[Methods in Molecular Biology] Carbohydrate Microarrays Volume 808 || Immobilization of Polyacrylamide-Based Glycoconjugates on Solid Phase in Immunosorbent Assays

167

Yann Chevolot (ed.), Carbohydrate Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 808,DOI 10.1007/978-1-61779-373-8_12, © Springer Science+Business Media, LLC 2012

Chapter 12

Immobilization of Polyacrylamide-Based Glycoconjugates on Solid Phase in Immunosorbent Assays

Oxana E. Galanina, Alexander A. Chinarev, Nadezhda V. Shilova, Marina A. Sablina, and Nicolai V. Bovin

Abstract

Our experience in coating of solid surfaces with glycans, mainly for obtaining routine glycoarrays based on immunological plates, is summarized. Three polystyrene coating techniques are described: direct physical adsorption, covalent binding, and immobilization using the biotin tag. Protocols for studies on anticarbo-hydrate antibodies are considered, with special emphasis on the application niches of different immobiliza-tion techniques as related to the specificity of each method of glycan-binding protein assay, as well as the problems of background binding and quantitative estimation of the results.

Key words: Glycoarrays, ELISA, Glycopolymers, Immobilization techniques, Physical adsorption, Covalent immobilization, Biotin–streptavidin bridge, Polyacrylamide

Abbreviations

Atri A trisaccharide, GalNAca1-3(Fuca1-2)Galb-AP Alkaline phosphataseBtri B trisaccharide, Gala1-3(Fuca1-2)Gal-Biot BiotinBSA Bovine serum albuminELISA Enzyme-linked immunosorbent assayGlyc Glycoside residueGPC Gel-permeationIg Anti-mouse antibodiesHRPO Horse radish peroxidasem.w. Molecular weightPAA Poly(acrylamide)PBS Phosphate saline bufferpNPA poly(4-nitrophenylacrylate)pNSA poly(N-oxisuccinimidylacrylate)Str StreptavidinTLC Thin-layer chromatography

168 O.E. Galanina et al.

Glycans constitute a complex and diverse family of biomolecules involved in a broad range of physiological (cell development and differentiation, cell recognition and cell-to-cell adhesion, cell motility, etc.) and pathological (malignant transformations, auto-immune diseases, host–pathogen interactions, etc.) processes, many important characteristics of which remain poorly studied (1–3).

For profiling glycan-binding proteins and studying interac-tions involving them, enzyme-linked immunosorbent assay (ELISA) and related assays based on the classical multiwell plates are generally used. Although many researchers note that ELISA is not sensitive enough and time consuming, its major advantages are simplicity and flexibility of the procedure. This allows the method to be adjusted to a specific research task, e.g., selection of the direct binding or inhibition conditions; choice of the optimal detection method (colorimetric or fluorescent); instant variation of glycan combinations, coating concentrations, and surface density; and choice between the monovalent and multivalent types of their pre-sentation. ELISA requires at least one order of magnitude lower concentrations of the analyzed proteins to be than, e.g., the printed glycan array (PGA) and suspension (Bioplex) array. It is notewor-thy that the assay does not require sophisticated equipment. Glycan ELISA has its own application niche; it is optimal for miniformat glycan profiling when 5–15 glycans are necessary for characteriza-tion of a glycan-binding protein. In practice, ELISA has proved to be a good complementary “partner” for PGA, when PGA is used as a screening tool, followed by ELISA for accurate verification of the results. Apart from research purposes, ELISA can be easily adapted for medical applications. The assay can be performed man-ually or, if necessary, robotized (4–7).

Typically, interactions involving glycans are weak; this impels researchers to use carbohydrates in the multivalent form. In par-ticular, glycoconjugates based on linear polyacrylamides (PAAs) with attached-side carbohydrate groups (Glyc) solve this problem. The polymer backbone plays two roles: first, a flexible linker between Glyc residues facilitating their optimal arrangement for multivalent binding to an antibody or lectin irrespective of the dis-tance between their carbohydrate-binding domains (8, 9); second, a long spacer between glycans and the support. On the contrary, monomeric glycans arrayed on a solid surface are often hardly capable of multivalent binding with glycan-recognizing proteins because of suboptimal density of Glyc residues and length of their bonds with the surface; i.e., this type of glycan arrays can be only technically regarded as multivalent (10–12).

