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>Paganelli, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specific+IgE+antibodies+in+the+diagnosis+of+atopic+disease.+Clinical+evaluation+of+a+new+in+vitro+test+system%2C+UniCAP%2C+in+six+European+allergy+clinics%5BTitle%5D)+AND+%22Allergy%22%5BJournal%5D)">Specific IgE antibodies in the diagnosis of atopic disease. Clinical evaluation of a new in vitro test system, UniCAP, in six European allergy clinics.</a> Allergy. 53 (8), 763-768 (1998).</li>
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<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>
<li>Eckels, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quality+control%2C+analysis+and+secure+sharing+of+Luminex%28R%29+immunoassay+data+using+the+open+source+LabKey+Server+platform%5BTitle%5D)+AND+%22Bmc+Bioinformatics%22%5BJournal%5D)">Quality control, analysis and secure sharing of Luminex(R) immunoassay data using the open source LabKey Server platform.</a> Bmc Bioinformatics. 14 (1), 145(2013).</li>
<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>
<li>Wood, R. A., Segall, N., Ahlstedt, S., Williams, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Accuracy+of+IgE+antibody+laboratory+results%5BTitle%5D)+AND+%22Annals+of+Allergy+Asthma+%26+Immunology%22%5BJournal%5D)">Accuracy of IgE antibody laboratory results.</a> Annals of Allergy Asthma & Immunology. 100 (2), 288-289 (2008).</li>
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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 symptoms. With regard to the reproducibility study done on three allergens, all of them showed good results, with an overall repetition rate of 88.89%, which means it met the maximum allowable error.
Using System 1 as reference, the current study evaluated the clinical diagnosis efficacy of the System 2. With a serum sIgE level of 0.35 IU/mL as cutoff2, a sample with sIgE &#62; 0.35 IU/mL implies that the patient is sensitive to the allergen, and the higher the titer, the better correlation to the patient&#8217;s symptoms18. The results show that the concordance rate of the three inhalant allergens were all more than 90%. In addition to the results for food allergens, which had a concordance of ~40.74%-72.39%, the total concordance was 81.75% (757/926). The kappa value of D. pteronyssinus, D. farina, and cat dander were 0.778, 0.663, and 0.860 (p &#60; 0.001), respectively. The kappa values for food allergens were all below 0.4. For the three major inhalant and food allergens, a significant correlation of quantitative results was seen between the two systems (rSpearman &#8776; 0.681-0.969, p &#60; 0.01).
It was noticed that while the rS coefficient for peanut was 0.969, the kappa value of the consistency evaluation index was only 0.349 (95% CI, 0.129-0.569). Such discrepancy might be due to the low prevalence of peanut sensitivity in the region and, hence, most of the recruited sera were negative for that specific IgE. Many studies have indicated that a significant discrepancy could be seen between sIgE titer and clinical symptoms of food allergens. The use of different assay systems for food-specific IgE determination could also create big variations19. This might be due to the fact it is not the raw ingested food which triggers the allergic symptoms, but the modified components generated during cooking or digestion. The use of different raw materials by different manufacturers to make the allergens can also contribute to the result discrepancy20.
The microfluidic cartridge is composed of five major parts: five storage tanks, five reagent delivery channels, five unidirectional pumps, a single reaction zone in which allergen extracts can be immobilized, and a waste tank to collect all reaction by-products. Based on the assay need, up to 40 allergen extracts can be dotted on the reaction zone. Controlled by the PC, the five unidirectional pumps guide and coordinate the flow of serum sample, washing buffer, blocking reagent, conjugates, and subtracts, to finish a two-step enzyme-linked immunosorbent assay. After the reaction is completed, the chemiluminescence reaction images are captured by a low-resolution cooled CCD camera and the signals are processed by the PC to establish the calibration curve and to calculate quantitative and semiquantitative sIgE results.
The current study shows that the two systems demonstrate good consistency. However, compared with the System 1, the System 2 is easier to use and has a lower demand for operator training. Since each microfluidic cartridge has its own quality control curve, the reliability of the system is greatly enhanced. Other advantages of the system include a light and small footprint, an expandable modular setup, and the ease with which it is connected to a PC for operation control. All these advantages greatly reduce the setup and running cost, and at the same time, they do not jeopardize the accuracy and speed requirements in daily clinical practice, which makes the system particularly suitable for allergy screening in primary care hospitals in China. Nevertheless, one major drawback of the microfluidic system is that it is not a fully automatic system, and frequent intervention is needed during the operation. It still cannot replace the systems that need to process a big number of samples daily.
Due to the lack of enough positive sera for certain allergens, this study did not cover all the 19 allergens available in the microfluidic cartridge, but just six common ones available in southern China. More study is needed to elaborate on whether the evaluation is also applicable to other allergens.

