Name: immunoglobulin g n-glycan analysis by ultra-performance liquid chromatography
Uploaded: 2020-01-18T12:00:00
Description: Watch this Scientific Journal Video about Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography at JoVE.com

This work was supported by grants from National Natural Science Foundation of China (81673247 &#38; 81872682) and Australian-China Collaborative Grant (NH &#38; MRC - APP1112767 -NSFC 81561128020).

All the subjects included in the protocol have been approved by the Ethics Committee of the Capital Medical University, Beijing, China12. Written informed consent was obtained from each subject at the beginning of the study.
1. IgG isolation
<ol>
	<li>Prepare the chemicals including binding buffer (phosphate buffered saline, PBS): 1x PBS (pH = 7.4), neutralizing buffer: 10x PBS (pH = 6.6-6.8), eluent: 0.1 M formic acid (pH = 2.5), neutralizing solution for eluent: 1 M...

<ol>
	<li>Kolarich, D., Lepenies, B., Seeberger, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycomics,+glycoproteomics+and+the+immune+system.">Glycomics, glycoproteomics and the immune system.</a> Current Opinion in Chemical Biology. 16, 214 (2012).</li><li>Cummings, R., Pierce, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Challenge+and+Promise+of+Glycomics.">The Challenge and Promise of Glycomics.</a> Chemistry Biology. 21 (1), (2014).</li><li>Shade, K. T. C., Anthony, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+Glycosylation+and+Inflammation.">Antibody Glycosylation and Inflammation.</a> Antibodies. 2, 392 (2013).</li><li>Gudelj, I., Lauc, G., Pezer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoglobulin+G+glycosylation+in+aging+and+diseases.">Immunoglobulin G glycosylation in aging and diseases.</a> Cellular Immunology. 333, 65 (2018).</li><li>Huffman, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+performance+of+four+methods+for+high-throughput+glycosylation+analysis+of+immunoglobulin+G+in+genetic+and+epidemiological+research.">Comparative performance of four methods for high-throughput glycosylation analysis of immunoglobulin G in genetic and epidemiological research.</a> Molecular Cellular Proteomics. 13, 1598 (2014).</li><li>Stockmann, H., Adamczyk, B., Hayes, J., Rudd, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated,+high-throughput+IgG-antibody+glycoprofiling+platform.">Automated, high-throughput IgG-antibody glycoprofiling platform.</a> Analytical Chemistry. 85, 8841 (2013).</li><li>Kristic, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycans+are+a+novel+biomarker+of+chronological+and+biological+ages.">Glycans are a novel biomarker of chronological and biological ages.</a> Journals of Gerontology. Series A, Biological Sciences Medical Sciences. 69, 779 (2014).</li><li>Nikolac, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+association+between+galactosylation+of+immunoglobulin+G+and+body+mass+index.">The association between galactosylation of immunoglobulin G and body mass index.</a> Progress in Neuropsychopharmacology Biological Psychiatry. 48, 20 (2014).</li><li>Liu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+changes+of+immunoglobulin+G+N-glycosylation+in+blood+lipids+and+dyslipidaemia.">The changes of immunoglobulin G N-glycosylation in blood lipids and dyslipidaemia.</a> Journal of Translational Medicine. 16, 235 (2018).</li><li>Lemmers, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgG+glycan+patterns+are+associated+with+type+2+diabetes+in+independent+European+populations.">IgG glycan patterns are associated with type 2 diabetes in independent European populations.</a> Biochimica Biophysica Acta General Subjects. 1861, 2240 (2017).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Association+Between+Glycosylation+of+Immunoglobulin+G+and+Hypertension:+A+Multiple+Ethnic+Cross-Sectional+Study.">The Association Between Glycosylation of Immunoglobulin G and Hypertension: A Multiple Ethnic Cross-Sectional Study.</a> Medicine (Baltimore). 95, e3379 (2016).</li><li>Liu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+stroke+is+associated+with+the+pro-inflammatory+potential+of+N-glycosylated+immunoglobulin+G.">Ischemic stroke is associated with the pro-inflammatory potential of N-glycosylated immunoglobulin G.</a> Journal of Neuroinflammation. 15, 123 (2018).</li><li>Russell, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+N-glycosylation+of+immunoglobulin+G+as+a+novel+biomarker+of+Parkinson's+disease.">The N-glycosylation of immunoglobulin G as a novel biomarker of Parkinson's disease.</a> GLYCOBIOLOGY. 27, 501 (2017).</li><li>Bones, J., Mittermayr, S., O'Donoghue, N., Guttman, A., Rudd, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra+performance+liquid+chromatographic+profiling+of+serum+N-glycans+for+fast+and+efficient+identification+of+cancer+associated+alterations+in+glycosylation.">Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associated alterations in glycosylation.</a> Analytical Chemistry. 82, 10208 (2010).</li><li>Pucic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+isolation+and+glycosylation+analysis+of+IgG-variability+and+heritability+of+the+IgG+glycome+in+three+isolated+human+populations.">High throughput isolation and glycosylation analysis of IgG-variability and heritability of the IgG glycome in three isolated human populations.</a> Molecular Cellular Proteomics. 10, M111 (2011).</li><li>Berruex, L. G., Freitag, R., Tennikova, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+antibody+binding+to+immobilized+group+specific+affinity+ligands+in+high+performance+monolith+affinity+chromatography.">Comparison of antibody binding to immobilized group specific affinity ligands in high performance monolith affinity chromatography.</a> Journal of Pharmaceutical and Biomedical Analysis. 24, 95 (2000).</li><li>Ren, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+IgG+galactosylation+as+a+promising+biomarker+for+cancer+screening+in+multiple+cancer+types.">Distribution of IgG galactosylation as a promising biomarker for cancer screening in multiple cancer types.</a> Cell Research. 26, 963 (2016).</li></ol>