1. Introduction

16912 Immobilization of Polyacrylamide-Based Glycoconjugates…

In this paper, we describe a set of techniques using various types of PAA-based glycoconjugates for coating polystyrene ELISA plates; the general strategy of the glycopolymer use in ELISA is shown in Fig. 1.

Atri-O(CH2)3NH2 and Btri-O(CH2)3NH2 were obtained from Lectinity, Inc. (Russia).

Biot-NH(CH2)6NH2 was obtained from Lectinity, Inc. (Russia).

2. Materials

2.1. Carbohydrates

2.2. Biotin

Fig. 1. The use of glycopolymers in ELISA. A glycan from the library of w-aminoalkyl glycosides is quantitatively coupled to fully activated polyacrylic acid prepared by radical polymerization of nitrophenylacrylate or N-acryloyl succinimide. There are three ways of further use of the resultant glycopolymers containing active ester groups. First, they can be covalently immobilized on aminated surfaces (including polystyrene) through the formation of amide bonds. Second, they can be physically adsorbed in the form of Glyc–PAA. Third, they can be coated onto a streptavidin-modified surface in the form of a biotinylated glycopolymer.


Poly(4-nitrophenylacrylates), pNPA, biot1-pNPA, and poly (N-oxisuccinimidyl acrylate), pNSA, were prepared as described before (11, 13).

Regular chemicals were obtained from Fluka (USA), Aldrich (USA), and Merck (Germany).

1. PBS: 137 mM NaCl, 2.7 mM KCl, 10.0 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4.

2. ELISA-coating buffer: 15 mM Na2CO3, 35 mM NaHCO3, pH 9.6.

3. ELISA washing buffer: 0.1% Tween-20 in PBS. 4. ELISA blocking buffer: 3% BSA in PBS. 5. Colorimetric solution: 0.1 M sodium phosphate, 0.1 M citric

acid buffer, 0.04% O-phenylenediamine, pH 4.7. 6. Fluorescent-revealing solution: 10−4 M 4-methylumbellyferyl

phosphate disodium salt in the coating buffer.

1. Mouse monoclonal antibodies B8 (against Btri) and A16 (against Atri) were obtained from All-Russian Hematology Research Center (Moscow, Russia).

2. Streptavidin–horseradish peroxidase (Str–HRPO) conjugate was the product of GE Healthcare (UK).

3. Anti-mouse IgG + IgM (H + L)–alkaline phosphatase (Ig–AP) conjugate was the product of AP Biotech. Inc. (UK).

1. Reacti-Bind TM Streptavidin coated High Binding Capacity black plates (#15503) were purchased from ThermoScientific (USA).

2. MaxiSorp transparent immunoplates (#439454) were pur-chased from NUNC (Denmark).

3. NH2-modules were obtained from Costar (USA).

1. Thin layer chromatography (TLC) aluminum sheets covered with silicagel 60 (Merck, Germany).

2. Sephadex LH-20 gel (Pharmacia BioTech, Austria). 3. Eluent A for TLC: EtOH/BuOH/Py/H2O/AcOH,

10:1:1:1:0.3. 4. Eluent B for TLC: MeOH/1 M Py·AcOH, 3:1. 5. Eluent C for gel-permeating chromatography (GPC): MeCN/

H2O, 1:1. 6. Charring reagent for TLC plates: 7% H3PO4.

Victor2 multilabel counter (PerkinElmer, USA).

2.3. Activated Polyacrylic Acids

2.4. Other Commercial Reagents and Solvents

2.5. Buffers

2.6. Antibodies

2.7. 96-Well Plates

2.8. Chromatography

2.9. Equipment


Three approaches to immobilization of Glyc–PAA are considered here: physical (noncovalent) adsorption, chemical (covalent) immobilization, and immobilization via biotin–Str bridge.

Physical adsorption due to Van der Waals interactions between glycopolymer molecules and the plate surface is the simplest and the least sophisticated method. The adsorption efficiency depends on the m.w. of the glycopolymer; the number of contacts and interaction strength between glycopolymer molecules and the sur-face increase with an increase in the m.w. (13).

