Mac-2 Binding Protein, Human

This kit is a solid phase sandwich ELISA using 2 kinds of highly specific antibodies.

Tetra Methyl Benzidine (TMB) is used as a coloring agent (Chromogen). The strength of coloring is proportional to the amount of Human Mac-2bp.

Materials needed but not supplied

• Plate reader (450nm)
• Micropipette and tip
• Graduated cylinder and beaker
• Deionized water
• Refrigerator (as 4°C)
• Graph (semilogarithmic) paper
• Paper towel
• Tube for dilution of Standard
• Washing bottle for precoated plate
• Disposable test tube for “2, Labeled antibody Conc.” and “6, Chromogen”

Preparation

1) Preparation of wash buffer
“8, Wash buffer Conc.” is a concentrated (40X) buffer. Adjust the temperature of “8, Washing buffer Conc.” to room temperature and then, mix it gently and completely before use. Dilute 50ml of “8, Wash buffer Conc.” with 1,950ml of deionized water and mix it. This is the wash buffer for use. This prepared wash buffer shall be stored in refrigerator and used within 2 weeks after dilution.

2) Preparation of Labeled antibody
“2, Labeled antibody Conc.” is a concentrated (30X). Dilute “2, Labeled antibody Conc.” with “5, Solution for Labeled antibody” in 30 times according to required quantity into a disposable test tube. Use this resulting solution as Labeled antibody.

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 the resulting solution by 100μl 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 firmly sealed vial.

3) Preparation of Standard
Put just 0.5 mL of deionized water into the vial of “3, Standard” and mix it gently and completely. This solution is 200ng/ml human Mac-2bp standard.

4) Dilution of Standard
Prepare 8 tubes for dilution of “3, Standard”. Put 230μl each of “4, EIA buffer” into the tube.Specify the following concentration of each tube.”

Put 230μl of Standard solution into tube-1 and mix it gently. Then, put 230μl of tube-1 mixture into tube-2. Dilute two times standard solution in series to set up 8 points of diluted standard between 100ng/ml and 0.78ng/ml. Tube-9 is the test sample blank as 0ng/ml.

See following picture.

5) Dilution of test sample
Test samples should be diluted with “4, EIA buffer” suitably.
Serum or plasma samples have to be diluted with “4, EIA buffer” accordingly.
The recommended dilution for them is from 500 to 1,000-fold. In case of the absorbance of sample is over than the assay range, it is necessary to dilute it more.

<Example of 500-fold dilution of serum or plasma>

1. Add 20μl of serum or plasma to 380μl of “4, EIA buffer” in a tube and mix them well.
2. Pipette 20μl of 20-fold diluted serum or plasma from the tube of above first dilution and add it to 480μl of “4, EIA buffer” in another tube, and mix them well.
   (In the case of 1,000-fold dilution, pipette 10μl of 20-fold diluted sample and add it to 490μl of “4, EIA buffer”)
3. This 500-fold diluted serum or plasma should be applied as a test sample according to the measurement procedure.

Measurement procedure

All reagents shall be brought to room temperature approximately 30 minutes before use. Then mix it gently and completely before use. Make sure of no change in quality of the reagents. Standard curve shall be prepared simultaneously with the measurement of test samples.

1. Determine wells for reagent blank. Put 100μl each of “4, EIA buffer” into the wells.
2. Determine wells for test sample blank, test sample and diluted standard.
   Then, put 100μl each of test sample blank (tube-9), test sample and dilutions of standard (tube-1-8) into the appropriate wells.
3. Incubate the precoated plate for 60 minutes at 4°C after covering it with plate lid.
4. Wash the plate with the prepared wash buffer and remove all liquid.
5. Pipette 100μl of labeled antibody solution into the wells of test samples, diluted standard and test sample blank.
6. Incubate the precoated plate for 30 minutes at 4°C after covering it with plate lid.
7. Wash the plate with the prepared wash buffer and remove all liquid.
8. Take the required quantity of “6, Chromogen” and put it into a disposable test tube. Then, pipette 100μl from the test tube into every well. Please do not return the rest of used chromogen in the test tube into “6, Chromogen” bottle in order to avoid contamination.
9. Incubate the precoated plate for 30 minutes at room temperature in the dark.
   The solution of Chromogen will turn blue.
10. Add 100μl of “7, Stop solution” to all wells. Mix the solution by tapping the side of precoated plate. The solution will turn yellow by addition of “7, Stop solution”.
11. Remove any dirt or drop of water on the bottom of the precoated plate and confirm there is no bubble on the surface of the solution. Then, run the plate reader and conduct measurement at 450nm against a reagent blank.
    The measurement shall be done within 30 minutes after addition of “7, Stop solution”.

Calculation of Test Result

Subtract the absorbance of test sample blank from all data, including standards and unknown samples before plotting. On a semi-logarithmic paper the concentration of the standards (x-axis, logarithmic) are plotted against their corresponding absorbance (y-axis, linear). Draw the best smooth curve through these points. Read the concentration for unknown samples from the standard curve. In automated method, 4 parameter logistics can generally give a good fit.

The typical standard curve is shown above. This curve cannot be used to derive test results. Please run a standard curve for each assay.

Performance Characteristics

Dilution linearity

Added Recovery Assay

Intra – Assay

Inter – Assay

Sensitivity

0.08ng/ml

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.