Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

Published 5/04/2012
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Summary

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

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Lu, C., Wonsidler, J. L., Li, J., Du, Y., Block, T., Haab, B., et al. Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples. J. Vis. Exp. (63), e3791, doi:10.3791/3791 (2012).

Abstract

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.

Introduction

Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of α-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

Protocol

1. Print an Antibody Microarray for the Assay

  1. Dilute all antibodies to 0.5 mg/ml in phosphate buffer saline, ph 7.2 (PBS).
  2. Aliquot 40 μl of each antibody into the 384-well source plate.
  3. Load the 384-well source plate onto the Scienion sciFLEXARRAYER microarrayer.
  4. Load 20 PATH microarray slides onto the microarrayer as target.
  5. Set the microarrayer to print 48 identical subarrays, in which 27 antibodies and control proteins are spotted in triplicate in a 9x9 pattern (Figure 1E, 1F).
  6. Start the microarrayer to print the antibody microarray slides.
  7. Collect the antibody microarray slides, and store them in slides cassette with desiccant. Vacuum seal the cassette in a plastic bag by using vacuum sealer (Foodsaver).
  8. Store the sealed microarray slides at 4 °C in refrigerator.

2. Chemically Block the Antibody Microarray to Prevent GBP Binding to the Capture Antibodies

The microarray assay starts once the microarray slides are chemically blocked and lasts for about 8 hours. Once started the microarray assay has to be completed (Steps 2 to 8).

  1. Take the microarray slides out of the refrigerator, and equilibrate them to room temperature for 30 minutes.
  2. Remove the slide from the storage box and briefly rinse them in phosphate buffer saline pH 7.2 with 0.1% Tween 20 (PBST0.1) once in a slide washing basin, and then in 15 mM sodium acetate buffer pH 5.0 with 0.1% Tween (CBT0.1) in a sequential fashion. Incubate the slides in CBT0.1 for 10 minutes in slide washing basin.
  3. Prepare fresh 150 mM NaIO4 in 15 mM sodium acetate buffer pH 5.0 (CB), and keep it in into a slide washing basin in a refrigerator while avoiding light before use.
  4. Remove the slide from the CB, and put it into the basin containing fresh NaIO4 with the antibody side facing up. Cover the basin with aluminum foil to avoid light, and incubate the slide basin for 2 hours with gentle shaking at 4 °C in a refrigerator.
  5. Prepare 300 mL of 10 mM hydrazide glutamic acid (the blocker) in CB.
  6. Remove the slide from the basin, and briefly rinse it in CB 3 times for 5 minutes each time in slide washing basin.
  7. Incubate the slides in the blocker in a washing basin for 2 hours at room temperature with gentle shaking.
  8. Remove the slides from the basin, and wash them with PBST0.1 for 3 minutes.

3. Block Non-specific Bindings to the Microarray with Bovine Serum Albumin (BSA)

  1. Prepare 300 ml of 1% BSA in phosphate buffer saline pH 7.2 with 0.5% Tween (PBST0.5) in a slide washing basin, and incubate the microarray slide in the basin for 1 hour at room temperature with gently shaking.
  2. Rinse the slides in PBST0.1 three times for 3 minutes each time.
  3. Put the slide on a slide rack, and spin at 1,200 x g on a centrifugation for 2 minutes to dry the microarray slide.

4. Imprint Wax Grid onto the Microarray Slide to Separate each Subarray

  1. Pre-heating the wax imprinter at 70 °C for 5 minutes.
  2. Load the blocked microarray slide into the wax imprinter with antibody side facing to the wax. Gently pull the handle to imprint wax onto the slide evenly.