UPLC serves as a relative quantitative analysis method5,15. The results indicate that UPLC is a stable detection technology with good reproducibility and relative quantitative accuracy. The amount of glycans in each peak is expressed as a percentage of the total integrated area using UPLC, which is the relative value. The relative quantification improves the comparability of test samples. In addition, 96 well protein G plates are used to purify IgG with 96 sample...

N-glycosylation of human proteins is a common and essential post-translational modification1 and may help predict the occurrence and development of diseases relatively accurately. Due to the complexity of its structure, it is expected that there are more than 5,000 glycan structures, providing great potential as diagnostic and predictive biomarkers for diseases2. N-glycans attached to immunoglobulin G (IgG) have been shown to be essential for IgG&#39;s function, and IgG N-glycosylation participates in the balance between the pro- and anti-inflammatory systems3. Differential IgG N-glycosylation is involved in disease development and progression, representing both a predisposition and functional mechanism involved in disease pathology. The inflammatory role of IgG N-glycosylation has been associated with aging, inflammatory diseases, autoimmune diseases, and cancer4.
With the development of detection technology, the following methods are most widely used in high throughput glycomics: hydrophilic interaction chromatography (HILIC) ultra-performance liquid chromatography with fluorescence detection (UPLC-FLR), multiplex capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and liquid chromatography electrospray mass spectrometry (LC-ESI-MS). These methods have overcome previous shortcomings of low flux, unstable results, and poor sensitivity specificity5,6.
UPLC is widely used to explore the association between IgG N-glycosylation and certain diseases (i.e., ageing7, obesity8, dyslipidemia9, type II diabetes10, hypertension11, ischemic stroke12, and Parkinson&#39;s disease13). Compared to the other three abovementioned methods, UPLC has the following advantages5,14. First, it provides a relative quantitative analysis method, and the data analysis that involves total area normalization improves the comparability of each sample. Second, the cost of equipment and required expertise are relatively low, which makes it easier to implement and transform glycosylation biomarkers into clinical applications. Presented here is an introduction to UPLC so it can be more widely used.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-aminobenzamide, 2-AB</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well collection plate</td>
			<td>AXYGEN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well filter plate</td>
			<td>Pol</td>
			<td>0.45 um GHP</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well monolithic plate</td>
			<td>BIA Separations</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate rotor</td>
			<td>Eppendorf Co., Ltd, Germany</td>
			<td>T_1087461900</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Huihai Keyi Technology Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Shenggong Biological Engineering Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium formate</td>
			<td>Beijing Minruida Technology Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Constant shaking incubator/rocker</td>
			<td>Zhicheng analytical instrument manufacturing co., Ltd, China</td>
			<td>ZWY-10313</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran Calibration Ladder/Glycopeptide column</td>
			<td>Watts technology Co., Ltd, China</td>
			<td>BEH column</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium phosphate</td>
			<td>Shenggong Biological Engineering Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electric ovens</td>
			<td>Tester instruments Co., Ltd</td>
			<td>202-2AB</td>
			<td></td>
		</tr>
		<tr>
			<td>Empower 3.0</td>
			<td>Waters technology Co., Ltd, America</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Huihai Keyi Technology Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlycoProfile 2-AB Labeling kit</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Junrui Biotechnology Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-speed centrifuge</td>
			<td>Eppendorf Co., Ltd, Germany</td>
			<td>5430</td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low temperature centrifuge</td>
			<td>Eppendorf Co., Ltd, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low temperature refrigerator</td>
