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All procedures involving sample collection have been performed in accordance with the institute&#39;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.
<ol>
	<li>Search the AIR-SKLRD&#39;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Study flow and measurements of interest
NOTE: The microfluidic system needs 100 &#181;L 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&#160;g, the upper layer was collected for testing. Unused serum was stored at -80 &#176;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.
<ol>
	<li>Primarily test the serum samples for sIgEs to whole allergens of&#160;Dermatophagoides pteronyssinus&#160;(d1),&#160;Dermatophagoides farinae&#160;(d2),&#160;Blomia tropicalis&#160;(d201), cat dander (e1), dog dander (e5), Bermuda grass (g2), timothy grass (g6), cockroaches (i6),&#160;Aspergillus fumigatus&#160;(m3),&#160;Candida albicans&#160;(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&#160;Table of Materials) and measured by a chemiluminescence analyzer.</li>
	<li>Randomly select three samples from among the samples with enough serum (at least 900 &#181;L) 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 &#181;L of serum).</li>
</ol>
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.
<ol>
	<li>Preparation of the equipment
	<ol>
		<li>Turn on the PC and the analyzer power. 
		NOTE: The power switch is on the left of the base.</li>
		<li>Start the LabIT program on the PC. If the&#160;Dark Frame&#160;warning window pops up, click&#160;OK&#160;to run&#160;the LEAK Test. Afterward, click the center logo to enter the operation interface. 
		NOTE: The system will remind the user to run&#160;the LEAK Test&#160;if idle for more than 24 hours.</li>
		<li>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 &#176;C &#177; 1 &#176;C in about 10 min, and the CCD Temp should drop to -15 &#176;C &#177; 1 &#176;C.</li>
		<li>Run the&#160;LEAK Test&#160;after the CCD Temp has dropped to -15 &#176;C &#177; 1 &#176;C. Before running the LEAK test, make sure no other items are left inside the instrument and close the door. Click&#160;Tools&#160;|&#160;System Test&#160;|&#160;LEAK Test. Do not open the door during testing. When the test is finished, the report window will pop up.</li>
	</ol>
	</li>
	<li>Sample loading
	<ol>
		<li>Add 620 &#181;L of wash buffer, 120 &#181;L of blocking buffer, 60 &#181;L of conjugates A and B, 60 &#181;L of substrate A and B, and 100 &#181;L of serum samples to the corresponding reagent tank on the microfluidic cartridge.</li>
	</ol>
	</li>
	<li>Incubation
	<ol>
		<li>Click on&#160;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&#160;Analyzer&#160;and&#160;Run&#160;to start the analysis.</li>
	</ol>
	</li>
	<li>Measurement
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
Table 1: Patient demographic characteristics.&#160;In total, 293 subjects who fulfilled the inclusion criteria were found, with a median&#160;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.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Characteristic</td>
			<td>No.(%)</td>
		</tr>
		<tr>
			<td>Gender, n(%)</td>
			<td></td>
		</tr>
		<tr>
			<td>Female</td>
			<td>123(41.98%)</td>
		</tr>
		<tr>
			<td>Male</td>
			<td>170(58.02%)</td>
		</tr>
		<tr>
			<td>Age, year, n(%)</td>
			<td></td>
		</tr>
		<tr>
			<td>Median (25%,75%)</td>
			<td>23(8,36)</td>
		</tr>
		<tr>
			<td>&#8804;10</td>
			<td>97(33.11%)</td>
		</tr>
		<tr>
			<td>11-20</td>
			<td>37(12.63%)</td>
		</tr>
		<tr>
			<td>21-40</td>
			<td>101(34.47%)</td>
		</tr>
		<tr>
			<td>&#62;41</td>
			<td>58(19.80%)</td>
		</tr>
		<tr>
			<td>Diagnosis, n(%)</td>
			<td></td>
		</tr>
		<tr>
			<td>Allergic rhinitis</td>
			<td>92(31.40%)</td>
		</tr>
		<tr>
			<td>Allergic asthma</td>
			<td>117(39.93%)</td>
		</tr>
		<tr>
			<td>Allergic rhinitis with asthma</td>
			<td>36(12.29%)</td>
		</tr>
		<tr>
			<td>Others</td>
			<td>48(16.38%)</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agnitio BioIC Analyzer</td>
			<td>Agnitio Science &#38; Technology(Taiwan, China)</td>
			<td>BA-G2000</td>
			<td></td>
		</tr>
		<tr>
			<td>BioIC Allergen specific-IgE Detection Kit</td>
			<td>Agnitio Science &#38; Technology(Taiwan, China)</td>
			<td>DR17A12</td>
			<td></td>
		</tr>
		<tr>
			<td>BioIC Cartrideg Placement plate</td>
			<td>Agnitio Science &#38; Technology(Taiwan, China)</td>
			<td>T20SET</td>
			<td></td>
		</tr>
		<tr>
			<td>BioIC Reagent Dispenser</td>
			<td>Agnitio Science &#38; Technology(Taiwan, China)</td>
			<td>DS-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Image two-dimensional barcode machine</td>
			<td>Agnitio Science &#38; Technology(Taiwan, China)</td>
			<td>NLS-HR200</td>
			<td></td>
		</tr>
		<tr>
			<td>VORTEX-5 Vortex Mixer</td>
			<td>Haimen Kylinbell Lab Lastruments Co., Ltd.</td>
			<td>VORTEX-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

a microfluidic cartridge-based detection system for allergen-specific immunoglobulin e

Source: Huang, Z., et al. <a href="https://app.jove.com/v/59100">Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE).</a>&#160;J. Vis. Exp.&#160;(2019)
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.

