Mac-2 Binding Protein, Mouse

Materials needed but not supplied

• Plate reader
• Micropipette and tip
• Test tubes for dilution
• Measuring cylinder and beaker
• Deionized water
• Plate washer
• Paper towel
• Collecting container (i.e. clean disposable test tube)
• Refrigerator
• Incubator (37℃±1℃)

Measurement Principle

This kit is a solid phase sandwich ELISA (Enzyme-linked Immunosorbent Assay).

As a primary antibody is coated on a plate, samples and standard are added into the wells for 1st reaction. After the reaction, HRP-conjugated secondary antibody is added into the wells for 2nd reaction. After washing away unbound the secondary antibody, Tetra Methyl Benzidine (TMB) is added to the wells and color develops.

Preparation

1) Preparation of wash buffer
Dilute “8, Wash buffer conc.” 40 fold with deionized water. The diluted one is used for the assay as a wash buffer. Adjust the required quantities if needed.

2) Preparation of labeled antibody

Dilute “2, Labeled antibody conc.” 30 fold with “5, Solution for labeled antibody” using a prepared collecting container. Example)

In case you use one strip (8 well), the required quantity of Labeled antibody is 800μl. (Dilute 30μl of “2, Labeled antibody Conc.” with 870μl of “5, Solution for labeled antibody” and mix it. And use 100μl the mixed solution in each well.)

This operation should be done just before applying labeled antibody.

The remaining “2, Labeled antibody Conc.” should be stored at 4°C in a firmly sealed vial.

3) Preparation of standard

Add 0.5ml of deionized water into the vial of “3, Standard” and completely dissolve it. Concentration of the standard is 100ng/ml. The standards enclosed in this kit can be frozen and stored after reconstitution. However the freeze-thaw shall not be repeated.

Prepare 7 test tubes for dilution of the standard and adding 230μl of the EIA buffer into each tube.

Put 230μl of 100ng/ml standard into the tube 50ng/ml (Tube-1) and gently mix it. Afterword, put 230μl of the mixed liquid of tube-1 into the tube 0.78ng/ml (Tube-2) and gently mix it. Dilute two fold standard solutions in series to set up 7 points of diluted standard between 0.78ng/ml and 50ng/ml.

4) Preparation of test samples

Dilute test samples with “4, EIA buffer” contained in this kit as follows.

Mouse serum:                        50 fold.

Mouse EDTA plasma:           50 fold

Cell culture supernatant:      more than 4 fold

Tissue extract:                        5 fold.

Measurement Procedure

1. Add test sample blank
   Determine wells for test sample blank. Put 100μl each of “4, EIA buffer” into the wells.

2. Add prepared test samples and standard
   Put 100μl prepared test samples and 100μl prepared standard into appropriate wells.

3. Incubation with plate lid (1st reaction).

4. Washing
   Wash the plate with the prepared wash buffer and remove all liquid.

5. Add prepared labeled antibody
   Put 100μl prepared labeled antibody into the wells.

6. Incubation with plate lid (2nd reaction).

7. Washing
   Wash the plate with the prepared wash buffer and remove all liquid completely.

8. Add “6, Chromogen – TMB solution”
   Put 100μl the TMB solution into the wells.

9. Incubation in dark

10. Add “7, Stop solution”
    Put 100μl the Stop solution into the wells.

11. Determination of optical density (O.D.)Remove any dirt or drop of water on the bottom of the plate and confirm there is no bubble on the surface of the liquid. Then, measure the both O.D. of standard and the test samples against a test sample blank.Measurement wavelength: 450nm. In case of 2 wavelengths:Main wavelength is 450nm. Sub-wavelength is between 600 and 650nm.

Table for Measurement Procedure

Calculation of Test Result

1        Plot the concentration of the standard on the x-axis and its O.D. on the y-axis. Draw a standard curve by applying appropriate regression curve on each plot (i.e. quadratic regression of double logarithm conversion).

2        Read the concentration by applying the absorbance of the test samples on a standard curve.

3        Calculate the concentration of the test samples by multiplying dilution ratio of test samples on the value.

Example of standard curve and measured value

Added recovery assay

Intra-assay

Inter-assay

Mac-2 binding protein (Mac-2bp, galectin-3 binding protein, galectin-3bp, M2BP, and 90K), is a highly N-glycosylated, secreted protein, identified as a ligand of Galectin-3. It is considered that through interaction with Galectin-3, Mac-2bp promotes homotypic cell-cell contact or regulates cell adhesion. And it has been reported that Mac-2bp levels in blood have associations with various cancers, an indicator or metastatic tumors and chronic hepatic diseases such as NASH (Non-Alcoholic Steatohepatitis).  Some reports suggest that fluctuations in N-glycosylation on Mac-2bp produced by hepatocytes mirror the progression of liver disease and fibrosis. These reports identify Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA+-M2BP or M2BPGi), a lectin-specific variant of Mac-2 bp, as a biomarker useful for the assessment of disease severity in non-alcoholic fatty liver disease (NAFLD).  Other reports provide conflicting data that total Mac-2bp can detect early stages of fibrosis compare to WFA+-M2BP (M2BPGi).

Mac-2bp has been also promising to be an emerging useful tool for researching NASH in animal models.  New data (below) shows increased serum levels of Mac-2 binding protein are detected in mice fed NASH Meal after 4 and 8 weeks.

Explanation of Method of Feeding

Normal Meal

The meal that is used as a normal meal at an animal testing center

was fed to mice.

NASH Meal

High fat and high cholesterol food (7.5% fatty acid, 1.25% cholesterol, 0.5% and 0.5% cholic acid) was fed to mice for 4 weeks or 8 weeks.