A Microfluidic Cartridge-Based Detection System for Allergen-Specific Immunoglobulin E

Overview

This video demonstrates a protocol for detecting serum-specific immunoglobulin E using a microfluidic cartridge-based chemiluminescence system. Serum IgEs interact with distinct allergens immobilized in the reaction zone of the cartridge, which are subsequently detected with peroxidase-conjugated anti-IgE antibodies and a chemiluminescent substrate, allowing simultaneous detection of multiple allergen-specific IgEs.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Basic information of the study group

NOTE: The Allergy Information Repository of the State Key Laboratory of Respiratory Disease (AIR-SKLRD) is a large serum bank established inside the Guangzhou Institute of Respiratory Hospital (GIRH). Initiated in the last decade, the AIR-SKLRD has already started collecting and storing serum samples from patients with allergic diseases and their clinical information (Table 1). The current study was performed with sera from the AIR-SKLRD.

1. Search the AIR-SKLRD's database for sera collected from January 2015 to June 2018 and select the patients with allergic disease who were found to be sensitive to the common allergens in the region.
2. Ensure that all selected patients have allergic-related diseases, such as allergic rhinitis and or asthma, allergic dermatitis, and/or urticaria, and that the serum of these patients contains multiple serum allergen-specific immunoglobulin E (sIgE) sensitizations of common allergens in this region, detected by the System 1.
3. Exclude patients with incomplete medical records, those lost to follow-up, those who refuse to give informed consent regarding the use of their serum samples for scientific purposes, those with an identified immunodeficiency, those currently on immunotherapy or immunomodulatory agents, or those found to have parasitic infections.
4. Ensure no treatment or drug prescription was given before the serum collection to minimize interference with the laboratory findings. All serum samples that did not fulfill the criteria were rejected.

2. Study flow and measurements of interest

NOTE: The microfluidic system needs 100 µL of serum to determine 19 allergens. Venous blood (5 mL) was collected from each patient using a vacuum blood vessel containing separating gel. After centrifuging for 10 min at 1,000 x g, the upper layer was collected for testing. Unused serum was stored at -80 °C. Before testing, the serum was kept at room temperature for 30 minutes and shaken with a vortex mixer. Repeated freeze-and-thaw cycles were avoided.

1. Primarily test the serum samples for sIgEs to whole allergens of Dermatophagoides pteronyssinus (d1), Dermatophagoides farinae (d2), Blomia tropicalis (d201), cat dander (e1), dog dander (e5), Bermuda grass (g2), timothy grass (g6), cockroaches (i6), Aspergillus fumigatus (m3), Candida albicans (m5), ragweed (w1), egg white (f1), milk (f2), wheat (f4), peanut (f13), soybean (f14), almond (f20), crab (f23), and shrimp (f24). Follow the instructions given in section 3.
   NOTE: sIgE determination was done with the allergen-specific IgE assay kit (see the Table of Materials) and measured by a chemiluminescence analyzer.
2. Randomly select three samples from among the samples with enough serum (at least 900 µL) for a reproducibility study. Keeping all the conditions unchanged, measure the three sera for allergen sIgEs daily for 9 consecutive days (i.e., 100 x 9 = 900 µL of serum).

3. Semi-automation test procedure of the microfluidic system

NOTE: System 2 integrates automatic microfluidic technology, protein microarray, cold light analysis, parallel IgE analysis, and image processing technology. The testing protocol is divided into four parts: preparation of the equipment, sample loading, incubation, and measurement.

1. Preparation of the equipment
   1. Turn on the PC and the analyzer power.
      NOTE: The power switch is on the left of the base.
   2. Start the LabIT program on the PC. If the Dark Frame warning window pops up, click OK to run the LEAK Test. Afterward, click the center logo to enter the operation interface.
      NOTE: The system will remind the user to run the LEAK Test if idle for more than 24 hours.
   3. Check the Reaction Temp and CCD (charge-coupled device) Temp at the lower right corner of the screen. The Reaction Temp should rise to 37 °C ± 1 °C in about 10 min, and the CCD Temp should drop to -15 °C ± 1 °C.
   4. Run the LEAK Test after the CCD Temp has dropped to -15 °C ± 1 °C. Before running the LEAK test, make sure no other items are left inside the instrument and close the door. Click Tools | System Test | LEAK Test. Do not open the door during testing. When the test is finished, the report window will pop up.
2. Sample loading
   1. Add 620 µL of wash buffer, 120 µL of blocking buffer, 60 µL of conjugates A and B, 60 µL of substrate A and B, and 100 µL of serum samples to the corresponding reagent tank on the microfluidic cartridge.
3. Incubation
   1. Click on Cartridge ID, use the barcode scanner to scan the serial number of the cartridge, enter the sample ID, put the cartridge into the analyzer, close the door, and click Analyzer and Run to start the analysis.
4. Measurement
   1. Export the results to statistical software (e.g., Excel) after the measurement.
      NOTE: After 30 min of incubation, the analyzer automatically performs the measurement and reports the result.

Table 1: Patient demographic characteristics. In total, 293 subjects who fulfilled the inclusion criteria were found, with a median age of 23 (interquartile range from 8 to 36 years old). Among them, 170 (58.02%) were male and 123 (41.98%) were female. Also, 92 (31.40%) of them had allergic rhinitis, 117 (39.93%) had allergic asthma, 36 (12.29%) had comorbidity of rhinitis and asthma, and 48 (16.38%) had other allergic diseases, such as a food allergy or skin allergies.

Characteristic	No.(%)
Gender, n(%)
Female	123(41.98%)
Male	170(58.02%)
Age, year, n(%)
Median (25%,75%)	23(8,36)
≤10	97(33.11%)
11-20	37(12.63%)
21-40	101(34.47%)
>41	58(19.80%)
Diagnosis, n(%)
Allergic rhinitis	92(31.40%)
Allergic asthma	117(39.93%)
Allergic rhinitis with asthma	36(12.29%)
Others	48(16.38%)

Materials

Name	Company	Catalog Number	Comments
Agnitio BioIC Analyzer	Agnitio Science & Technology(Taiwan, China)	BA-G2000
BioIC Allergen specific-IgE Detection Kit	Agnitio Science & Technology(Taiwan, China)	DR17A12
BioIC Cartrideg Placement plate	Agnitio Science & Technology(Taiwan, China)	T20SET
BioIC Reagent Dispenser	Agnitio Science & Technology(Taiwan, China)	DS-1
Image two-dimensional barcode machine	Agnitio Science & Technology(Taiwan, China)	NLS-HR200
VORTEX-5 Vortex Mixer	Haimen Kylinbell Lab Lastruments Co., Ltd.	VORTEX-5