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)

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 for food allergens.
Repeatability of the microfluidic system 
Repeatability results for cat dander, dog dander, and cockroach, based on nine rounds of testing, were 32.98 &#177; 8.94, 1.61 &#177; 0.48, and 0.76 &#177; 0.18, respectively, and the consistency levels were 100% (9/9), 100% (9/9), and 67% (6/9). Distribution of the results is shown with the Levey-Jennings curve in Figure 2. All data are within the range of X &#177; 2 x SD, which is consistent with the maximum allowable clinical error14.
Comparison of two systems 
Qualitative results showed that cat dander had the highest concordance to System 1 (95.33%, 243/150). The lowest concordance was seen in shrimp (40.75%, 88/216). The total concordance among inhalant allergens ranged from 92.00% to 95.33%. For food allergens, the concordance range was 40.74%&#8211;72.39%. The highest sensitivity for inhalants was seen in Dermatophagoides farinae (93.94%), with a 100% specificity. Among food allergens, the highest sensitivity was seen in peanut (54.55%), with a specificity of 80.65%. Table 2 also shows that all the evaluation results for inhalant allergens were superior to food allergens. Since the AUC values showed a range from 0.613 to 0.984 and the AUC for the three inhalant allergens was greater than 0.950, it can be concluded that the System 2 has a high accuracy with reference to System 1.
Consistency analysis for the two systems showed that the kappa values for the three inhalants were between 0.727&#8211;0.876, with the highest value seen in cat dander as 0.876 (95% CI, 0.786&#8211;0.965). They were all better than the kappa values for food allergens which, in general, fell &#60;0.400. The lowest kappa value was 0.112 in shrimp (95% CI, 0.062&#8211;0.162) (Table 3). Spearman&#8217;s correlation analysis showed that the best correlation was seen in peanut and cat dander, with correlation coefficients as r = 0.942 (95% CI, 0.907&#8211;0.965; p &#60; 0.0001) and r = 0.927 (95% CI, 0.900&#8211;0.947; p &#60; 0.0001), respectively.
In Figure 3, a scatter plot is constructed with System 2&#8217;s results along the x-axis and System 1&#8217;s along the y-axis to show the distribution of the sIgE concentration results from the two systems for D. pteronyssinus, D. farina, cat dander, milk, shrimp, and peanut. For a concordance and discordance analysis, the allergens that showed &#177; 1 class difference were D. pteronyssinus (91.60%, 229 vs. 250), D. farinae (81.25%, 91 vs. 112), cat dander (98.00%, 147 vs. 150), milk (83.58%, 112 vs. 134), shrimp (59.72%, 129 vs. 216), and peanut (76.56%, 49 vs. 64). The combined total concordance rate was 81.75% (757 vs. 926).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59100/59100fig1.jpg" /> 
Figure 1: The positivity rates of the detection of 19 common allergens by the microfluidic assay. d1 -&#160;Dermatophagoides pteronyssinus, d2 - Dermatophagoides farinae, d201 - Blomia tropicalis, e1 - cat dander, e5 - dog dander, g2 - Bermuda grass, g6 - timothy grass, i6 - cockroaches, m3 - Aspergillus fumigatus, m5 - Candida albicans, w1 - ragweed, f1 - egg white, f2 - milk, f4 - wheat, f13 - peanut, f14 - soybean, f20 - almond, f23 - crab, and f24 - shrimp. <a href="https://www.jove.com/files/ftp_upload/59100/59100fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59100/59100fig2.jpg" /> 
Figure 2: Levey-Jennings graphs of the three allergens repeatedly detected by the microfluidic system. (A) Cat hair, (B) dog hair, and (C) cockroach were selected for repeatability evaluation. The black, green, yellow, and red lines represent the mean (X), the mean &#177; the standard deviation (X &#177; SD), the mean + the standard deviation times two (X &#177; 2SD), and the mean + the standard deviation times three (X &#177; 3SD) of multiple measurements, respectively. <a href="https://www.jove.com/files/ftp_upload/59100/59100fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59100/59100fig3.jpg" /> 
