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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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)

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 tha...

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. ...

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

Research

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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

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

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

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 ...

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 ...

Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay (CBA) is a widely used method to detect anti-AQP4 IgG in human serum with high sensitivity and specificity. Briefly, serum anti-AQP4 IgG is captured by AQP4-transfected cell that is fixed on the biochip then detected by a fluorescein-labelled secondary antibody. Fluorescence microscopy is utilized to visualize the fluorescence, and the intensity of fluorescence is evaluated by at least two experienced clinicians. A final diagnosis of NMOSD can be made based on the combination of anti-AQP4 IgG detection results, clinical manifestations, and neuroradiological findings. According to previous studies, CBA is more sensitive and specific than other anti-AQP4 IgG detection methods, and it can be applied to both clinical diagnosis and studies of NMOSD. The method has limitations; for example, an international scale to evaluate serum anti-AQP4 IgG titers is still lacking. Here, a detailed protocol for human serum anti-AQP4 IgG detection using CBA is described.

Cell-based assay is a widely used method to detect serum anti-aquaporin-4 immunoglobulin G. This method could be applied to clinical diagnosis and scientific researches of neuromyelitis optical spectrum disorders.

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay ...

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 ...

Humanized Mediator Enzyme Release Assay: An Immunochemical Method to Screen Allergic Potential of Test Antigen by Monitoring Basophil Degranulation Response In Vitro

Centrifuge the pre-incubated Ag8/serum suspension, and transfer 50 microliters of the centrifuged Ag8/serum suspension to each well containing humanized RBL cells without disturbing the Ag8 cell pellet.
Use sensitized, unstimulated, cells without antigen, as no-antigen control for indication of the bottom signal plateau or background, and do not sensitize the background and maximum lysis control wells. Cover the plate with the lid, and incubate overnight at 37 degrees Celsius and 5% to 7% carbon dioxide.
Aspirate the sera-containing cell medium. Invert and tap the plate on the absorbent paper to empty the plate for washing humanized RBL cells. Wash the cells three times by adding 200 microliters of Tyrode&#39;s buffer per well, and incubate for approximately 30 seconds per wash, for the first two washes. After adding Tyrode&#39;s buffer for the third time, aspirate the buffer and leave the solution in the wells, until ready to add the antigen dilution.
Transfer 100 microliters of antigen solution to each well containing the pre-sensitized, humanized RBL cells, but do not stimulate the maximum lysis and non-sensitized background cells with antigen.
Add 100 microliters of Tyrode&#39;s buffer in the maximum lysis control wells, non-sensitized background control wells, and sensitized no-antigen wells. Incubate the cells for one hour at 37 degrees Celsius and 5% to 7% carbon dioxide.
Treat the maximum lysis control wells with 10 microliters of 10% Triton X-100 per well, and mix properly to lyse the cells completely for 100% release of beta-hexosaminidase. Add 50 microliters of substrate solution into a new non-binding 96-well plate.
Transfer 50 microliters of supernatant from the wells of humanized RBL cells-containing plate into the new plate containing the substrate solution, and incubate the plate for one hour at 37 degrees Celsius to allow conversion of the fluorogenic substrate.
Add 100 microliters of stopping solution per well, and measure the fluorescence as described in the text manuscript.

Biological Techniques

Assay Techniques

Cell-based assays

EoE Sub Articles

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

First, bring the patient serum samples, the biochip slide, the PBS powder, and the Tween-20 from the refrigerated cell-based assay kit to the room temperature. Then, prepare more than 200 milliliters of PBS wash buffer containing 0.2% Tween-20 for each biochip slide.
Use the wash buffer to make 10-fold serial dilutions of each serum sample. Prepare positive and negative controls, according to the manufacturer&#39;s instructions. Add 30 microliters of 10-fold serial dilutions of the sample, the positive control, and the negative control to five individual reaction fields on the reagent tray.
Hold the biochip slide by its sides and remove its protective cover without touching its reaction fields. Put the slide on top of the reagent tray, and incubate the setup at room temperature for 30 minutes. Gently rinse the biochip slide with the wash buffer. Then, immerse it in a 500-milliliter beaker filled with 100 milliliters of the wash buffer, and leave it at the room temperature for 10 minutes.
Take out the biochip slide from the wash buffer. Use a paper towel to carefully wipe away any remaining buffer from outside of the reaction fields. Let the slide air-dry for 1 to 2 minutes.
In the dark room, add 25 microliters of fluorescent secondary antibody to each reaction field of the reagent tray. Put the slide on top of the reagent tray, and incubate the setup at room temperature for 30 minutes. Gently rinse the biochip slide with the wash buffer.
Then, immerse it in a 500-milliliter beaker filled with 100 milliliters of the fresh wash buffer, and leave it at room temperature for 10 minutes. Take out the biochip slide from the wash buffer. Use a paper towel to carefully wipe away any remaining buffer from outside of the reaction fields.
Let the slide air-dry for 1 to 2 minutes. Carefully add 1 drop of the embedding medium to each reaction field on the biochip slide. Finally, seal the biochip slide with a coverslip glass, while avoiding air bubbles, and proceed with imaging.