			<td>Qingdao Haier Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Manifold 96-well plate</td>
			<td>Watts technology Co., Ltd, China</td>
			<td>186001831</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Huihai Keyi Technology Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q pure water meter</td>
			<td>Millipore Co., Ltd, America</td>
			<td>Advantage A10</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Shenggong Biological Engineering Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PH tester</td>
			<td>Sartorius Co., Ltd, Germany</td>
			<td>PB-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, PBS</td>
			<td>Shenggong Biological Engineering Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf Co., Ltd, Germany</td>
			<td>4672100, 0.5-10&#956;l &#38; 10-100&#956;l &#38; 20-200&#956;l &#38; 1000&#956;l</td>
			<td></td>
		</tr>
		<tr>
			<td>PNGase F enzyme</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Shenggong Biological Engineering Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Propan-2-ol</td>
			<td>Huihai Keyi Technology Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Shenggong Biological Engineering Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium cyanoborohydride (NaBH3CN)</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Shanghai Yuanxi instrument Co., Ltd</td>
			<td>B-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer liquid gun</td>
			<td>Smer Fell Science and Technology Co., Ltd, China</td>
			<td>4672100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Amresco, America</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-low temperature refrigerator</td>
			<td>Thermo Co., Ltd, America</td>
			<td>MLT-1386-3-V; MDF-382E</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-performance liquid chromatography</td>
			<td>Watts technology Co., Ltd, China</td>
			<td>Acquity MLtraPerformance LC</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Pump</td>
			<td>Watts technology Co., Ltd, China</td>
			<td>725000604</td>
			<td></td>
		</tr>
		<tr>
			<td>Volatilizing machine/Dryer</td>
			<td>Eppendorf Co., Ltd, Germany</td>
			<td>T_1087461900</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Changzhou Enpei instrument Co., Ltd, China</td>
			<td>NP-30S</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-bath</td>
			<td>Tester instruments Co., Ltd</td>
			<td>DK-98-IIA</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing balance</td>
			<td>Shanghai Jingke Scientific Instrument Co., Ltd.</td>
			<td>MP200B</td>
			<td></td>
		</tr>
	</tbody>
</table>

immunoglobulin g n-glycan analysis by ultra-performance liquid chromatography

Glycomics is a new subspecialty in omics system research that offers significant potential in discovering next-generation biomarkers for disease susceptibility, drug target discovery, and precision medicine. Alternative IgG N-glycans have been reported in several common chronic diseases and suggested to have great potential in clinical applications (i.e., biomarkers for diagnosis and prediction of diseases). IgG N-glycans are widely characterized using the method of hydrophilic interaction chromatography (HILIC) ultra-performance liquid chromatography (UPLC). UPLC is a stable detection technology with good reproducibility and high relative quantitative accuracy. In addition, the structure of IgG N-glycan is clearly separated, and glycan composition and relative abundance in plasma are characterized.

As shown in Figure 1, IgG N-glycans were analyzed into 24 initial IgG glycan peaks (GPs) based on peak position and retention time. The N-glycan structures are available through mass spectrometry detection according to a previous study (Table 1)15. To ensure that the results were comparable, total area normalization was applied, in which the amount of glycans in each peak was expressed as a percentage...

Watch this Scientific Journal Video about Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography at JoVE.com

Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography

Immunoglobulin G (IgG) N-glycan is characterized using hydrophilic interaction chromatography UPLC. In addition, the structure of IgG N-glycan is clearly separated. Presented here is an introduction to this experimental method so that it can be widely used in research settings.

Glycomics is a new subspecialty in omics system research that offers significant potential in discovering next-generation biomarkers for disease ...