5. Apply Serum Samples onto the Microarray Slide

  1. During Step 2.4, prepare serum samples for either glyco profiling assay in one sample (5.1.1), or single glyco epiptope measurement among multiple samples (5.1.2).
    1. In an experiment for the glycan profilings of multiple serum glycoproteins in one serum sample by using multiple GBPs (see sample experiment 1), one serum samples will be applied onto all subarrays. In this case, 40 μl serum ample is diluted into 360 μl of PBS containing 0.1% Tween-20, 0.1% Brij 35, 100 μg/mL of mouse IgG, 100 μg/mL of rat IgG, 100 μg/mL of rabbit IgG, 100 μg/mL of goat IgG and 100 μg/ml of donkey IgG. This volume is sufficient for applying 6 μl of diluted serum solution onto each subarray.
    2. In an experiment for the one glycan measurement on multiple serum proteins among multiple serum samples by using one GBP detections (see sample experiment 2). In this case, 1 μl serum ample is diluted into 9 μl of PBS containing 0.1% Tween-20, 0.1% Brij 35, 100 μg/mL of mouse IgG, 100 μg/mL of rat IgG, 100 μg/mL of rabbit IgG, 100 μg/mL of goat IgG and 100 μg/mL of donkey IgG. This volume is sufficient for applying 6 μL of diluted serum solution onto each subarray.
  2. After wax imprint in Step 4, carefully apply 6 μL of diluted sample or control samples (PBST0.1) to each subarray of the slide. Incubate the slide in a humidified cassette with wet paper towels at room temperature for 1 hour.
  3. Rinse the slide with PBST0.1 three times for 3 minutes each time.
  4. Dry the slide by spinning it at 1200 x g for 2 minute.

6. Apply Biotinylated GBP (Lectin or Anti-glycan Antibody) onto the Slide

  1. During Step 2.4, prepare 10μg/ml of biotinylated lectins/GBPs in PBST0.1.
    1. In glycan profiling experiment that probe one sample with multiple lectins (sample experiment 1), prepare 350 μL of biotinylated lectin that is sufficient for all subarrays.
    2. In single glycan epitope/biomarker screening in multiple samples by using multiple lectins, prepare 10 μl of each biotinylated lectin that is sufficient for one subarray.
  2. Apply 6 μL of the diluted biotinylated lectin(s) to each subarray of the slide, and incubate in the humidified slide box with wet paper towels at room temperature for 1 hour.
  3. Rinse the slides with PBST0.1 three times for 3 minutes each time.
  4. Dry the slide by spinning it at 1200 x g in centrifuge for 2 minute.

7. Apply Dye Labeled NeutrAvidin for Fluorescence Detection

  1. Prepare 350 μL of Dylight 549 labeled NeutrAvidin that is sufficient for all subarrays.
  2. Apply 6 μL of Dylight 549 labeled NeutrAvidin onto each subarray, and incubate the slide in the humidified slide cassette at room temperature for 1 hour.
  3. Rinse the slide with PBST0.1 three times for 3 minutes each time.
  4. Dry the slide by spinning the it at 1200 x g in centrifuge for 2 minute.

8. Obtain Microarray Slide Image by Scanning the Slide

  1. Scan the slide by using a fluorescence microarray scanner at 10 μm resolution. The laser and PMT settings should be as strong as possible, but no saturation spot is observed.

9. Data Extraction and Analysis

  1. Open the image in ArrayPro 3.2.
  2. Set up the array template according to the array map that shows the antibody spots locations. Carefully align each template circle onto the corresponding spot in the image.
  3. Extract the intensity of each spot into an Excel file for further analysis.

10. Representative Results

Sample Experiment 1

Glycosylation profiling of multiple serum glycoproteins in hepatocellular carcinoma patient serum sample by using chemically blocked antibody microarray with multiple lectins detection.

The goal of this experiment is to explore the individual glycosylation profile of 20 glycoproteins in hepatocellular carcinoma (HCC) patient serum sample by using chemically blocked antibody microarray with lectin detection. An antibody microarray, that contains 48 identical subarrays which include 26 antibodies and biotin-BSA, was designed and manufactured as described in Step 1. These 26 antibodies were against 20 serum glycoproteins that identified as promising early diagnosis value for HCC patients by using lectin based immunoprecipitation combined with mass spectrometric protein identification12, 32 as shown in Table 1. The pattern and arrangement of the antibody spots printed in triplicate in one representative subarray are shown in Figure 1E and 1F, respectively. Two identical microarray slides, one was not chemically blocked (Figure 1A), while the other one was (Figure 1B), were used to carry out the same glycosylation profiling experiment in order to demonstrate the importance of the chemically blocking procedure to the analysis. For the chemically blocked slide (Figure 1B), the experiment started at the Step 2; for the none chemically blocked slide (Figure 1A), the experiment started from Step 3. The experiment was carried by following all the steps described in the protocol except for Step 5.1.2 and 6.1.2. In the Step 5.2, a PBST0.1 control sample was applied onto subarrays in column 1 and 3, and a pooled HCC serum sample was applied onto subarrays in column 2 and 4, respectively (as shown in Figure 1G). This comparison is to show the effectiveness, efficiency of the procedure, as well as the antigen binding affinity of the antibodies after chemically blocking. 22 biotinylated lectins (as shown in Table 1) that specific to different glycans18, 20 were applied onto each subarray as shown in Figure 1G for glycosylation profiling. Images of the chemically blocked (Figure 1B) and non-chemically blocked (Figure 1A) microarrays after the glycosylation profiling assay by following the protocol. As shown in the subarrays in column 1 and 3 in non-chemically blocked microarrays (Figure 1A and Figure 1C), on which only PBST0.1 was applied, most lectins bound to capture antibodies, and showed very high background that comparable to those subarrays in column 2 and 4, on which the serum sample was applied. It is impossible to obtain glycan profile information from this microarray slide. On the contrary, when the same experiment was done on a chemically blocked antibody microarray slide, the subarrays in column 1 and 3, on which only PBST0.1 was applied, most lectins showed no or very low bindings to capture antibodies; while high antigen bindings were still observed in subarrays in column 2 and 4, on which serum sample was applied (Figure 1B and 1D). These result showed the chemically blocking procedure was a critical step fore the measurement of glycans on antibody-captured glycoproteins. By following the protocol, glycosylation profiles of 22 glycoproteins in HCC serum can be obtained.

