Application

This kit can be used to measure the lipase (LPS) content in serum, plasma and tissue samples. This kit (50 Assays) can detect 48 samples.

Detection principle

Micelle of Emulsion formed by triglyceride and water have milky appearance because of the absorption and scattering of incident light. Triglyceride will hydrolyze under the Lipase (LPS) and the micelle will split. Thus, the rate of turbidity reduction was measured by colorimetry, and the activity of lipase was indirectly determined.

Experimental instrument

Micropipettor, 37℃ water-bath, Spectrophotometer (420 nm )

Sample preparation

1. Serum/plasma: detect directly.

2. Tissue: Weigh the tissue sample accurately and mince the tissues to small pieces, then  homogenized in normal saline on ice, the volume of normal saline (mL): the weight of the tissue (g) =4:1. The tissue homogenate is centrifuged for 10 min at 2500 rpm and collect the supernatant for detect. Meanwhile, determine the concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).

Detection procedures

1. For serum (plasma) sample

(1) Set the spectrophotometer at 420 nm and set to zero with Reagent 2 with a 1 cm diameter cuvette.

(2) Preheat the Reagent 1 at 37℃ for more than 5 min.

(3) Add 50 μL of fresh sample to the tubes, then take 2 mL of preheated Reagent 1 into the tube. Mix immediately and record the time at the same time.

(4) Transfer the mixed solution to the cuvette rapidly, measure the OD value at 420 nm at 30 second (A1).

(5) Transfer the colorimetric-solution to the original tube and incubate in 37℃ water-bath for 10 min accurately, and then pour the liquid to the cuvette, measure the OD value at 420 nm at 630 second (A2).

(6) Measurement of the standard tube: Add 50 μL of normal saline to 2 mL of Reagent 1, measure the OD value at 420 nm (As). (Note: As value is equivalent to the absorbance of 454umol/L standard.)

2. For tissue sample

(1) Set the spectrophotometer at 420 nm and set to zero with Reagent 2 with a 1 cm diameter cuvette.

(2) Preheat the Reagent 1 at 37℃ for more than 5 min.

(3) Add 25 μL of 20% tissue homogenate supernatant to the tubes, then take 25 μL of Reagent 4 and 2 mL of preheated Reagent 1 into the tube. Mix immediately and record the time at the same time.

(4) Transfer the mixed solution into the cuvette rapidly, measure the OD value at 420 nm at 30 second (A1).

(5) Transfer the colorimetric-solution to the original tube and incubate in 37℃ water-bath for 10 min accurately, and then pour the liquid to the cuvette, measure the OD value at 420 nm at 630 second (A2).

(6) Measurement of the standard tube: Add 50 μL of normal saline to 2 mL of Reagent 1, measure the OD value at 420 nm (As). (Note: As value is equivalent to the absorbance of 454umol/L standard.)

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 validity of kit is 6 months.

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

5. The cuvette must be wash with deionized water, and then measure the OD value.

6. The absorbance of some sample will increase after reacting with substrate. It is the samples which repeated freezing-thawing or samples with increasing IgM (such as rheumatoid factor).

7. The lipase content in pancreas is very high, so it is recommended to take a pre-experiment to determine the best sampling concentration (△A≤0.4, △A=A1-A2).