Figure 3: Scatter plots of six allergen sIgE concentrations measured by System 1. System 1 (Y-axis) and the System 2&#160;(X-axis). Each line in the plot represents class cutoffs (class 0: 0.35, class 1: 0.35&#8211;0.7, class 2: 0.7&#8211;3.5, class 3: 3.5&#8211;17.5, class 4: 17.5&#8211;50, class 5: 50&#8211;100, and class 6: &#62;100 IU/mL). Shaded boxes are concordant areas in the concentration class. <a href="https://www.jove.com/files/ftp_upload/59100/59100fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<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 1: Patient demographic characteristics. In total, 293 subjects were found who fulfilled the inclusion criteria, with an 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 rowspan="2"></td>
			<td rowspan="2">Sample size</td>
			<td colspan="2">CAP +</td>
			<td colspan="2">CAP -</td>
			<td rowspan="2">Total agreement</td>
			<td rowspan="2">SE</td>
			<td rowspan="2">SP</td>
			<td rowspan="2">PPV</td>
			<td rowspan="2">NPV</td>
			<td rowspan="2">AUC(95%, CI)</td>
		</tr>
		<tr>
			<td>BioIC +</td>
			<td>BioIC -</td>
			<td>BioIC +</td>
			<td>BioIC -</td>
		</tr>
		<tr>
			<td>d1</td>
			<td>250</td>
			<td>196</td>
			<td>20</td>
			<td>0</td>
			<td>34</td>
			<td>92.00%</td>
			<td>90.74%</td>
			<td>100.00%</td>
			<td>100.00%</td>
			<td>62.96%</td>
			<td>0.975(0.947 to 0.991)</td>
		</tr>
		<tr>
			<td>d2</td>
			<td>112</td>
			<td>93</td>
			<td>6</td>
			<td>0</td>
			<td>13</td>
			<td>94.64%</td>
			<td>93.94%</td>
			<td>100.00%</td>
			<td>100.00%</td>
			<td>68.42%</td>
			<td>0.984(0.941 to 0.999)</td>
		</tr>
		<tr>
			<td>e1</td>
			<td>150</td>
			<td>34</td>
			<td>5</td>
			<td>2</td>
			<td>109</td>
			<td>95.33%</td>
			<td>87.18%</td>
			<td>98.20%</td>
			<td>94.44%</td>
			<td>95.61%</td>
			<td>0.968(0.925 to 0.990)</td>
		</tr>
		<tr>
			<td>f2</td>
			<td>134</td>
			<td>16</td>
			<td>27</td>
			<td>10</td>
			<td>81</td>
			<td>72.39%</td>
			<td>37.21%</td>
			<td>89.01%</td>
			<td>61.54%</td>
			<td>75.00%</td>
			<td>0.744(0.661 to 0.815)</td>
		</tr>
		<tr>
			<td>f13</td>
			<td>64</td>
			<td>18</td>
			<td>15</td>
			<td>6</td>
			<td>25</td>
			<td>67.19%</td>
			<td>54.55%</td>
			<td>80.65%</td>
			<td>75.00%</td>
			<td>62.50%</td>
			<td>0.731(0.606 to 0.834)</td>
		</tr>
		<tr>
			<td>f24</td>
			<td>216</td>
			<td>36</td>
			<td>127</td>
			<td>1</td>
			<td>52</td>
			<td>40.74%</td>
			<td>22.09%</td>
			<td>98.11%</td>
			<td>97.30%</td>
			<td>29.05%</td>
			<td>0.613(0.545 to 0.678)</td>
		</tr>
		<tr>
			<td colspan="12">d1-Der. p1, d2- Der. f1, e1-Cat dander, f2-Milk, f13-Peanut, f24-Shrimp. CAP-ImmunoCAP, +-positive, &#8211;-negative, SE-sensitivity, SP-specificity, PPV-positive predictive value, NPV-negative predictive value, AUC-area under the ROC curve. For AUC values, the 95% interval value (95%, CI) is also shown in the table.</td>
		</tr>
	</tbody>
</table>
Table 2: Clinical performance between the two systems. d1 -&#160;D. pteronyssinus, d2 - D. farina, e1 - cat dander, f2 - milk, f13 - peanut, and f24 - shrimp. CAP - ImmunoCAP, + - positive, &#8211; - negative, SE - sensitivity, SP - specificity, PPV - positive predictive value, NPV - negative predictive value, AUC - area under the ROC curve. For the AUC values, the 95% interval value (95%, CI) is also shown in the table.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Kappa(95%,CI)</td>
			<td>Spearman&#39;rho(95%,CI)</td>
		</tr>
		<tr>
			<td>d1</td>
			<td>0.727(0.617 to 0.838)</td>
			<td>0.896(0.869 to 0.918)</td>
		</tr>
		<tr>
			<td>d2</td>
			<td>0.783(0.617 to 0.948)</td>
			<td>0.731(0.631 to 0.807)</td>
		</tr>
		<tr>
			<td>e1</td>
			<td>0.876(0.786 to 0.965)</td>
			<td>0.927(0.900 to 0.947)</td>
		</tr>
		<tr>
			<td>f2</td>
			<td>0.293(0.122 to 0.463)</td>
			<td>0.681(0.579 to 0.763)</td>
		</tr>
		<tr>
			<td>f13</td>
			<td>0.349(0.129 to 0.569)</td>
			<td>0.969(0.949 to 0.981)</td>
		</tr>
		<tr>
			<td>f24</td>
			<td>0.112(0.062 to 0.162)</td>
			<td>0.833(0.788 to 0.870)</td>
		</tr>
	</tbody>
</table>
Table 3: Correlation and agreement between the two systems. d1 -&#160;D. pteronyssinus, d2 - D. farina, e1 - cat dander, f2 - milk, f13 - peanut, and f24 - shrimp. For kappa and Spearman&#8217;s Rho values, the 95% interval value (95%, CI) is also shown in the table.

