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Assay method for the lectin activity of mannan-binding protein ~ELLBA-1
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Assay method for the lectin activity of mannan-binding protein ~ELLBA-1

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Introduction Protocol References Credit lines
Category
Sugar binding proteins
Protocol Name

Assay method for the lectin activity of mannan-binding protein ~ELLBA-1

Authors
 Kawasaki, Nobuko
Research Center for Glycobiotechnology, Ritsumeikan University
KeyWords
Reagents
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PV-Man (poly[N-p-vinylbenzyl-O-β-D-mannopyranosyl-(1,4)-D-mannamide]) from Seikagaku Corp., Tokyo, Japan dissolved in H2O (1 mg/mL)

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TBS; 50 mM Tris-HCl buffer, pH 7.5, 150mM NaCl

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TBS containing 0.05% Tween20

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TBS containing 10 mM CaCl2 *1

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TBS containing 2 mM EDTA *2

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TBS containing 10 mM CaCl2 , 0.05% Tween20 *3

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TBS containing 2 mM EDTA, 0.05% Tween20 *4

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TBS containing 1 % bovine serum albumin (BSA), 10 mM CaCl2, 0.1 % NaN3 *5

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TBS containing 1 % BSA, 2mM EDTA, 0.1 % NaN3 *6

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Blocking solution: TBS containing 3% BSA, 0.1 % NaN3

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Substrate solution: p-nitrophenylphosphate 2Na dissolved (1 mg/mL) in 50 mM Na2CO3, 0.5 mM MgCl2 *7

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Reference MBP: purified recombinant human MBP or MBP purified from human serum

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Mouse anti-human MBP monoclonal antibody (10 μg/mL): HYB 131-01 from BioPorto Diagnostics A/S, Gentofte, Denmark

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Alkaline phosphatase–conjugated goat anti-mouse IgG from Vector Laboratories Inc., Burlingame, CA etc.

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Microtiter plates (FALCON 35-3072: BD, Franklin Lakes, NJ)

Instruments
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Microtiter plate reader
Methods
1.

Quantification of MBP bound to PV-Man-coated plates

1) 

 Coat a microtiter plate with PV-Man at 100 μg/mL in H2O (50 μL/well) for 18 h at 4°C.

(The rest of the procedure is performed at room temperature, unless otherwise stated.)

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2) 

 Block with reagent 10 for 2 h at room temperature.

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3) 

 Wash the wells with reagent *3.

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4) 

 Incubate the wells for 2 h at room temperature with a sample containing MBP (50 μL/well) diluted with reagent *5.

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5) 

 Wash the wells with reagent *3.

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6) 

 Add 10 μg/mL of monoclonal antibody to human MBP in reagent *5 (50 μL/well) to the wells.

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7) 

 Incubate for 18 h at 4°C.

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8) 

 Wash the wells with reagent *3.

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9) 

 Incubate the wells with alkaline phosphatase-conjugated anti-mouse IgG diluted 1000-fold in reagent *5 (50 μL/well) for 2 h at room temperature.

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10) 

 Wash the wells with reagent *1.

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11) 

 Add reagent *7 to each well (100 μL/well).

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12) 

 Incubate at room temperature for 30–90 min.

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13) 

 Read the A405 nm of each well with a microtiter plate reader.

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Notes

ELLBA: Enzyme-linked lectin binding assay

Figure & Legends

Figure & Legends

Fig. 1. Enzyme-linked lectin binding assaying of MBP

The amount of recombinant human MBP bound to PV-Man-coated plates was determined by the ELLBA-1 method in the presence of 10 mM CaCl2 (solid circles) or 2 mM EDTA (open circles).

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Date of registration：2015-03-26 16:31:54
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