Name: Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE) (Video) | JoVE Journal
Uploaded: 2019-04-21T14:00:00.000+00:00
Duration: 7 min 10 s
Description: Learn microfluidic cartridge-based chemiluminescence for detecting serum-specific IgE in allergy diagnosis. This low-cost method requires minimal serum volume and enables rapid detection of respiratory, food, and skin allergens.

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5 minutes

The authors thank Professor Mei Jiang for her help in the statistical analysis and Mr. Hammer Tsui in the preparation of the manuscript. This study was supported by the Guangzhou Science and Technology Foundation (201804020043) and the National Natural Science Foundation of China (NSFC 81572063 and NSFC 81802076). The funding groups agreed with the study design, data analysis, manuscript preparation, and decision to publish. No other funding was received for this study.

This study and the use of human serum samples were approved by the Ethics Committee of The First Affiliated Hospital of Guangzhou Medical University (GYYY-2016-73). All participants have given their written consent independently or through their parents (in the case of children).
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 to collect and store serum samples from patients with allergic diseases, together with their clinical information (Table 1)4,5. The current study was performed with sera from the AIR-SKLRD.
<ol>
	<li>Search the AIR-SKLRD&#8217;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 prior to the serum collection so as to minimize interference to the laboratory findings. All serum samples that do 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 for determining 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 &#176;C. Prior to testing, the serum was kept at room temperature for 30 min and was 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 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.</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., a total of 100 x 9 = 900 &#181;L of serum).</li>
</ol>
3. Semi-automation test procedure of the microfluidic system
NOTE: The System 2 is the integration of 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 Dark Frame warning window pops up, click OK to run LEAK Test. Afterward, click the center logo to enter the operation interface. 
		NOTE: The system will remind the user to run LEAK Test if it is idle for more than 24 h.</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 LEAK Test after the CCD Temp has dropped to -15 &#176;C &#177; 1 &#176;C. Before running the LEAK test, make sure there are no other items 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.</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 Cartridge ID, use the barcode scanner to scan the serial number of the cartridge, enter the sample ID, put the cartridge into the analyzer and close the door, and click Analyzer and Run 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>
	<li>Switching off of the analyzer
	<ol>
		<li>For routine maintenance, after finishing the test, remove the cartridge and wipe the analyzer&#8217;s internal heating iron and electromagnet lightly with 75% alcohol. 
		NOTE: Do not press hard or shake the electromagnet.</li>
		<li>Close the LabIT window. The temperature monitoring window will pop up. It will automatically close when the CCD warms up to the 5 &#176;C protection mode. By then, it will be safe to turn off the power of the analyzer and PC. 
		NOTE: Do not manually close the temperature monitoring window before the CCD Temp has risen to 5 &#176;C, and do not turn off the power of the analyzer nor the PC during the CCD warm-up.</li>
	</ol>
	</li>
</ol>
4. Definition of sIgE reactivity
NOTE: For an undiluted serum specimen, the detection range of the System 2 is 0.21&#8211;100 IU/mL.
<ol>
	<li>Based on the threshold value of 0.35 IU/mL, consider an sIgE level exceeding 0.35 IU/mL to be positive6,7. Rate the reactivity of the sIgE tests as8: class 1 (&#8805;0.35 and &#60;0.70 IU/mL), class 2 (&#8805;0.70 and &#60;3.50 IU/mL), class 3 (&#8805;3.50 and &#60;17.50 IU/mL), class 4 (&#8805;17.50 and &#60;50.00 IU/mL), class 5 (&#8805;50.00 and &#60;100.00 IU/mL), and class 6 (&#8805;100.00 IU/mL).</li>
</ol>
5. Statistical analysis
<ol>
	<li>Use a histogram to show the positive rate of the 19 allergens (Figure 1) and use the Levey-Jennings curve to demonstrate the repeatability of the detection system (Figure 2)9.</li>
	<li>Select the three most common inhalant allergens and food allergens (in total, six allergens) and compare the results to the I System 1 to evaluate its clinical diagnostic performance10,11. Include the concordance rate, sensitivity, specificity, positive and negative predictive values, and the area under the receiver operating characteristic (ROC) curve (AUC) as the evaluation criteria.</li>
	<li>Apply Spearman&#8217;s correlation analysis12 to describe correlations between the two systems and use kappa value for consistency. Categorize the kappa value as almost perfect (0.8&#8211;1.0), substantial (0.6&#8211;0.8), moderate (0.4&#8211;0.6), fair (0.2&#8211;0.4), or poor (&#60;0.2)13. Use SPSS 23.0 and MedCalc 11.0 statistical analysis and define P &#60; 0.05 as statistical significance.</li>
</ol>