Watch this Scientific Journal Video about Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E sIgE at JoVE.com

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

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.

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. ...

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Universidad Científica del Sur

Research

JoVE Journal

Medicine

16.3K Views.  First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University. 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.

Video: Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E sIgE

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Bioengineering

Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) expression. These are acidic sugars that have a 9-carbon backbone and that cap vertebrate glycans on cell surfaces. Two of the major Sia forms in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). Humans cannot produce endogenous Neu5Gc due to the inactivation of the gene encoding cytidine 5&#39;monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). Foreign Neu5Gc is acquired by human cells through the dietary consumption of red meat and dairy and subsequently appears on diverse glycans on the cell surface, accumulating mostly on carcinomas. Consequently, humans have circulating anti-Neu5Gc antibodies that play diverse roles in cancer and other chronic inflammation-mediated diseases and that are becoming potential diagnostic and therapeutic targets. Here, we describe a high-throughput sialoglycan microarray assay to assess such anti-Neu5Gc antibodies in the human sera. Neu5Gc-containing glycans and their matched pairs of controls (Neu5Ac-containing glycans), each with a core primary amine, are covalently linked to epoxy-coated glass slides. We exemplify the printing of 56 slides in a 16-well format using a specific nano-printer capable of generating up to 896 arrays per print. Each slide can be used to screen 16 different human sera samples for the evaluation of anti-Neu5Gc antibody specificity, intensity, and diversity. The protocol describes the complexity of this robust tool and provides a basic guideline for those aiming to investigate the response to Neu5Gc dietary carbohydrate antigen in diverse clinical samples in an array format.

A sialoglycan microarray assay can be used to evaluate anti-Neu5Gc antibodies in human sera, making it a potential high-throughput diagnostic assay for cancer and other chronic inflammation-mediated human diseases.

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) ...

Behavior

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels ...

Immunology and Infection

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the individual immune condition determines the success in combating internal and external potential threat signals, but also the existence of a particular pattern of circulating anti-glycan antibodies (and their serological level variation) could be a significant marker of the onset and progression of certain pathological conditions. Here, we describe a Printed Glycan Array (PGA)-based methodology that offers the opportunity to measure hundreds of glycan targets with very high sensitivity; using a minimal amount of sample, which is a common restriction present when small animals (rats, mice, hamster, etc.) are used as models to address aspects of human diseases. As a representative example of this approach, we show the results obtained from the analysis of the repertoire of natural anti-glycan antibodies in BALB/c mice. We demonstrate that each BALB/c mouse involved in the study, despite being genetically identical and maintained under the same conditions, develops a particular pattern of natural anti-carbohydrate antibodies. This work claims to expand the use of PGA technology to investigate repertoire (specificities) and the levels of circulating anti-carbohydrates antibodies, both in health and during any pathological condition.

This work shows the potential of printed glycan array (PGA) technology for the analysis of circulating anti-carbohydrate antibodies in small animals.

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

An Assay to Detect Serum Anti-aquaporin-4 Immunoglobulin G

Source: Liu, C. et al., <a href="https://app.jove.com/v/59014">Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay.</a>&#160;J. Vis. Exp.&#160;(2019)
The video demonstrates a cell-based assay for detecting anti-aquaporin-4 immunoglobulin G using a biochip containing untransfected cells and transfected cells expressing aquaporin-4. This method has applications in diagnosing neuromyelitis optica spectrum disorders, confirmed by strong green fluorescence on the biochip.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

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.

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