This article describes a microfluidic method for analyzing allergen-specific immunoglobulin E (IgE) in serum samples. The process involves using a cartridge with various reagents and a chemiluminescence analyzer to quantify IgE levels.
Begin with a microfluidic cartridge equipped with storage tanks and unidirectional pumps that deliver reagents through channels into a reaction zone.
The reaction zone contains multiple spots immobilized with distinct allergens.
Load wash buffer, blocking buffer, peroxidase-conjugated secondary antibodies, and a chemiluminescent peroxidase substrate into the designated tanks.
Add a serum sample containing allergen-specific immunoglobulin E or IgE.
Initiate the analysis inside a chemiluminescence analyzer.
Within the reaction zone, the blocking buffer flows and blocks non-specific interaction sites.
The introduction of serum allows the IgEs to interact with the immobilized allergens.
Washing eliminates unbound IgEs.
Next, peroxidase-conjugated secondary antibodies flow in and interact with the immobilized IgEs.
Subsequent washing removes unbound antibodies.
The analyzer then introduces the chemiluminescent substrate and hydrogen peroxide .
The peroxidase oxidizes the substrate, emitting luminescence .
The luminescence intensity in each spot provides a quantifiable indication of distinct allergen-specific IgEs in the serum.
To test serum samples for serum immunoglobulin E antibodies to whole allergens, randomly select three samples with at least 900 microliters of serum, and take the chip from the kit, and number it. Then, turn on the computer and the analyzer power. Start the LabIT program on the computer.
If the dark frame warning window pops up, click OK to run a leak test. Then, click the center logo to enter the operation interface. Check the reaction temperature and the charge-coupled device or CCD temperature. The reaction temperature should rise to 37 plus or minus 1 degrees Celsius in about 10 minutes, and the CCD temperature should drop to minus 15 plus or minus 1 degrees Celsius.
When the CCD temperature has dropped to minus 15 plus or minus 1 degrees Celsius, confirm that there are no other items left inside the instrument and close the door. Then click Tools, System Test, Leak Test. When the test is finished, the report window will pop up.
Next, load 620 microliters of wash buffer, 120 microliters of blocking buffer, 60 microliters of conjugates A and B, 60 microliters of substrates A and B, and 100 microliters of each serum sample into the corresponding reagent tanks on the microfluidic cartridge. After clicking cartridge ID, use the barcode scanner to scan the serial number of the cartridge and enter the sample ID. Then, place the cartridge into the analyzer, close the door, and click analyzer, and run to start the analysis.
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