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

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Basic information of the study ...

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&#160; 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.

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Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

56 Views. Source: Huang, Z., et al. Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE). J. Vis. Exp. (2019) 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 chemiluminesc...

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

A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen allergens, data on this topic are limited in southern China. Any available data were obtained by self-report questionnaires, skin prick tests, and total or specific IgE tests using crude extracts. For many reasons, these methods are unreliable. Serum sIgE reactivity to Bermuda grass, Timothy grass, and Humulus scandens allergens in a cohort of patients from Greater Guangzhou (southern China&#39;s largest city and its outskirts) with allergic rhinitis and/or asthma were examined using a fully-automated immunoassay analyzer as a component-resolved diagnostics (CRD) tool.
For the first time, a considerably high prevalence of Bermuda grass sIgE positivity was demonstrated in Chinese southerners with allergic rhinitis and/or asthma. In these patients, a subtle prevalence of sensitization to Timothy grass and Humulus scandens was also noted, which may arise from cross-reactivity, as the latter two are not common in the region. This was also supported by the detection of allergen components.
Fully-automated immunoassay analyzers may offer satisfactory consistency between regions, laboratories, and institutions and over time. The automaticity of the instrument may enable a standardized detection that would not have been readily revealed before the advent of CRD.
This is a study that uses a CRD approach to investigate sensitization to grass pollen allergens in southern China. It adds to current evidence in the literature. Future studies are needed to validate these findings. However, although CRD is a useful tool, the findings made with the fully-automated immunoassay analyzer should not substitute for other laboratory investigations, clinical evaluations, and physician expertise.

This work describes a protocol that uses a component-resolved approach to study sensitization to grass pollen allergens in a cohort of patients from southern China with allergic rhinitis and/or asthma.

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen ...

JoVE Journal

Medicine

Humanized Mediator Release Assay as a Read-Out for Allergen Potency

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells and basophils, upon stimulation with serial dilutions of putative allergens. Therefore, these assays represent an essential tool that mimics the in vivo degranulation process, which occurs upon allergen exposure in sensitized patients or in skin prick tests. Additionally, these assays are usually employed to investigate the allergenic potential of proteins and the reactivity of patients' sera's reactivity. Herein, we describe a simple 2-day protocol using an immortalized rat basophil leukemia cell line transfected and humanized with the human high-affinity IgE plasma-membrane receptor (Fc&#949;RI). This variant of the mediator release assay is a robust, sensitive, and reproducible in vitro cell-based system without the need to immobilize the antigen to solid matrices. The protocol consists of the following steps: (1) complement inactivation of human sera, (2) harvesting, seeding, and passive sensitization of the cells, (3) stimulation with antigen to cause mediator release, and (4) measuring of &#946;-hexosaminidase activity as a surrogate for the released inflammatory mediators, such as histamine. The assay represents a useful tool to assess the capacity of the allergen-IgE cross-linking to trigger cell degranulation and can be implemented to standardize allergen extracts, to compare patients' reactivity to minor or major allergens and to allergenic extracts (pollen, cat dander, etc.), to investigate the potency of allergen homologs, isoforms, and fold-variants (e.g., hypoallergenicity), as well as the effects of ligands on the allergenic activity. A more recent application includes the use of the assay to monitor the treatment efficacy in the course of allergen immunotherapy.

Here we present the mediator release assay, using a rat basophilic leukemia cell line transfected with the human IgE receptor, to simulate the degranulation of effector cells typically observed in type 1 allergic reactions. This method investigates the biological activity of allergens in a highly sensitive, reproducible, and tailorable manner.

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells ...

Immunology and Infection

Basophil Activation Test for Allergy Diagnosis

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and specific IgE (sIgE) determination in the evaluation of IgE-mediated allergic reactions to food, insect venom, drugs, as well as some forms of chronic urticaria. However, the role of this technique in the diagnostic algorithms is highly variable and not well determined.
BAT is based on the determination of basophil response to allergen/drug cross-linking IgE activation through the measurement of activation markers (such as CD63, CD203c) by flow cytometry. This test can be a useful and complementary tool to avoid controlled challenge tests&#160;to confirm allergy diagnosis, especially in subjects experiencing severe life-threatening reactions. In general, the performance of BAT should be considered if i) the allergen/drug produces false positive results in ST; ii) there is no allergen/drug source to use for ST or sIgE determination; iii) there is discordance between patient history and ST or sIgE determination; iv) symptoms suggest that ST may result in systemic response; v) before considering a CCT to confirm the culprit allergen/drug. The main limitations of the test are related to non-optimal sensitivity, especially in drug allergy, the need to perform the test no longer than 24 h after sample extraction, and the lack of standardization between laboratories in terms of procedures, concentrations, and cell markers.

The basophil activation test is a complementary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions based on the detection of basophil activation in the presence of a specific stimulus through the measure of activation markers by flow cytometry.

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and ...

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

Transcript

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

A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma

Humanized Mediator Release Assay as a Read-Out for Allergen Potency

Basophil Activation Test for Allergy Diagnosis