the PM10 particles are detected and the population of particles become located approximately in the middle of the forward scatter axis, and in the lower half of the sideward scatter axis. Restart the analysis and record the data.
Then, use the fluorescence voltage controller to adjust for the secondary antibody fluorescence voltage, and another fluorescence voltage like FITC. When this is completed, restart the analysis and record the data. Make sure that all of the particles are visible in the lower half of both of the fluorescence axes.
Consecutively examine each sample and store the forward scatter, sideward scatter, secondary antibody fluorescence and the FITC data of at least 10, 000 particles per sample. One way to quantify the absorbed allergen content is to analyze the APC fluorescence intensity of all particles. For this purpose, edit the statistics by clicking on the FITC median and the APC median button.
Then, compare the respective values and the negative control with those in the specifically stained sample. Another way to quantify the absorbed allergen content is to determine the percentage of PM10 particles with bound anti-Bet v 1 antibody. This is accomplished by first setting a gate around the particles considered as false positive.
Next, click on the specifically stained sample and using the same gate, resolve the APC positive particles in the specific sample. During the high pollen season, a large number of PM10 particles have bound Bet v 1 antigen. The recognition of Bet v 1 is specific, as when the primary antibody blocked with a recombinant version of the Bet v 1 antigen, the signal significantly drops.
When the PM10 particles collected during the high pollen season were compared with those from the low pollen season, a significant difference of a scene in the percentage of Bet v 1 positive PM10 particles. The particles collected during the high pollen season were much more likely to contain the Bet v 1 antigen. Once mastered, the flow cytometric analysis of the sampled PM10 can be done in four to five hours if it is performed properly.
Please keep in mind that flow cytometry only detects particles greater than 5 microliters. If you follow this procedure, it is important to remember that we invented this method for determining the Bet v 1 allergen load of PM10 particles, and this is fundamental difference from the ELISA method where you deal with dissolved antigen. If the Bet v 1 content of ambient air can defer from the pollen count, allergic symptoms might correlate to the allergen level.
Together with clinical data, this method enables us to examine whether the allergic reactions to birch pollen correspond to the allergen level in PM10. With other suitable antibodies on hand, this method might be extended to the detection of other antigens on ambient air particles such as LPS or mold or dust mite allergens.
