We thank Yueming Su for his assistance in preparing the figure. This study was funded by the Guangdong Foundation of Science and Technology (Project No.: 2014A020212352), the&#160;Guangzhou Education Bureau (1201630044; 1201630393) and the National Natural Science Foundation of China (NSFC 81572063; NSFC 81601394).

The study protocol, including human serum sample use, was approved by the Ethics Committee, First Affiliated Hospital of Guangzhou Medical University. All participants offered written informed consent, either independently or via their parents (in the case of children). The protocol has been registered online (Chinese Clinical Trial Registry, Reg No.: ChiCTR-DCC-13004003, refer to http://www.chictr.org/cn/).
1. Identification of the Study Population
NOTE: The...

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The authors have nothing to disclose.

Fully-automated immunoassay analyzers may offer satisfactory consistency between regions, laboratories, and institutions and over time. Such an automatic procedure has been found to substantially support bench research and clinical studies, enable the standardized detection of many allergens and/or allergen components, and identify probable allergen cross-reactivity that would not have been readily revealed before the advent of CRDs. Moreover, the whole testing procedure can be completed with strict quality control, allo...

Grass pollen accounts for nearly 50% of immunoglobulin E (IgE)-related allergies and, because of its airborne nature, imposes a global risk of allergic airway diseases, particularly asthma and allergic rhinitis1,2,3,4. Prevention against grass pollen allergies in a region relies on information about the local distribution of the pollen allergens. Unfortunately, the spectrum of the sensitizing allergens varies from place to place&#8212;an observation generally known as regional specificity.
In China, a vast East-Asian territory, regional specificity of grass pollen sensitization is further complicated by limited data currently available from the southern part of this country over the past decades5. Such a paucity in data has led to a difficulty in showing the overall grass pollen sensitization in this large country6. Additionally, the very limited data on Chinese southerners were largely obtained from self-report questionnaires, skin prick tests (rather than laboratory serum tests), total IgE measurements (rather than allergen-specific IgE (sIgE)), and sIgE detection to allergens (rather than to allergen component molecules).
The self-reporting of allergic symptoms can be unreliable when specific allergens are concerned. Skin prick tests, although widely accessible, involve the use of non-standardized, crude allergen extracts, which themselves may contain other sensitizing components that confound the outcome interpretation. The reliability of the skin prick test also critically depends on skillful manipulation by allergists or specialist nurses, recent histamine use, and patient compliance (sometimes very difficult for young children). Total IgE is useful for screening an allergy but does not sensibly determine the offending allergens in a patient. Moreover, almost all allergens contain a number of allergen component molecules. Sensitization to the major components (i.e., Der p 1 and Der p 2 from house dust mites) may suggest a &#34;genuine&#34; allergy (allergy to a specific allergen, rather than false positivity due to cross-reactions with other allergens), but sensitization to certain ones (i.e., the cross-reactive Der p 10, a tropomyosin) may not7.
Before component-resolved diagnostics (CRD) was introduced, these disappointing setbacks impeded allergen studies in China. CRD precisely detects the components of an allergen by using recombinant or purified allergen molecules, thereby making the measurement easily quantifiable and characterizable8. In past decades, allergy studies frequently employed crude extracts of allergens that inevitably contain a number of irrelevant components. Many of these components are inert, but occasionally, some could lead to confounding positive reactions. The use of recombinant or purified allergen molecules in CRD can circumvent this and therefore provide better test accuracy. When processed in microarrays, a CRD approach rapidly screens or identifies sensitization to hundreds of allergen molecules in individual subjects. A well-designed CRD study on grass pollen sensitization in southern China is currently lacking. Ideally, such a study should include a local population of considerable geographic coverage, account for grass pollens commonly reported in China, and address the technical aspects mentioned above. To reach a practical level of clinical relevance and to facilitate the recruitment of serum samples, early investigation on sensitization to grass pollens as inhalant allergens may as well be focused on patients with allergic respiratory disorders, rather than on the general population.
This work describes a method used to test serum sIgE reactivity to Bermuda grass, Timothy grass, and Humulus scandens in patients from Greater Guangzhou (southern China&#39;s largest city and its outskirts) with allergic rhinitis and/or asthma using the CRD approach5. These findings on sensitization to these subtropical and temperate grass pollens in southern China add important evidence to that currently in the literature.

<table border="0" cellpadding="0" cellspacing="0" width="661">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">ImmunoCAP 1000 Instrument</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">12-3800-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Phadia Information Data Manager Software Package</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">12-3801-11</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Software Package, ImmunoCAP 1000</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">12-3801-12</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g2 
			(Bermuda grass)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-4131-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g6 
			(Timothy grass)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-4100-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">w22 
			(Japanese Hop)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-4452-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g216 
			(nCyn d 1)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-4972-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g205 
			(rPhl p 1)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5234-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g208 
			(nPhl p 4)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5288-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g215 
			(rPhl p 5b)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5338-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g209 
			(rPhl p 6)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5289-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g210 
			(rPhl p 7)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5290-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g211 
			(rPhl p 11)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5291-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g212 
			(rPhl p 12)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5292-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:157px;">o214 
			Cross-reacting carbohydrate determinants (CCDs)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5339-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Specific IgE Conjugate 400</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9310-08</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Specific IgE Calibrator Strip</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9459-08</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Specific IgE Curve Control Strip</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9312-08</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Specific IgE Anti-IgE ImmunoCAP</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-4417-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">ImmunoCAP Specific IgE Negative Control</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9445-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">ImmunoCAP Specific IgE Control L</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9528-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">ImmunoCAP Specific IgE Control M</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9529-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">ImmunoCAP Specific IgE Control H</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9530-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Development Solution</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9439-08</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Stop Solution</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">34-2271-58</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Washing Solution</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9202-08</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">FluoroC</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9264-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Maintenance Solution Kit</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9476-01</td>
			<td style="width:177px;"></td>
		</tr>
	</tbody>
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a component-resolved diagnostic approach for a study on grass pollen allergens in chinese southerners with allergic rhinitis and/or asthma