Experiment 2

Screen for altered fucosylation on specific serum glycoproteins as biomarkers to discriminated liver cirrhosis and hepatocellular carcinoma patients.

The goal of this experiment is to screen for altered fucosylation on specific serum glycoproteins as biomarkers that discriminate liver cirrhosis and hepatocellular carcinoma (HCC) patients. Different from the Experiment 1, in which only one serum sample was applied onto every subarrays and probed with various lectins, in this assay, total 40 different serum samples from HCC and cirrhosis patients were applied onto each subarray, and probed with one lectin (AAL). Statistical analysis, such as T test, receiver-operating characteristic (ROC) curve, was done to evaluate the distributions or diagnostic performance of the glycan epiptope/biomarker on each individual protein in all serum samples. We used the same antibody microarray manufactured in Experiment 1 except for anti-CA19-9 and anti-Lewis X antibodies in this study. The experiment was carried out from Sept 2 to Step 9 except for the Step 5.1.1 and 6.1.1. Total 40 serum samples from 20 cirrhosis and 20 HCC patients were applied at random subarray of the 48 subarrays along with the control PBS samples as negative control. Fucosylation of each the captured proteins was then detected by using biotinylated Fucose-specific lectin. The microarray image shown in Figure 1 demonstrated the lectin AAL only bound to serum proteins captured on the microarray (Figure 2D) instead of captured antibodies (Figure 2E). The AAL binding intensities of all the spots were then extracted, and analyzed by using T test and ROC curves to evaluate the performance of the fucosylation (AAL binding intensity) of each serum protein on the discrimination between the HCC and cirrhosis groups. The results showed that the fucosylation of GP73 protein gave the best discrimination between the two groups with a p = 0.03 and area-under-curve of ROC curve equals to 0.72. This experiment demonstrated this procedure is a rapid, efficient method for the glycan epitope/biomarker screening on multiple samples within multiple proteins.

