Assay method for the lectin activity of mannan-binding protein ~ELLBA-2
Mannan-binding protein (MBP), also called mannose-binding protein (MBP) or mannan-binding lectin (MBL), is a Ca2+-dependent (C-type) mammalian lectin specific for mannose, N-acetylglucosamine and fucose. MBP activates complement through the lectin pathway, and is an important serum component associated with innate immunity (Kawasaki T. 1999). MBP also binds to human colorectal carcinoma cells.
This chart shows the relation between a family of mannan-binding protein (MBP)-associated protocols.
Isolation of mannan-binding protein from human serum (plasma) ~Method 1 Isolation of mannan-binding protein from human serum (plasma) ~Method 2 Isolation of mannan-binding protein from human serum (plasma) ~Method 3
Assay method for the lectin activity of mannan-binding protein ~ELLBA-1* Assay method for the lectin activity of mannan-binding protein ~ELLBA-2 Assay method for the lectin activity of mannan-binding protein ~ELLBA-3
*PV-Man used in ELLBA-1 is not commercially available at present.
Determination of mannan-binding protein by enzyme-linked immunosorbent assaying (ELISA)
This method can be used for the estimation of the MBP content not only in the purified samples
but also in the crude samples like human serum or plasma.
Characteristics of each method were evaluated in a 3-point scale (see below).
Mannan from S. cerevisiae (Sigma-Aldrich (St. Louis, MO))
Biotin-AC5-hydrazide obtained from Dojindo Laboratories (Kumamoto, Japan), or biotin-LC-hydrazide from Thermo Scientific (Rockford, IL)
15 mM glycerol *1
100 mM sodium acetate buffer, pH 5.5 *2
10 mM sodium acetate buffer, pH 5.5 *3
TBS: 10 mM Tris/HCl, 145 mM NaCl, pH 7.4 *4
TBST: TBS containing 0.05％Tween *5
TBS containing 1% BSA *6
TBS containing 10 mM CaCl2 *7
TBST containing 10 mM CaCl2 *8
TBS containing 2 mM EDTA *9
TBST containing 2 mM EDTA *10
Mouse anti-human MBP monoclonal antibody (1 μg/mL): HYB 131-01 from BioPorto Diagnostics A/S (Gentofte, Denmark). Dilute the antibody (1 mg/ml) 1:10 with reagent 9, followed by further dilution 1:100 with reagent 10 (or reagent *9) (i.e., final dilution 1:1000, 1 μg/mL)
HRP-conjugated polyclonal rabbit anti-mouse immunoglobulins from DAKO Denmark A/S (Glostrup, (Denmark)), etc. Dilute the antibody 1/20 with reagent *6, followed by further dilution 1:100 with reagent 10 (or reagent *9) (i.e., final dilution 1:2000, 1 μg/mL) *11
HRP-development kit consisting of substrate, TMBZ (tetramethylbenzidine), and stop reagent (0.5M H2SO4) (Sumitomo Bakelite (Tokyo, Japan)) *12
Streptavidin-coated plate: Nunc streptavidin-coated plate and module from Thermo Scientific (Rockford, IL)
Microtiter plate reader
Quantification of MBP-binding to biotinylated mannan using streptavidin-coated plates: Biotinylation of mannan
React mannan (1mg/mL) with 1.5 mM NaIO4 in reagent *2 for 30 min on ice.
Stop the reaction with reagent *1.
Dialyze the mixture against reagent *3 overnight at 4 °C.
Incubate the dialyzed material with 5 mM biotin-AC5-hydrazide (biotin-LC-hydrazide) dissolved in reagent *2 for 2 h at 23 °C.
Dialyze the reaction product against reagent *3 overnight at 4 °C to remove non-reacted biotin.
Quantification of MBP-binding to biotinylated mannan using streptavidin-coated plates: Assaying of MBP binding to biotinylated-mannan on a streptavidin-coated plate
Wash the wells with 300 μL of reagent 8 per well 3 times.
Add 1 μg of biotinylated-mannan dissolved in 100 μL of reagent *4 to each well.
React the biotinylated-mannan with streptavidin-coated wells at RT for 2 h with gentle shaking.
Wash the wells with 300 μL of reagent *5 3 times.
Block the wells by adding 200 μL of reagent 9 for 2h~overnight at 4 °C.
Add 1 μg of MBP dissolved in 100 μL of reagent *7 (or reagent *9 for the control wells), and react overnight or for 2h with gentle shaking at 4 °C.
Wash the wells with 300 μL of reagent *8 (or reagent *10 for the control wells) 3 times.
Add 100 μL of mouse anti-human MBP monoclonal antibody (1.0 μg/mL) and react for 2 h with gentle shaking.
Add secondary antibody (reagent *11) at 100 μL per well and react for 1 h with gentle shaking.
Add 100 μL of TMBZ substrate (in reagent *12) per well.
Add 100 μL of stop solution (in reagent *12) per well to stop the color development.
Read absorbance at 450 nm with a plate reader.
ELLBA: Enzyme-linked lectin binding assay
Methods 1. Biotinylation of mannan:
Store the biotinylated-mannan at 4 °C.
Methods 2. Assaying of MBP binding to biotinylated-mannan on a streptavidin-coated plate:
One μg of MBP bound to 1 μg of biotinylated-mannan showed A405 nm≈0.9 with a plate reader.
Kawasaki T (1999) Structure and biology of mannan-binding protein, MBP, an important component of innate immunity. Biochim Biophys Acta 1473: 186-195. [PMID : 10580138]
Nadesalingam J, Bernal A L, Dodds A W, Willis A C, Mahoney D J, Day A J, Reid K B M, and Palaniyar N (2003) Identification and characterization of a novel interaction between pulmonary surfactant protein D and decorin. J Biol Chem 278; 25678–25687. [PMID : 12730206]
Nakamura N, Nonaka M, Ma BY, Matsumoto S, Kawasaki N, Asano S, Kawasaki T (2009) Characterization of the interaction between serum mannan-binding protein and nucleic acid ligands. J Leukoc Biol 86: 737-748. [PMID : 19465640]