Two glycoconjugates differing in m.w., Glyc–PAA30 and Glyc–PAA2000 (see Notes 1 and 2), were synthesized (the protocols were developed for blood group A trisaccharide derivatives, Atri–PAA) as shown in Fig. 2a and coated onto the plates. The coating procedure included incubation of the glycoconjugate solutions in wells and sub-sequent washing off of nonadsorbed material. It was found that only as little as about 2% of the added low-m.w. glycopolymer is adsorbed, whereas this value for high-m.w. glycopolymer is ~20% (11).

The difference in the efficiency of immobilization for low- and high-m.w. Glyc–PAAs is illustrated in Fig. 3. Addition of equal amounts of Glyc into wells yields a stronger optical signal in the case of Glyc–PAA2000; the deference between glycoconjugate sig-nals is greater at a low coating concentration (<50 pmol Glyc per well). The maximum adsorption for the high- and low-m.w. glyco-conjugates is observed, respectively, at 50 and 200 pmol of Glyc residue per well. Thus, the use of the high-m.w. glycoconjugate allows reducing the amount of carbohydrates required for coating by a factor of 10–20.

The difference observed in mAb binding with glycopolymers is explained as follows. The average size of coiled glycopolymer mol-ecules adsorbed on polystyrene is known to be ~100 Å for 30-kDa conjugates and ~500 Å for 2,000-kDa ones (11), whereas the dis-tance between Fab fragments of antibodies varies from 100 to 150 Å for IgG and may be even more for IgM molecules (14). Thus, in the case of Atri–PAA30, binding of an antibody with several neighboring glycopolymer molecules on the surface is much more probable compared to an antibody with only one glycopolymer (Fig. 2b). An increase in the density of Atri–PAA30 molecules on the surface leads to an increase in the detected signal. On the contrary, the size of Atri–PAA2000 molecules allows them to bind one or sev-eral antibodies simultaneously: a signal is detectable even at a low density of Atri–PAA2000 (Fig. 2c).

Chemical immobilization of glycopolymers on the surface of aminated polystyrene is accomplished via formation of covalent

3. Methods


amide bonds (Fig. 4). Activated polyacrylic acids with pendant Glyc residues, Glyc–pNPA30 and Glyc–pNSA2000 (see Notes 1 and 2), are used without isolation. The coating procedure consists of consecutive incubations of plates in a glycopolymer solution (the coupling stage) and then in an aqueous ammonia solution (the quenching stage), which is followed by washing off of the unbound

Fig. 2. Polyacrylamide glycoconjugates for physical adsorption on polystyrene plates. (a) Synthesis: One of activated polyacrylic acids differing in m.w. (pNPA or pNSA) is bound with Glyc-O(CH2)3NH2 and then treated with ethanolamine to quench the remaining activated ester groups. The average sizes of molecules and distances for (b) low- and (c) high-m.w. glycoconjugates on the surface are shown.


Fig. 3. Physical adsorption of (a) high- and (b) low-m.w. glycoconjugates (Atri–PAA2000 and Atri–PAA30, respectively) on 96-well, transparent polystyrene plates. The efficacy of adsorp-tion was demonstrated by colorimetric detection using anti-Atri mAbs.

Fig. 4. Coating of an aminated surface with glycopolymers.


material. As a result, the plates are coated with PAA glycoconju-gates differing in m.w. (30 and 2,000 kDa).

A stronger binding is observed for the high-m.w. glycoconju-gate; the sorption is maximum at a concentration of 75 pmol of Glyc per well, whereas in the case of the low-m.w. glycoconjugate, a concentration of 200 pmol per well is required (Fig. 5).

A similar immobilization protocol allows glycopolymers to be attached to other aminated surfaces, e.g., coronary angioplasty stents made of stainless steel coated with nanodiamonds and then aminated plasmachemically over the diamond layer (Plasmachem GmbH, Germany). To solve the problem of smooth muscle cell overgrowth and to make the stent surface attractive for selectin-bearing endothelial cells, the stent surface is modified with carbo-hydrates capable of mediating endothelial cell adhesion (15). The efficacy of chemical coating of the stents with SiaLea–pNPA30 was proved by ELISA with anti-SiaLea mAbs (Fig. 6).