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<li>Luo, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((grass+pollen+allergens+and+components+detected+in+a+southern+Chinese+cohort+of+patients+with+allergic+rhinitis+and%2For+asthma%5BTitle%5D)+AND+%22Molecular+Immunology%22%5BJournal%5D)">grass pollen allergens and components detected in a southern Chinese cohort of patients with allergic rhinitis and/or asthma.</a> Molecular Immunology. 78 (2016), 105-112 (2016).</li>
<li>Zeng, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Longitudinal+profiles+of+serum+specific+IgE+and+IgG4+to+Dermatophagoides+pteronyssinus+allergen+and+its+major+components+during+allergen+immunotherapy+in+a+cohort+of+southern+Chinese+children%5BTitle%5D)+AND+%22Molecular+Immunology%22%5BJournal%5D)">Longitudinal profiles of serum specific IgE and IgG4 to Dermatophagoides pteronyssinus allergen and its major components during allergen immunotherapy in a cohort of southern Chinese children.</a> Molecular Immunology. 74 (2016), 1-9 (2016).</li>
<li>Lee, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specific+IgE+measurement+using+AdvanSure%28R%29+system%3A+comparison+of+detection+performance+with+ImmunoCAP%28R%29+system+in+Korean+allergy+patients%5BTitle%5D)+AND+%22Clinica+Chimica+Acta%22%5BJournal%5D)">Specific IgE measurement using AdvanSure(R) system: comparison of detection performance with ImmunoCAP(R) system in Korean allergy patients.</a> Clinica Chimica Acta. (9-10), 914-919 (2012).</li>
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<li>Bland, J. M., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Statistical+methods+for+assessing+agreement+between+two+methods+of+clinical+measurement%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Statistical methods for assessing agreement between two methods of clinical measurement.</a> Lancet. 327 (8476), 307-310 (1986).</li>
<li>Carletta, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+Agreement+on+Classification+Tasks%3A+The+Kappa+Statistic%5BTitle%5D)+AND+%22Computational+Linguistics%22%5BJournal%5D)">Assessing Agreement on Classification Tasks: The Kappa Statistic.</a> Computational Linguistics. 22 (2), 249-254 (1996).</li>
<li>Shyur, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+multiple+allergen-specific+IgE+by+microfluidic+immunoassay+cartridge+in+clinical+settings%5BTitle%5D)+AND+%22Pediatric+Allergy+and+Immunology%22%5BJournal%5D)">Determination of multiple allergen-specific IgE by microfluidic immunoassay cartridge in clinical settings.</a> Pediatric Allergy and Immunology. 21, 623-633 (2010).</li>
<li>Cesana, B. M., Antonelli, P., Gallazzi, E., Marino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+measurement+methods%3A+an+endless+application+of+wrong+statistical+methods%5BTitle%5D)+AND+%22Intensive+Care+Medicine%22%5BJournal%5D)">Comparison of measurement methods: an endless application of wrong statistical methods.</a> Intensive Care Medicine. 37 (6), 1038-1040 (2011).</li>
<li>Park, K. H., Lee, J., Sim, D. W., Lee, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+Singleplex+Specific+IgE+Detection+Immunoassays%3A+ImmunoCAP+Phadia+250+and+Immulite+2000+3gAllergy%5BTitle%5D)+AND+%22Annals+of+Laboratory+Medicine%22%5BJournal%5D)">Comparison of Singleplex Specific IgE Detection Immunoassays: ImmunoCAP Phadia 250 and Immulite 2000 3gAllergy.</a> Annals of Laboratory Medicine. 38 (1), 23-31 (2018).</li>
<li>Teppo, H., Revonta, M., Haahtela, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Allergic+rhinitis+and+asthma+have+generally+good+outcome+and+little+effect+on+quality+of+life+-+a+20-year+follow-up%5BTitle%5D)+AND+%22Allergy%22%5BJournal%5D)">Allergic rhinitis and asthma have generally good outcome and little effect on quality of life - a 20-year follow-up.</a> Allergy. 66 (8), 1123-1125 (2011).</li>
<li>Fischer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevalence+of+type+I+sensitization+to+alpha-gal+in+forest+service+employees+and+hunters%5BTitle%5D)+AND+%22Allergy%22%5BJournal%5D)">Prevalence of type I sensitization to alpha-gal in forest service employees and hunters.</a> Allergy. 72 (10), 1540-1547 (2017).</li>
<li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+multicentre+study+assessing+the+prevalence+of+sensitizations+in+patients+with+asthma+and%2For+rhinitis+in+China%5BTitle%5D)+AND+%22Allergy%22%5BJournal%5D)">A multicentre study assessing the prevalence of sensitizations in patients with asthma and/or rhinitis in China.</a> Allergy. 64 (7), 1083-1092 (2009).</li>
<li>Ahlstedt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Understanding+the+usefulness+of+specific+IgE+blood+tests+in+allergy%5BTitle%5D)+AND+%22Clinical+%26+Experimental+Allergy%22%5BJournal%5D)">Understanding the usefulness of specific IgE blood tests in allergy.</a> Clinical & Experimental Allergy. 32 (1), 11-16 (2002).</li>
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</ol>

The authors have nothing to disclose.