Ultra-performance liquid chromatography is established technology for accurate analysis of IgG n-glycan Due to its sensitivity seems to be and it has ability to provide a specific information of glycan This method is easy to use and has number of different relatively low cost of increments, high reproducibility The ability to glycan isomers. The measurements of protein in plasma could be attractive. in medicine.also associated with IgG in glycose base and Such and biologic containing the population. You know that of impure state is used to test IgG in glycanes. Actually, it can test n-glycanes of protein.This is needed in a cross-bound protein of two pure state can be applied to the Protein The OR experimental process is relatively simple while the experimental process need to be standardized. The end-to-end experimental process takes three days to operate, and the experimental steps need to be memorized in order to master the experimental skills. To prepare the samples, thaw the frozen plasma sample.Then, centrifuge at 80 times G for 10 minutes. Leave the protein G monolithic plate at room temperature for 30 minutes. Transfer 100 micrometers of sample into each well of the two-milliliter collection plate.Add ultra pure water as one control sample. Dilute the samples with one X PBS by one to seven volume by volume ratio. Clean a 0.45 micron hydrophilic polypropolene filter plate with 200 microliters of ultra pure water.Repeat the cleaning two additional times. Transfer the diluted samples into the filter plate, and filter the samples into the collective plate using a vacuum pump with pressure at 266.6 to 399.9 Pascals. To prepare the protein G monolithic plates, first discard the storage buffer.Clean the monolithic plates with two milliliters of ultra pure water, two milliliters of one X PBS, one milliliter of 0.17 molar formic acid, two milliliters of 10 X PBS, and two milliliters of one X PBS sequentially. Remove the following liquid using a vacuum pump. Using a multichannel pipet, transfer the filtered samples to the protein G monolithic plate for IgG binding and cleaning.Then, add two milliliters of one X PBS to clean the monolithic plates. Remove the PBS using a vacuum pump, and repeat the cleaning two additional times. Illute IgG with one milliliter of 0.17 molar formic acid, and filter the samples into the collection plate by vacuum pump.Then, add 170 microliters of one molar ammonium bicarbonate into the collection plate. Detect the IgG concentration using an absorption spectropetometer at the optimal wavelength of 280 nanometers. Place the extracted IgG in an oven to dry at 60 degrees Celsius for three hours.Determine the amount to be observed according to its concentration as described in the manuscript, and preserve it in minus 80 degrees Celsius. Gather the dried IgG, and store chemicals including 1.33%SDS, four percent IgePal and 5%PBS at room temperature. Prepare indoglycosidase F enzyme by diluting 250 units of enzyme with 250 microliters of ultra pure water.To denature the IgG, add 30 microliters of 1.33%SDS and mix by vortexing. Transfer the sample into a 65 degree Celsius oven for 10 minutes. Then, remove it from the oven, and let it cool for 15 minutes.Add 10 microliters of four percent IgePal into the same sample, and place it on a shaking incubator for five minutes. Add 20 microliters of five X PBS and 30 to 35 microliters of 0.1 molar sodium hydroxide to regulate the pH to 8.0, and mix by vortexing. Add four microliters of endoglycosidase F enzyme, and mix by vortexing.Then, incubate for 18 to 20 hours in a 37 degrees Celsius water bath. The next day, place the released gylcans in an oven at 60 degrees Celsius to dry for two and a half to three hours. Save the release glycans at minus 80 degrees Celsius until further measurement.Prepare the two amino benzimide labeling reagent with 24.5 microliters of DMSO, 10.5 microliters of acetic acid, 0.7 milligrams of two amino benzimide, and six milligrams of sodium cyanoborohydride. Next, in a dark room, label the glycans by adding 35 microliters of two amino benzimide labeling reagent. Transfer the labeled glcans to an oscillator for five minutes, and then, transfer it to the oven for three hours at 65 degrees Celsius.After that, transfer it to room temperature for 30 minutes. The IgG n-glycase, we'll label it with in order to be detected by the fluorescent detect of impure state. Pretreat a 0.2 micron GHP filter plate with 200 microliters of 70%ethanol, 200 microliters of ultra pure water, and 200 microliters of 96%aceto nitrile at four degrees Celsius.Then, remove the waste using a vacuum pump. To purify the two amino benzimide labeled glycans, add 700 microliters of 100%acetonitrile to the sample, and transfer it to a shaking incubator for five minutes. Centrifuge at 134 times G for five minutes at four degrees Celsius.Transfer the supernatent to a 0.2 microns GHP filter plate for two minutes, and remove the filtrate using the vacuum. Wash the two amino benzimide labeled glycan with 200 microliters of 96%acetonitrile at four degrees Celsius, and remove the filtrate using a vacuum pump five to six times. Illute two amino benzimide labeled glycan with 100 microliters of ultra pure water three times.Transfer the two amino benzimide labeled glycan into an oven to dry at 60 degrees Celsius for three and a half hours. Save the labeled n-glycans at minus 80 degrees Celsius until further measurement. First, open the software to control the mobile phases.Wash UPLC instruments with 50%solvent B and 50%solvent C at the flow rate of 0.2 milliliters per minute for 30 minutes. Then, wash with 25%solvent A and 75%solvent B at a flow rate of 0.2 milliliters per minute for 20 minutes. Then, add a flow rate of 0.4 milliliters per minute.Dissolve the labeled n-glycans with 25 microliters of a mixture of 100%acetonitrile and ultra pure water at a two to one volume by volume ratio. Then, centrifuge at 134 times G for five minutes at four degrees Celsius, and use a dry pipe to load 10 microliters of the supernatent into the UPLC instruments. Separate the labeled n-glycans at flow rate of 0.4 milliliters per minute with a linear gradient of 75%to 62%acetonitrile for 25 minutes.Then, perform an analytic run by Dexteran calibration ladder glycopeptide column on a UPLC at 60 degrees Celsius. Detect n-glycan fluorescents at X citation and emission wavelengths of 330 nanometers and 420 nanometers, respectively. As shown in this figure, IgG n-glycans were analyzed into 24 initial IgG glycan peaks based on peak position and retention time.The n-glycan structures are available in 24 types through mass spectrometry detection. To asses the repeatability and stability of the method, the standard sample was tested in parallel six times. The glycan peaks with relatively small proportions showed high measurement errors of more than 10%of coefficient of variation.In the piece and important to forming to an established of IgG n-glycan structure, especially for establishing terminal Therefore, instead of 3.3 IgG n-glycase found in is critical in this the importance of IgG. Glycans from are known to be different. With the developments in glycan prototomics, combined in glycans to explore information to this such that It can be used as a potential for today's diagnosis.After this technique development, it provides a new way to explore the