ID Name of the reagent Abbreviation Company Catalog #
L1 Biotinylated Concanavalin A ConA Vector Laboratories BK-1000
L2 Biotinylated Sambucus Nigra Lectin SNA Vector Laboratories B-1305
L3 Biotinylated Lens Culinaris Agglutinin LCA Vector Laboratories BK-2000
L4 Biotinylated Ricinus Communis Agglutinin I RCA Vector Laboratories BK-1000
L5 Biotinylated Aleuria Aurantia Lectin AAL Vector Laboratories B-1395
L6 Biotinylated Erythrina Cristagalli Lectin ECL Vector Laboratories BK-3000
L7 Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin II GSL II Vector Laboratories BK-3000
L8 Biotinylated Wheat Germ Agglutinin WGA Vector Laboratories BK-1000
L9 Biotinylated Phaseolus vulgaris Erythroagglutinin PHA-E Vector Laboratories BK-2000
L10 Biotinylated Phaseolus vulgaris Leucoagglutinin PHA-L Vector Laboratories BK-2000
L11 Biotinylated Peanut Agglutinin PNA Vector Laboratories BK-1000
L12 Biotinylated Pisum Sativum Agglutinin PSA Vector Laboratories BK-2000
L13 Biotinylated Dolichos Biflorus Agglutinin DBA Vector Laboratories BK-1000
L14 Biotinylated Datura Stramonium Lectin DSL Vector Laboratories BK-3000
L15 Biotinylated Sophora Japonica Agglutinin SJA Vector Laboratories BK-2000
L16 Biotinylated Soybean Agglutinin SBA Vector Laboratories BK-1000
L17 Biotinylated Solanum Tuberosum (Potato) Lectin STL Vector Laboratories BK-3000
L18 Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin I GSL I Vector Laboratories BK-2000
L19 Biotinylated Vicia Villosa Lectin VVL Vector Laboratories BK-2000
L20 Biotinylated Lycopersicon Esculentum (Tomato) Lectin LEL Vector Laboratories BK-3000
L21 Biotinylated Ulex Europaeus Agglutinin I UEA I Vector Laboratories BK-1000
L22 Biotinylated Jacalin JACALIN Vector Laboratories BK-3000
A1 Goat F(ab')2 Fragment anti-human IgM, Fc5μ antibody IgM Jackson Immuno Research 109-006-129
A2 Donkey F(ab')2 Frag anti-human IgG (H+L) antibody AB1 Jackson Immuno Research 709-006-149
A3 Mouse anti-human IgG F(ab')2 monoclonal antibody AB3 Jackson Immuno Research 209-005-097
A4 Goat anti-human alpha 2 macroglobulin polyclonal antibody A2M GeneTex GTX62924
A5 Rabbit anti-human alpha-1-antitrypsin polyclonal antibody A1AT Lee Biosiences CA1T-80A
A6 Mouse anti-human alpha-1-antitrypsin monoclonal antibody A1AT Sigma Aldrich SAB4200198
A7 Rabbit anti-human alpha-1-antitrypsin polyclonal antibody ACT NeoMarkers RB-367-A1
A8 Rabbit anti-human alpha-1-antichymotrypsin polyclonal antibody ACT Fisher Scientific RB9213R7
A9 Mouse anti-human transferrin monoclonal antibody Transferrin GeneTex GTX101035
A10 Rabbit anti-human transferrin polyclonal antibody Transferrin GeneTex GTX77130
A11 Goat anti-human apolipoprotein J polyclonal antibody ApoJ Abcam ab7610
A12 Mouse anti-human GP73 monoclonal antibody GP73 Abbott 14H4-23
A13 Mouse anti-human GP73 monoclonal antibody GP73 SANTA CRUZ BIOTECHNOLOGY INC sc-101275
A14 Rabbit anti-human alpha-1 fetoprotein polyclonal antibody AFP GenWay GWB-41C966
A15 Mouse anti-human alpha-1 fetoprotein monoclonal antibody AFP Fitzgerald 10-A05A
A16 Mouse anti-human hemopexin monoclonal antibody Hemopexin Assaypro 60190-05011
A17 Mouse anti-human glypican-3(1G12) monoclonal antibody GPL3 Santa Cruz Bio sc-65443
A18 Mouse anti-human Kininogen (LMW) monoclonal antibody Kininogen Assaypro 20333-05011
A19 Rabbit anti-human MMP-21 monoclonal antibody MMP21 Epitomic 1955-1
A20 Mouse anti-human CEACAM-1 monoclonal antibody CEACAM R&D Systems MAB1180
A21 Rat anti-human DPPIV/CD26 monoclonal antibody DPPIV R&D Systems MAB22441
A22 Mouse anti-human PIVKA II monoclonal antibody PIVICA Crystal chem 8040
A23 Mouse anti-carcinoembryonic antigen CEA US biological C1300
A24 Mouse anti-CA125 Cancer Antigen CA125 US biological C0050-01D
A25 Mouse anti -CA19-9 Cancer antigen CA19-9 US biological C0075-18
A26 Mouse anti-Lewis x monoclonal antibody Lewis X Calbiochem 434631
bio Biotinylated BSA (positive control) Bio Home-made N/A

Table 1. List of lectins and antibodies used in this protocol.

Name of the reagent s/equipments Company Catalogue number
Non contact microarrayer BioDot Inc sciFLEXARRAYER
384 microplate Fisher 14-230-243
FoodSaver FoodSaver V3835
Ultrathin nitrocellulose coate microarray slides Gentel PATH
Slide Imprinter (optional) The Gel Company WSP60-1
Shaker Fisher 15-453-211
Centrifuge Eppendorf 5804 000.013
Slide washing basin/Slide Staining Dish with Removable Rack Fisher 08-812
Slide incubation chamber/microscope slide box Fisher 03-448-5
Brij 35, 30 w/v% solution in water Acros Organics AC32958-0025
Tween-20 Fisher P337-100
Sodium Periodate (NaIO4) Sigma 311448
L-Glutamic acid γ-hydrazide Sigma G-7257
Sodium Acetate Anhydrous (CH3COONa) Sigma S2889
Bovine Serum Albumin (BSA) Lampire Biological Labs 7500804
Phosphate Buffer Saline (PBS) (10X) Denville Scientific CP4390-48
Dylight 549 conjugated NeutrAvidin Thermo 22837
Protease Inhibitor Cocktail Tablets Roche 4693159001
ChromPure Human IgG, Fc fragment Jackson Immunoresearch 009-000-008
ChromPure Human IgG, whole molecule Jackson Immunoresearch 009-000-003
ChromPure Mouse IgG, whole molecule Jackson Immunoresearch 015-000-003
ChromPure Mouse IgG, Fc fragment Jackson Immunoresearch 015-000-008
ChromPure Rabbit IgG, whole molecule Jackson Immunoresearch 011-000-003
ChromPure Donkey IgG, whole molecule Jackson Immunoresearch 017-000-003
Microarray Scanner Tecan LS Reloaded

Table 2. List of equipments and reagents used in this protocol.

Scheme 1
Scheme 1. A scheme showing the lectin antibody microarray based glycan biomarker discovery process. 1 (Step 2 to 4): Block the antibody microarray with the blocker (Glu-hydrazide) and BSA; 2 (Step 5): apply serum samples and capture specific glycoproteins with specific antibodies; 3 (Step 6): apply biotinylated lectin(s); 4 (Step 7): Probe the biotinylated AAL with Dylight 549 labeled NeutrAvidin for microarray imaging.

Figure 1
Figure 1. Microarray images of the Sample Experiment 1 glycosylation profiling of multiple serum glycoproteins in HCC patient serum sample by using chemically blocked antibody microarray with multiple lectin detection. Two identical microarray slides, (A) none chemically blocked, or (B) chemically blocked as described in Step 2, both went through all the steps from 2 to 9 for glycosylation profiling, as well as comparison purpose. (A) and (B) are the microarray images scanned at Step 8 in a resolution of 10 micron. (C) the zoom in image of the first two rows of the none chemically blocked microarray slide (A); (D) the zoom in image of the first two rows of the non chemically blocked microarray slide (B)); (E) the diagram of the antibody arrangement within each subarray; (F) array maps: the location of each antibody within the subarray, each antibody name represents 3 spots; (G) Serum sample and lectin location: a diagram shows which subarray each serum sample and lectin was applied onto.

Figure 2
Figure 2. Microarray images of the sample experiment 2 screen for altered fucosylation on specific serum glycoproteins as biomarkers that discriminate liver cirrhosis and HCC patients. The microarray assay was carried out as described in the Sample Experiment 2 section. (A) The whole slide image of the microarray slide from Step 8; (B) the diagram of the antibody arrangement within each subarray; (C) array maps: the location of each antibody within the subarray, each antibody name represents 3 spots; (D) a zoom-in image of a subarray that were incubated with serum sample; (E) a zoom-in image of a subarray that were incubated with PBS control.

Figure 3
Figure 3. Glycan profiling results from sample experiment 1. Each bar graph represent lectin binding profile (or glycan profiles) of one of the 20 tested protein. Total 22 different lectins were used to analyze the glycan profile of each protein. Please click here to see a larger version of this figure.

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Discussion

1. Target protein and capture antibody selection

Prior to the antibody microarray assay, some reagents and materials are needed to be considered and prepared. To design an antibody microarray for glycan profiling or glycan biomarker screening, a panel of antibodies specific to glycoprotein candidates should be determined according to the literature or from previous results. These antibodies were usually purchased from different vendors such as R&D Systems etc. IgGs are preferred capture antibodies since our previous tests showed that some IgM and IgE may completely lose their antigen binding affinities after the chemical modification.

2. Design and manufacture of antibody microarray

Manufacture of the antibody microarray is an optional step which needs professional and expensive microarrayer, and well-trained personnel for operation. However, customized antibody microarray manufacture can be readily done from a service provider such as Serome Biosciences Inc. For a robust result, we recommend a non-contact microarrayer, such as the Scienion sciFLEXARRAYER ultra low volume non-contact microarrayer which was used in our antibody microarray manufacture. High binding capacity microarray slides, such as PATH (Gentel Bio Inc. WI) or Slide H (Shott, PA).

3. Select and Prepare Glycan Binding Proteins (GBPs) for glycosylation profiling

GBPs that target different mono- or oligosaccharides can be found in the literature and through a search engine developed by the Haab laboratory 29 and maintained at the Translational Genomics Institute (TGen): https://vai-apps.tgen.org/haab/default.do;jsessionid=83A3F59423A92A49D76D8B4426FAC015?method=search. To select GBPs with high specificity and affinity to the targeted glycan epitopes, select the motif (epitope) from the drop down menu, and click "search." The GBPs specific to this motif (epitope) will be listed according to their logP value from high to low order. Higher logP indicates a stronger binding affinity and specificity to the glycan motif/epitope. Due to the inherent non-specific binding issue from lectin, this method is still not as optimal as the antibody based assay. Thus, we strongly recommend that anti-glycan antibodies are used, such as anti-Lewis x or anti-sialyl Lewis a antibodies, if they are available. Use of multiple GBPs for detection is another strategy to cross check different binding profiles and to obtain reliable binding data.

4. Data analysis

Because this method is used to detect native serum proteins, protein-protein complexes may be captured and detected. Western blot or mass spectrometry is good methods to validate the microarray data. In the meantime, detection of protein levels using the same microarray is another method to learn the details of the glycosylation alteration of the protein, such as whether the changes were due to the total protein level change or just that the glycosylation level increased on each of the proteins.

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Institute for Hepatitis and Virus Research.

Materials

Name Company Catalog Number Comments
Biotinylated Concanavalin A Vector Laboratories BK-1000 ConA
Biotinylated Sambucus Nigra Lectin Vector Laboratories B-1305 SNA
Biotinylated Lens Culinaris Agglutinin Vector Laboratories BK-2000 LCA
Biotinylated Ricinus Communis Agglutinin I Vector Laboratories BK-1000 RCA
Biotinylated Aleuria Aurantia Lectin Vector Laboratories B-1395 AAL
Biotinylated Erythrina Cristagalli Lectin Vector Laboratories BK-3000 ECL
Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin II Vector Laboratories BK-3000 GSL II
Biotinylated Wheat Germ Agglutinin Vector Laboratories BK-1000 WGA
Biotinylated Phaseolus vulgaris Erythroagglutinin Vector Laboratories BK-2000 PHA-E
Biotinylated Phaseolus vulgaris Leucoagglutinin Vector Laboratories BK-2000 PHA-L
Biotinylated Peanut Agglutinin Vector Laboratories BK-1000 PNA
Biotinylated Pisum Sativum Agglutinin Vector Laboratories BK-2000 PSA
Biotinylated Dolichos Biflorus Agglutinin Vector Laboratories BK-1000 DBA
Biotinylated Datura Stramonium Lectin Vector Laboratories BK-3000 DSL
Biotinylated Sophora Japonica Agglutinin Vector Laboratories BK-2000 SJA
Biotinylated Soybean Agglutinin Vector Laboratories BK-1000 SBA
Biotinylated Solanum Tuberosum (Potato) Lectin Vector Laboratories BK-3000 STL
Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin I Vector Laboratories BK-2000 GSL I
Biotinylated Vicia Villosa Lectin Vector Laboratories BK-2000 VVL
Biotinylated Lycopersicon Esculentum (Tomato) Lectin Vector Laboratories BK-3000 LEL
Biotinylated Ulex Europaeus Agglutinin I Vector Laboratories BK-1000 UEA I
Biotinylated Jacalin Vector Laboratories BK-3000 JACALIN
Goat F(ab')2 Fragment anti-human IgM, Fc5μ antibody Jackson Immuno Research 109-006-129 IgM
Donkey F(ab')2 Frag anti-human IgG (H+L) antibody Jackson Immuno Research 709-006-149 AB1
Mouse anti-human IgG F(ab')2 monoclonal antibody Jackson Immuno Research 209-005-097 AB3
Goat anti-human alpha 2 macroglobulin polyclonal antibody GeneTex GTX62924 A2M
Rabbit anti-human alpha-1-antitrypsin polyclonal antibody Lee Biosiences CA1T-80A A1AT
Mouse anti-human alpha-1-antitrypsin monoclonal antibody Sigma-Aldrich SAB4200198 A1AT
Rabbit anti-human alpha-1-antitrypsin polyclonal antibody NeoMarkers RB-367-A1 ACT
Rabbit anti-human alpha-1-antichymotrypsin polyclonal antibody Fisher Scientific RB9213R7 ACT
Mouse anti-human transferrin monoclonal antibody GeneTex GTX101035 Transferrin
Rabbit anti-human transferrin polyclonal antibody GeneTex GTX77130 Transferrin
Goat anti-human apolipoprotein J polyclonal antibody Abcam ab7610 ApoJ
Mouse anti-human GP73 monoclonal antibody Abbott Laboratories 14H4-23 GP73
Mouse anti-human GP73 monoclonal antibody SANTA CRUZ BIOTECHNOLOGY INC sc-101275 GP73
Rabbit anti-human alpha-1 fetoprotein polyclonal antibody GenWay GWB-41C966 AFP
Mouse anti-human alpha-1 fetoprotein monoclonal antibody Fitzgerald 10-A05A AFP
Mouse anti-human hemopexin monoclonal antibody Assaypro 60190-05011 Hemopexin
Mouse anti-human glypican-3(1G12) monoclonal antibody Santa Cruz Bio sc-65443 GPL3
Mouse anti-human Kininogen (LMW) monoclonal antibody Assaypro 20333-05011 Kininogen
Rabbit anti-human MMP-21 monoclonal antibody Epitomic 1955-1 MMP21
Mouse anti-human CEACAM-1 monoclonal antibody R&D Systems MAB1180 CEACAM
Rat anti-human DPPIV/CD26 monoclonal antibody R&D Systems MAB22441 DPPIV
Mouse anti-human PIVKA II monoclonal antibody Crystal chem 8040 PIVICA
Mouse anti-carcin–mbryonic antigen US biological C1300 CEA
Mouse anti-CA125 Cancer Antigen US biological C0050-01D CA125
Mouse anti -CA19-9 Cancer antigen US biological C0075-18 CA19-9
Mouse anti-Lewis x monoclonal antibody Calbiochem 434631 Lewis X
Biotinylated BSA (positive control) Home-made N/A Bio
Table 1. List of lectins and antibodies used in this protocol.
Non contact microarrayer BioDot Inc sciFLEXARRAYER
384 microplate Fisher Scientific 14-230-243
FoodSaver FoodSaver V3835
Ultrathin nitrocellulose coate microarray slides Gentel PATH
Slide Imprinter (optional) The Gel Company WSP60-1
Shaker Fisher Scientific 15-453-211
Centrifuge Eppendorf 5804 000.013
Slide washing basin/Slide Staining Dish with Removable Rack Fisher Scientific 08-812
Slide incubation chamber/microscope slide box Fisher Scientific 03-448-5
Brij 35, 30 w/v% solution in water Acros Organics AC32958-0025
Tween-20 Fisher Scientific P337-100
Sodium Periodate (NaIO4) Sigma-Aldrich 311448
L-Glutamic acid γ-hydrazide Sigma-Aldrich G-7257
Sodium Acetate Anhydrous (CH3COONa) Sigma-Aldrich S2889
Bovine Serum Albumin (BSA) Lampire Biological Labs 7500804
Phosphate Buffer Saline (PBS) (10X) Denville Scientific CP4390-48
Dylight 549 conjugated NeutrAvidin Thermo Fisher Scientific, Inc. 22837
Protease Inhibitor Cocktail Tablets Roche Group 4693159001
ChromPure Human IgG, Fc fragment Jackson ImmunoResearch 009-000-008
ChromPure Human IgG, whole molecule Jackson ImmunoResearch 009-000-003
ChromPure Mouse IgG, whole molecule Jackson ImmunoResearch 015-000-003
ChromPure Mouse IgG, Fc fragment Jackson ImmunoResearch 015-000-008
ChromPure Rabbit IgG, whole molecule Jackson ImmunoResearch 011-000-003
ChromPure Donkey IgG, whole molecule Jackson ImmunoResearch 017-000-003
Microarray Scanner Tecan Group Ltd. LS Reloaded
Table 2. List of equipments and reagents used in this protocol.

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References

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