Similar to the results from many other studies15,16,17, the results from the microfluidic system based on sera from 293 allergic patients showed that house dust mites (including D. pteronyssinus, D. farinae, and B. tropicalis) are the main inhalant allergens leading to allergic diseases in southern China, whereas for food, milk, peanut, shrimp, and crab are the commonest allergens that cause allergic s...

The prevalence of allergies has increased steadily in the past decades and is affecting 20%-30% of the global population1. Identification of the causative allergens has important significance in managing the diseases. In China, since registered in vivo skin prick tests are not available in the country, in vitro determination of serum-specific IgE is the most important and commonly used tool for type I allergy diagnoses2. This is similar to practices in the Western world, but although ImmunoCAP system (System 1), a fluorescence enzyme-linked immunosorbent base system, is perceived as the gold standard for in vitro allergy diagnosis3, its usage in China is very limited due to its high equipment and reagent price. Hence a new alternative allergy diagnosis system with a high price-performance ratio is badly needed.
The BioIC System (System 2) is a microfluidic cartridge-based system based on the chemiluminescence principle for multiplexed assays of serum-specific IgE. With a size of 7 cm x 4 cm, the microfluidic cartridge is composed of three layers of injection-molded plastic. The upper part is 3 mm-thick transparent polycarbonate which carries good stability during thermal assembly processes. Together with the 3 mm-thick bottom layer constructed from a copolymer of acrylonitrile, butadiene, and styrene (ABS), it sandwiches the 0.5 mm-thick middle layer made of silicone rubber. Being black in color, the middle layer offers lower background during chemiluminescence detection. On top of the silica gel, a thin layer of nitrocellulose membrane (NC membrane) is sprayed at the position corresponding to the reaction zone, which allows the spotting of different allergenic proteins. The purpose of this study is to evaluate the clinical performance of the microfluidic system for the multiplexed determination of allergen-specific IgE in serum.

<table><tbody><tr><td>Agnitio BioIC Analyzer</td><td>Agnitio Science &amp;amp; Technology(Taiwan, China)</td><td>BA-G2000</td><td></td></tr><tr><td>BioIC Allergen specific-IgE Detection Kit</td><td>Agnitio Science &amp;amp; Technology(Taiwan, China)</td><td>DR17A12</td><td></td></tr><tr><td>BioIC Cartrideg Placement plate</td><td>Agnitio Science &amp;amp; Technology(Taiwan, China)</td><td>T20SET</td><td></td></tr><tr><td>BioIC Reagent Dispenser</td><td>Agnitio Science &amp;amp; Technology(Taiwan, China)</td><td>DS-1</td><td></td></tr><tr><td>Image two-dimensional barcode machine</td><td>Agnitio Science &amp;amp; Technology(Taiwan, China)</td><td>NLS-HR200</td><td></td></tr><tr><td>Software Package, LabIT</td><td>Agnitio Science &amp;amp; Technology(Taiwan, China)</td><td>Version 2.4.12</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>

application of biochip microfluidic technology to detect serum allergen-specific immunoglobulin e (sige)

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE)

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. However, a specific immunoglobulin E (IgE) measurement system with a high price-performance ratio is lacking in mainland China, especially in the primary care hospitals. This paper describes the principle and operation procedures of using a microfluidic cartridge-based chemiluminescence system to detect allergen-specific IgE in serum. The results were compared with those from ImmunoCAP (System 1), the industrial standard, and the reproducibility of the system to detect patients sensitized to common allergens is evaluated. The results showed that in comparison with ImmunoCAP (System 1), the BioIC System (System 2) has good precision and sensitivity in detecting serum-specific IgE against various inhalant and food allergens but with a significantly lower cost. It can serve as a good alternative to System 1 in primary care hospitals in mainland China who have lower financial affordability.

Positive rates for 19 common allergens 
The results on 293 sera are shown in Figure 3. Among all the inhalant allergens, D. farinae had the highest positive rate (80.89%, 273/293), followed by D. pteronyssinus (78.84%, 231/293). Among the food allergens, crab has the highest positive rate (20.48%, 60/293), followed by 13.65% (40/293) for shrimp. The total positive rate for inhalant allergens was higher than that ...

This paper presents a protocol for detecting serum-specific immunoglobulin E with a microfluidic cartridge-based chemiluminescence system and the evaluation of its usefulness in allergy diagnoses.

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. ...

application-biochip-microfluidic-technology-to-detect-serum-allergen

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Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE)

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