immunoglobulin-g-n-glycan-analysis-ultra-performance-liquid

Research

JoVE Journal

Medicine

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Single-stage Dynamic Reanimation of the Smile in Irreversible Facial Paralysis by Free Functional Muscle Transfer

Unilateral facial paralysis is a common disease that is associated with significant functional, aesthetic and psychological issues. Though idiopathic facial paralysis (Bell&#8217;s palsy) is the most common diagnosis, patients can also present with a history of physical trauma, infectious disease, tumor, or iatrogenic facial paralysis. Early repair within one year of injury can be achieved by direct nerve repair, cross-face nerve grafting or regional nerve transfer. It is due to muscle atrophy that in long lasting facial paralysis complex reconstructive methods have to be applied. Instead of one single procedure, different surgical approaches have to be considered to alleviate the various components of the paralysis.
The reconstruction of a spontaneous dynamic smile with a symmetric resting tone is a crucial factor to overcome the functional deficits and the social handicap that are associated with facial paralysis. Although numerous surgical techniques have been described, a two-stage approach with an initial cross-facial nerve grafting followed by a free functional muscle transfer is most frequently applied. In selected patients however, a single-stage reconstruction using the motor nerve to the masseter as donor nerve is superior to a two-stage repair. The gracilis muscle is most commonly used for reconstruction, as it presents with a constant anatomy, a simple dissection and minimal donor site morbidity.
Here we demonstrate the pre-operative work-up, the post-operative management, and precisely describe the surgical procedure of single-stage microsurgical reconstruction of the smile by free functional gracilis muscle transfer in a step by step protocol. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly comprehend the procedure. We further discuss indications and limitations of the technique and demonstrate representative results.

The use of the masseteric nerve as donor nerve represents a single-stage alternative to the criterion standard two-stage procedure of cross-facial nerve grafting and free muscle transfer in facial paralysis. We provide a detailed description to safely perform this technique with a gracilis muscle transfer and discuss indications and limitations.

