AIM (Apoptosis Inhibitor of Macrophage), a protein produced by macrophage and present in the blood, interacts with both adipose cells and macrophages) . By inducing neutral fat breakdown in adipose cells and inhibits cellular death of macrophage, AIM shows a strong association with various lifestyle-related diseases such as obesity, arteriosclerosis, and diabetes2-5). In recent years, it is indicated that AIM plays a role in the pathogenesis of autoimmune disease associated with obesity 6).
As seen above, amount of AIM in the blood is related to pathological condition of many diseases problematic in modern society, and the measurement of AIM is suggested to be effective for diagnostic and therapeutic research of the lifestyle-related diseases. Precise measurement may be difficult depending on the antibody character since AIM is attached to IgM in the blood, however the antibody used in this kit is unaffected by IgM, which enables accurate measurement of AIM7).
This assay kit employs sandwich ELISA method utilizing mouse monoclonal antibody specific for human AIM (hAIM) protein. hAIM-specific antibody is pre-coated on the microplate. hAIM in standard or specimen (biological sample such as cell culture supernatant and serum) binds specifically to antibody attached on each well of microplate. Then, biotinylated hAIM-specific antibody and HRP-labeled Avidin are added.
Finally, colorimetric substrate (TMB) is added and reacted, which produces a color in proportion to the amount of hAIM in the sample. After stopping the enzyme reaction by adding sulfuric acid, the microwell absorbances are measured for the dominant wavelength at 450 nm (reference wavelength: around 650 nm) utilizing microplate reader. Plot the calibration curve using hAIM Standard, and hAIM concentration in the specimen is determined by finding the absorbance value on the calibration curve.
○ Antibody Coated Wells --- 1 (6 strips)
○ Biotinylated Antibody (30×) --- 1 (200 μL)
○ HRP-labeld Avidin (30×) --- 1 (200 μL)
○ Enzyme Diluent --- 1 (12 mL)
○ Standard/Sample Diluent --- 1 (30 mL)
○ Wash Concentrate (20×) --- 1 (30 mL)
○ TMB Substrate Reagent --- 1 (12 mL)
○ Stop Solution (sulfuric acid) --- 1 (12 mL)
○ hAIM Standard (freeze dried) --- 1
(add 250uL distilled water just before use)
MATERIALS REQUIRED BUT NOT PROVIDED
○ Microplate reader capable of measurement at 450 nm [reference wavelength at around 650 nm]
○ Precision pipettes or multichannel pipettes to deliver 10 μL -1000 μL volumes, and disposable tips
○ Tubes with low-protein absorbent to prepare standard dilutions
○ Measuring cylinder or beaker (500 mL) to prepare wash solution
○ Distilled water
○ Do not mix reagents from different kit lots.
○ Diluted reagents should not be stored for more than 24 hours.
○ Do not discontinue the operation during the experimentation. Also, do not leave the microplate dehydrated during operation.
○ Use microplate lid to prevent drying during operation if needed.
○ All standards and samples are recommended to be run in duplicate.
○ Biotinylated Antibody solution
Add 200 μL of 30×Biotinylated Antibody to 5800 μL Standard/Sample Diluent to prepare 6.0 mL solution.
○ HRP-labeled Avidin solution
Add 200 μL of 30×HRP-labeled Avidin to 5800 μL Standard/Sample Diluent to prepare 6.0 mL solution.
○ Wash Buffer
Add 25 mL of Wash Concentrate to 475 mL of distilled water to prepare 500 mL solution.
○ hAIM Standard
Reconstitute hAIM Standard with 250 μL of distilled water to prepare a 200 ng/mL stock standard. Add 120 μL Standard/Sample Diluent to 8 tubes, respectively. Perform serial doubling dilutions by adding 120 μL of each standard to the next tube to create concentrations as described below. #1; 100 ng/mL, #2; 50.0 ng/mL, #3; 25.0 ng/mL, #4; 12.5 ng/mL, #5; 6.25 ng/mL, #6; 3.125 ng/mL, #7; 1.5625 ng/mL, #8; 0 ng/mL
1. Pipette 300 μL of Wash Buffer to each well, and soak at 25°C for 20 minutes. (See Flowchart indicated below)
2. Wash wells by filling with 300 μL/well Wash buffer, and tap the plate to remove any residual buffer. Repeat wash 3 times.
3. Pipette 50 μL of each standard or sample into antibody coated wells, and incubate for 1 hour at 37°C.
4. Wash wells as in Step 2.
5. Pipette 50μL of Biotinylated Antibody solution into each well, and incubate for 1 hour at 37°C
6. Wash wells as in Step 2.
7. Add 50μL of HRP-labeled Avidin solution into each well, and incubate for 1 hour at 37°C.
8. Wash wells as in Step 2.
9. Add 100μL of TMB Substrate Reagent to each well. Incubate for 10 minutes at room temperature (25) in the dark.
10. Add 100μL of Stop Solution to each well.
11. Read absorbance at 450 nm (reference wavelength at 650nm) utilizing microplate reader.
CALCULATION OF RESULTS
hAIM concentration may be calculated by utilizing calculation program stored in most of the microplate readers. Also, hAIM concentration in the sample is determined by finding the absorbance value on the calibration curve.
Apoptosis Inhibitor of Macrophage (hAIM) ELISA kit
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.