A convenient method of glycopolymer immobilization on plates is based on the use of biotin–Str bridge, when a glycopolymer bearing a biotin tag is anchored to a Str-coated plate. We used two approaches to the synthesis of biotinylated glycopolymers. According to the first one, a biotin derivative containing an amino group in the linker (biot-NH(CH2)6NH2) and an w-aminoalkyl glycoside (Glyc-O(CH2)3NH2) are consecutively coupled to pNPA. Finally, the remaining active ester groups in the polymer are quenched using ethanolamine (Fig. 7a). The resultant glycopolymer contains several side-pendant Glyc and biotin residues (see Notes 2 and 3). In the second method, a biotin residue is introduced into the polymer scaffold as a single end group (Fig. 7b) with a fragment of

Fig. 5. Chemical immobilization of (a) high- and (b) low-m.w. glycoconjugates (Btri–PAA2000 and Btri–PAA30, respectively) on aminated polystyrene plates. The efficacy of coating was demonstrated using anti-Btri mAbs.


Fig. 6. Photograph of a SiaLea-modified stent placed into a well of a nonadsorbing plate (as a vessel) and stained with anti-SiaLea mAbs; the color is developed due to HRPO-labeled secondary antibodies.

Fig. 7. Biotinylated glycopolyacrylamides for coating onto Str plates: Synthesis of the glycopolymers with (a) side- and (b) end-pendant biotin residues. (c) Anchoring of glyco-polymers with one and several pendant biotins on the surface and their presentation for interaction with antibodies.


biotinylated initiator of polymerization. The resultant activated polyacrylic acid, pNPA–biot1, interacts with Glyc-O(CH2)3NH2, which is followed by treatment with ethanolamine. The resultant glycopolymer, Glyc–PAA30–biot1, contains several side-pendant Glyc residues and the only one end biotin (see Note 2).

The immobilization procedure includes incubation of the biotinylated glycopolymers, Glyc–PAA30–biot5 and Glyc–PAA30–biot1, with Str plates and washing off of nonadsorbed material; the procedure has been shown to give a quantitative yield (16).

The monobiotinylated glycopolymer Glyc–PAA30–biot1 slightly better bound mAbs compared to an analogue bearing several-side biotins, Glyc–PAA30–biot5 (Fig. 8). The adsorption was maximal for the monobiotinylated conjugate at a concentra-tion of 70 pmol/well, whereas for the pauci–biot analog it was maximal at 100 pmol/well. As the glycopolymers have the same m.w., the observed difference is likely to have resulted from dif-ferences in the anchoring on the surface and, hence, in the pre-sentation of the glycan for mAbs (16). In immobilized molecules of Glyc–PAA30–biot1, the single end biotin is hidden inside the streptavidin matrix. The monobiotinylated glycopolymer is rec-ommended for plate coating when additional biotin residues are expected to affect the results of the assay, i.e., when hydrophobic-ity of extra biotin residues could critically elevate the background signal of the assay or when (strept)avidin-based reagents are used not only for immobilization, but also at one of the next stages, as illustrated in Fig. 9. Finally, it should be noted that immobiliza-tion through Str–biotin bridge has appeared to be a less reagent-consuming method.

Fig. 8. Binding of anti-Btri mAbs by glycopolymers with (a) end- and (b) side-pendant biotin residues (Btri–PAA30–biot1 and Btri–PAA30–biot5) immobilized on an Str plate; fluorimetric detection.