Unilateral facial paralysis is a common disease that is associated with significant functional, aesthetic and psychological issues. Though idiopathic ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

VDJ-Seq: Deep Sequencing Analysis of Rearranged Immunoglobulin Heavy Chain Gene to Reveal Clonal Evolution Patterns of B Cell Lymphoma

Understanding tumor clonality is critical to understanding the mechanisms involved in tumorigenesis and disease progression. In addition, understanding the clonal composition changes that occur within a tumor in response to certain micro-environment or treatments may lead to the design of more sophisticated and effective approaches to eradicate tumor cells. However, tracking tumor clonal sub-populations has been challenging due to the lack of distinguishable markers. To address this problem, a VDJ-seq protocol was created to trace the clonal evolution patterns of diffuse large B cell lymphoma (DLBCL) relapse by exploiting VDJ recombination and somatic hypermutation (SHM), two unique features of B cell lymphomas.
In this protocol, Next-Generation sequencing (NGS) libraries with indexing potential were constructed from amplified rearranged immunoglobulin heavy chain (IgH) VDJ region from pairs of primary diagnosis and relapse DLBCL samples. On average more than half million VDJ sequences per sample were obtained after sequencing, which contain both VDJ rearrangement and SHM information. In addition, customized bioinformatics pipelines were developed to fully utilize sequence information for the characterization of IgH-VDJ repertoire within these samples. Furthermore, the pipeline allows the reconstruction and comparison of the clonal architecture of individual tumors, which enables the examination of the clonal heterogeneity within the diagnosis tumors and deduction of clonal evolution patterns between diagnosis and relapse tumor pairs. When applying this analysis to several diagnosis-relapse pairs, we uncovered key evidence that multiple distinctive tumor evolutionary patterns could lead to DLBCL relapse. Additionally, this approach can be expanded into other clinical aspects, such as identification of minimal residual disease, monitoring relapse progress and treatment response, and investigation of immune repertoires in non-lymphoma contexts.

This protocol describes an approach to interrogate the recombined immunoglobulin heavy chain VDJ regions of lymphomas by deep-sequencing and retrieve VDJ rearrangement and somatic hypermutation status to delineate clonal architecture of individual tumor. Comparing clonal architecture between paired diagnosis and relapse samples reveals lymphoma relapse clonal evolution modes.

Understanding tumor clonality is critical to understanding the mechanisms involved in tumorigenesis and disease progression. In addition, understanding ...

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

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.

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.

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

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules.
Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.

We present a robust, transferable and predictive in vitro exposure system for the screening and monitoring of airborne particles concerning their acute pulmonary cytotoxicity by exposing cultivated human lung cells at the air-liquid interface (ALI).

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to ...

Quality-Controlled Sputum Analysis by Flow Cytometry

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed using cytology-based methodologies. Its utility is limited because reading the slides is time-consuming and requires highly specialized personnel. Moreover, extensive debris and the presence of too many squamous epithelial cells (SECs), or cheek cells, often renders a sample inadequate for diagnosis. In contrast, flow cytometry allows for high-throughput phenotyping of cellular populations while simultaneously excluding debris and SECs.
The protocol presented here describes an efficient method to dissociate sputum into a single cell suspension, antibody stain and fix cellular populations, and acquire samples on a flow cytometric platform. A gating strategy that describes the exclusion of debris, dead cells (including SECs) and cell doublets is presented here. Further, this work also explains how to analyze viable, single sputum cells based on a cluster of differentiation (CD)45 positive and negative populations to characterize hematopoietic and epithelial lineage subsets. A quality control measure is also provided by identifying lung-specific macrophages as evidence that a sample is derived from the lung and is not saliva. Finally, it has been demonstrated that this method can be applied to different cytometric platforms by providing sputum profiles from the same patient analyzed on three flow cytometers; Navios EX, LSR II, and Lyric. Furthermore, this protocol can be modified to include additional cellular markers of interest. A method to analyze an entire sputum sample on a flow cytometric platform is presented here that makes sputum amenable for developing high-throughput diagnostics of lung disease.

This protocol describes an efficient method for dissociating sputum into a single cell suspension and the subsequent characterization of cellular subsets on standard flow cytometric platforms.

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed ...