Application

This kit can be used for detection of ACP activity in serum (plasma), tissue, cells and other sample. This kit (100 Assays) can detect 96 samples.

Detection significance

Acid phosphatase (ACP) is a kind of enzyme which catalyzes the hydrolysis of phosphate monoester to phosphoric acid under acidic conditions. There are different acid phosphatase isozymes in different organs. These isozymes differ greatly in tissue and chromosome origin, molecular weight, amino acid homology, sequence length, and resistance to L-tartrate or fluoride.

Detection principle

Acid phosphatase decomposes disodium phenyl phosphate under acidic conditions to produce free phenol and phosphoric acid. Phenol acts with 4-aminoantipyrine in alkaline solution, and oxidizes to a derivative of red quinone by potassium ferricyanide. The activity of the ACP can be calculated by measuring the OD value at 520 nm.

Experimental instrument

Test tube, Micropipettor, Vortex mixer, 37℃ water bath, Spectrophotometer (520 nm)

Sample preparation

1.   Serum/plasma: Detect the sample directly. If the concentration is beyond the linear range, please dilute the sample with normal saline before detection.

2.   Tissue: Weigh 0.02-1.0 g tissue sample, rinse with distilled water and dry with filter paper, then weigh the tissue and add 9 times the volume of PBS (0.01 M, pH7~7.4) according to the ratio of Weight (g): Volume (mL) =1:9. Mechanical homogenate the sample in ice water bath. Centrifuge at 3100 g for 10 min, then take the supernatant and preserve it on ice for detection. Meanwhile, determine the protein concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).

3.   Culture cell sample: Wash the cells with PBS (0.01 M, pH7~7.4) for 1~2 times. Centrifuge at 1000 g for 10 min and then discard the supernatant and keep the cell sediment. Add PBS at a ratio of cell number (106): PBS (μL) =1: 300-500. Sonicate or grind with hand-operated in ice water bath. Centrifuge at 10000 g for 10 min, then take the supernatant and preserve it on ice for detection. Meanwhile, determine the protein concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).

Operation steps

1.   Blank well: add 50 μL of double distilled water into a 5 mL EP tube.

Standard well: add 50 μL of 0.1 mg/mL Phenol standard application solution into a 5 mL EP tube.

Sample well: add 50 μL of Sample into a 5 mL EP tube.  

2.   Add 500 μL of Reagent 1 and 500 μL of Reagent 2 respectively and oscillate fully with the vortex mixer.

3.   Incubate at 37℃ for 30 min, then add 1000 μL of Reagent 3 and 1500 μL of Reagent 4 immediately, oscillate fully with the vortex mixer and stand at room temperature for 10 min.

4.   Set spectrophotometer to zero with double distilled water and measure the OD values of each tube at 520 nm with 1 cm diameter cuvette.

Note: It can be refer to the following operating table

Technical parameters

1.  The sensitivity of the kit is 0.27 U/100 mL.

2.  The detection range of the kit is 0.27-40 U/100 mL.

3.  The intra-assay CV is 2.8 % and the inter-assay CV is 7.1 %.

4.  The recovery of the kit is 100 %.

Notes

1.  This kit is for research use only.

2.  Instructions should be followed strictly, changes of operation may result in unreliable results.

3.  The valid of kit is 3 months.

4.  Do not use components from different batches of kit.