In summary, three approaches can be used for coating immu-nological plates with glycans in the form of Glyc–PAA; in all cases, the immobilized molecules are practically the same. What is the reason for selecting one of the methods for design of a specific assay? Physical adsorption has proved to be the simplest protocol; however, it has a serious drawback, namely, a high consumption of carbohydrates to be coated. Therefore, it is impracticable when only a minute quantity of glycan is available or the cost of the assay is the critical point. Biotin-mediated coating eliminates this disad-vantage and, in addition, is methodologically simple. One impor-tant characteristic of biot–Str immobilization should be noted. In the case of physical adsorption or the aforementioned covalent binding, the amount of glycoconjugate immobilized on the plate surface is not strictly proportional to their amount added. In con-trast, biotinylated glycoconjugate can be immobilized quantita-tively when the biot-to-Str ratio is <1 (in assays using the same plates as in this study, at concentrations of biotin of 0–50 pmol Glyc/well). Therefore, immobilization via biot–Str bridge allows a researcher to know the absolute amount of the coated material, which is necessary, e.g., when a correct affinity constant is calcu-lated. Limitations of the biot–Str technique are the high cost of commercially available plates and, sometimes, a low stability of the streptavidin monolayer. Chemical immobilization is undoubtedly the most sophisticated and time-consuming technique. It could be recommended for nonroutine assays, when the use of other meth-ods may lead to desorption because of unusual conditions, or the plate should be regenerated for repeated assays without loss of the coating glycoconjugate.

Fig. 9. Demonstration of the lack of available biot residues in the composition of Str complexed with end-biotinylated glycopolymer. Str plates are coated with a glycopolymer with side- or end-pendant biotin residues, Glyc–PAA30–biot5 (open bars) and Glyc–PAA30–biot1 (hatched bars), and developed with Str–AP.


1. Add a mixture of 3.2 mg (5.5 mmol) of Atri-O(CH2)3NH2 and 1.5 mL (11 mmol) of NEt3 in 100 mL of DMSO to a solution of 4.7 mg (27.5 mmol) of pNPA in 400 mL of DMSO.

2. Keep the mixture for 12 h at 40°C performing control for the reaction by TLC; eluent A: Rf 0.35 for Atri-O(CH2)3NH2, Rf 0 for Atri–PAA30 (see Subheading 3.2).

3. Add an excess of ethanolamine (90 mL) to the reaction mixture and keep it for 24 h at 40°C.

4. Purify the product by GPC (see Subheading 3.3). 5. Yield 85–95%; white solid.

1. Add a mixture of 3.2 mg (5.5 mmol) of Atri-O(CH2)3NH2 and 1.5 mL (11 mmol) of NEt3 dissolved in 100 mL of DMSO to a solution of 4.1 mg (27.5 mmol) of pNSA in 400 mL of DMSO.

2. Keep the mixture for 12 h at 40°C performing control for the reaction by TLC; eluent A: Rf 0.35 for Atri-O(CH2)3NH2, Rf 0 for Atri–PAA2000 (see Subheading 3.2).



1. Add a mixture of 1.4 mg (2.6 mmol) of Btri-O(CH2)3NH2 and 1.5 mL (11 mmol) of NEt3 dissolved in 100 mL of DMSO to a solution of 1.9 mg (12.8 mmol) of pNPA in 400 mL of DMSO.

2. Keep the mixture for 12 h at 40°C performing control for the reaction by TLC; eluent A: Rf 0.3 for Btri-O(CH2)3NH2, Rf 0 for Btri–pNPA30 (see Subheading 3.2).

3. Dilute 100 mL of Btri–pNPA30 reaction mixture with 900 mL of DMSO containing 0.2 mL of NEt3 (to concentration 0.94 nmol Btri/mL) and use the obtained solution for coating of aminated polystyrene plates (see Subheading 3.4.2).

1. Add a mixture of 1.4 mg (2.6 mmol) of Btri-O(CH2)3NH2 and 1.5 mL (11 mmol) of NEt3 dissolved in 100 mL of DMSO to a solution of 1.7 mg (12.8 mmol) of pNSA in 400 mL of DMSO.

2. Keep the mixture for 12 h at 40°C performing control for the reaction by TLC; eluent A: Rf 0.3 for Btri-O(CH2)3NH2, Rf 0 for Btri–pNSA2000 (see Subheading 3.2).

3. Dilute 100 mL of Btri–pNSA2000 reaction mixture with 900 mL DMSO containing 0.2 mL of NEt3 (to concentration 0.94 nmol Btri/mL) and use the obtained solution for coating of aminated polystyrene plates (see Subheading 3.4.2).