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

The fully-automated immunoassay analyzer used in this study has a solid-phase structure covered with covalently coupling allergens or allergen components that react with the molecules of interest (sIgE) in serum specimens from the patient. With an automatic program, the analyzer washes off all non-specific IgEs with 150 &#181;L of rinse solution and repeats this 5 times. Then it adds enzyme-labeled anti-IgE antibodies (50 &#181;L) to generate a complex. The enzyme-labeled anti-IgE antibod...

Watch this Scientific Journal Video about A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma at JoVE.com

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

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

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

The overall goal of this experiment is to investigate the sensitization to various grass pollen allergen components in southern China. This med can help answer key questions in the energy field about how individuals become sensitized to grass pollens. Main advantage of this technique is that the use of recombinant or purify allergen molecules.Can also convey the occurrence of confounding rashes, providing a better test accuracy. The implication of this technique is then to thwart the diagnosis of allergy, rhinitis, and asthma. As many of these patients are sensitized to common detectable allergens.Though this method can provide insight into allergy airway disease, it can also be a to other type of allergies such as food allergies. We first had the idea for this method when you found or CID for Chinese Demonstrating the procedure will be Nili Wei, a from my lab. After retrieving the serum samples of eligible patients from the biobank.Turn on the fully automated immunoassay analyzer and the built in information data management computer. With the primary power on, switch the instrument to the system power and wait three minutes for the built in software to start. Then add 140 microliters of serum to one vile per allergen and or allergen component test.And label each vile of serum with an identification number unique to each patient. From the information data management interface, open the request list window to confirm that serum IGE is the test method of choice. Then load the sample tubes into the sample racks.And the barcode labeled quality control tubes into the quality control racks. In the assay processing menu, select, load reagents to complete the loading of the sample and quality control racks, development solution, conjugate, calibraters, carrier, pipette tips, stop solution, and washing solution, according to the load list. Click load rinse solution and load washing solution.When all of the materials have been loaded select load and start and click okay. At the end of the measurement, the results will appear in the information data management interface. Select the data to be analyzed and click menu, approve, and save as to export the serum IGE measurement results as a spreadsheet file.Then import the spreadsheet file into an appropriate statistical software program. And use a specific module for Spearman's coefficient to analyze the correlations in sensitization between the grass pollens and the pollen components. Of the 258 respiratory allergy patients studied in this experiment, 22.5 percent tested positive to Bermuda grass, 13.6 percent to Timothy grass, and seven percent to Hummulus scandens.Interestingly, patients with allergic rhinits and or asthma demonstrated a remarkably higher rate of sensitization to Bermuda grass than did patients with other allergies. Among the patients with Bermuda grass class one serum IGE reactivity, the likelihood of positive tests for Cyn d 1, Timothy grass, or Humulus scandens allergens or cross reactive carbohydrate determinants was below 30 percent. In contrast, patients with class four Bermuda grass SIGE reactivity exhibited a 67 to 100 percent chance of concomitant positivity to these grass components.Among the 35 Timothy grass positive patients, the most frequently detected allergen component was Phl p four followed by Phl p one. Three patients each were positive for any one of Phl p five, six, seven, 11, or 12. Serum Phl P four SIGE levels correlated strongly with total Timothy grass allergen and cross reactive carbohydrate determinants SIGE levels.41.4 percent of Bermuda grass positive patients and 68.6 percent of Timothy grass sensitized patients were also SIGE positive to cross reactive carbohydrate determinants. With Spearman's rank correlation analyses demonstrating a significant association in sensitization between cross reactive carbohydrate determinants and other cross allergens. Following this procedure are the methods like the of immunoassay analyzers can be performed to answer additional questions about sensitization to grass pollen allergy in the center or nation-wide study.While attending this procedure, it's important to remember to insure the appropriate and correct calibration has been growed in the system. And this is essential for accurate test results. After watching this video, you should have a good understanding of how to investigate patient sensitization to components of grass pollen allergies.Don't forget that working with human serum samples can be extremely dangerous. And appropriate precautions against pathogen infections should always be taken while performing this procedure.

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JoVE Journal

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9.9K visualizações.  State Key Laboratory of Respiratory Disease. The overall goal of this experiment is to investigate the sensitization to various grass pollen allergen components in southern China. This med can help answer key questions in the energy field about how individuals become sensitized to grass pollens. Main advantage of this technique is that the use of recombinant or purify allergen molecules.Can also convey the occurrence of confounding rashes, providing a better test accuracy. The implication of this technique is then to thwart the d...