3.1. Preparation of Polyacrylamide-Based Glycoconjugates

3.1.1. Synthesis of Atri–PAA30 for Physical Adsorption on Polystyrene Plates (See Note 4)

3.1.2. Synthesis of Atri–PAA2000 for Physical Adsorption on Polystyrene Plates

3.1.3. Synthesis of Btri–pNPA30 for Chemical Immobilization on Aminated Polystyrene Plates

3.1.4. Synthesis of Btri–pNSA2000 for Chemical Immobilization on Aminated Polystyrene Plates


1. Add a mixture of 500 mg (1.3 mmol) of biot-NH(CH2)6NH2 and 0.4 mL (2.6 mmol) of NEt3 dissolved in 100 mL of DMSO to a solution of 4.7 mg (27.5 mmol) of pNPA in 300 mL of DMSO.

2. Add a mixture of 3 mg (5.5 mmol) of Btri-O(CH2)3NH2 and 1.5 mL (11 mmol) of NEt3 in 100 mL of DMSO to the obtained solution and keep it for 24 h at 40°C; absence of the free amin-oligands in the reaction mixture was confirmed by TLC; eluent A: Rf 0.3 for Btri-O(CH2)3NH2, Rf 0 for Btri–PAA30–biot5; elu-ent B: Rf 0.54 for biot-NH(CH2)6NH2; Rf 0 for Btri–PAA30–biot5 (see Subheading 3.2).



1. Add a mixture of 3 mg (5.5 mmol) of Btri-O(CH2)3NH2 and 1.5 mL (11 mmol) of NEt3 dissolved in 100 mL of DMSO to a solution of 4.7 mg (27.5 mmol) of pNPA–biot1 in 300 mL of DMSO.

2. Keep the mixture for 12 h at 40°C performing control for the reaction by TLC; eluent A: Rf 0.3 for Btri-O(CH2)3NH2, Rf 0 for Btri–PAA30–biot1; eluent B: Rf 0.54 for biot-NH(CH2)6NH2; Rf 0 for Btri–PAA30–biot1 (see Subheading 3.2).



1. Apply 5 mL of reaction mixture on a TLC plate and develop the plate using appropriate eluent (see Subheading 3.1.1–3.1.6).

2. Spray the charring reagent and heat the plate for 10 min at 150°C to visualize spots.

1. Apply reaction mixture on a glass column (75 × 1.5 cm) packed with Sephadex LH-20.

2. Elute the column with the eluent C. 3. Collect the first 50 mL of eluate and evaporate it to dryness. 4. Dissolve the residue in water and freeze dry the obtained

solution.

1. Add serially twofold diluted Atri–PAA solution in coating buf-fer (concentration 0.02–200 mg/mL or 2–2 × 105 pmol Atri per well) to the plate (100 mL per well); keep the plate for 1 h at 37°C (see Notes 5, 6, and 7).

3.1.5. Synthesis of Btri–PAA30–biot5 for Immobilization on Str Plates

3.1.6. Synthesis of Btri–PAA30–biot1 for Immobilization on Str Plates

3.2. Thin-Layer Chromatography

3.3. Preparative GPC

3.4. Plate Coating with Glycopolymers

3.4.1. Physical Adsorption


2. Wash the plate (hereinafter, the washing buffer, 200 mL per well).

3. Block the plate (the blocking buffer, 200 mL per well) and wash.

1. Add serially twofold diluted Btri–pNPA30 or Btri–pNSA2000 solu-tion in DMSO (concentration 0.02–200 mg/mL or 2–188 pmol Btri per well) to the aminated plate (100 mL per well) and incu-bate the plate for 24 h at room temperature.

2. Add 3 M aqueous ammonia to the plate (10 mL per well) and incubate for 24 h at room temperature.


4. Block the plate (the blocking buffer, 200 mL per well) and wash (See Note 7).

1. Rinse streptavidin-coated, black, 96-well plate (see Note 7) twice with PBS.

2. Add serial twofold dilution of coating solution in PBS (con-taining 0.002–80 mg/mL biotinylated glycoconjugate or, respectively, 2–9 × 104 pmol Btri per well) to the plate (100 mL per well); keep the plate for 1 h at 37°C.


4. Block the plate (the blocking buffer, 200 mL per well) and wash (See Note 7).

1. Add A16 mAbs (1:4,000 in PBS containing 0.3% BSA) to the 96-well transparent plate (100 mL per well).

2. Incubate the plate for 1 h at 37°C and wash with the washing buffer.

3. Add Ig–Biot (1:2,000 in PBS containing 0.3% BSA) to the plate (100 mL per well), incubate the plate for 1 h at 37°C, and wash with the washing buffer.

4. Add Str-HRPO (1:2,000 in PBS containing 0.3% BSA) to the plate (100 mL per well), incubate the plate for 1 h at 37°C, and wash with the washing buffer.

5. Just before use, add 0.03% H2O2 to a colorimetric solution and apply the obtained solution to the plate. Incubate the plate for 30 min at room temperature; stop the reaction by addition of 50 ml 1 M aqueous H2SO4. Read an absorbance at 492 nm in Victor2 counter.

6. Measure optical density (492 nm) by Victor2 counter. Carry out each assay in duplicate, and omit mAbs to obtain blank reaction.

3.4.2. Chemical Immobilization

3.4.3. Coating via Biotin–Str Bridge

3.5. ELISA

3.5.1. ELISA with Colorimetric Detection


1. Add B8 mAbs (1:100 in PBS containing 0.3% BSA) to the 96-well black plate (100 mL per well).

2. Incubate the plate for 1 h at 37°C and wash. 3. Add Ig–AP (1:5,000 in PBS containing 0.3% BSA) to the plate

(100 mL per well); incubate the plate for 1 h at 37°C and wash.

4. Add fluorescent-revealing solution to the plate (100 mL per well); incubate the plate for 30 min at room temperature.

5. Measure fluorescence intensity (355/460 nm) by Victor2 mul-tilabel counter (PerkinElmer, USA). Carry out each assay in duplicate, and perform blank reaction by omitting the mAbs. Subtract the blank reading from the final fluorescence to pro-vide the corrected fluorescence intensity values.

1. The meanings of apparent m.w. (in kDa) are shown in the gly-copolymer notations as the upper index. Degree of polymer-ization in the case of high-m.w. activated polyacrylic acid is reproduced with high accuracy; it could be monitored by ana-lytical GPC. For details, see ref. 16.

2. Molar fraction of the monomer units substituted with Glyc is 20 mol% for all the glycopolymers considered here; according to our previous experience, this is the optimal loading of poly-mer scaffolds affording to better binding with carbohydrate-recognizing antibodies (8, 9).

3. Molar fraction of the monomer units substituted with biotin in Glyc–PAA30–biot5 is 5 mol%, which corresponds to five biotin residues per molecule of the glycopolymer.

4. 30-kDa Glyc–PAAs can be available via home pages of CFG (http://www.functionalglycomics.org/), Lectinity Corp. (http://www.lectinity.com/), or GlycoTech Corp. (http://www.glycotech.com/).

5. Physical immobilization of Glyc–PAA is carried out at 37°C for 1 h or alternatively at 4°C for 12 h. These two regimens give rise to the same efficiency.

6. Coating buffer pH basically does not affect the efficiency of Glyc–PAA immobilization in the pH interval from 5.0 to 9.6. Salts-free water as a solvent gives twofold less rate of coating.

7. Serially diluted coating antigen and constant concentration of antibodies is recommended as a protocol optimization stage. Then, when the optimal antigen coating has been selected, the antibodies in test are serially diluted.

3.5.2. ELISA with Fluorimetric Detection

4. Notes


8. Storage and use of Str-coated plates should be in strict accor-dance with manufacturer’s manual in order to avoid loss of capac-ity. Otherwise, plates with similar quality and capacity could be prepared in-house by coating Str onto NUNC Maxisorb polysty-rene. 384-well avidin-coated plates could be used as well (6).

Acknowledgments

This study was supported by the Program of the Presidium of the RAS “Molecular and Cell Biology” (FP6 Project SP5B-CT- 2007-044512) and the RFBR grant #10-04-01693-a.

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