Human Lipase(LIP) ELISA Kit

This assay has high sensitivity and excellent specificity for detection of Human LIP.Nosignificant cross-reactivity or interference between Human LIP and analogues wasobserved

Note:Limited by current skills and knowledge, it is impossible for us to complete thecross- reactivity detection between Human LIP and all the analogues, therefore, crossreaction may still exist..

• a) Dilution of standard solutions: (This kit provides one standard originalconcentration. Users may independently dilute in small tubes following the chart below.)
  
  
• b) The number of stripes needed is determined by that of samples to be tested added bythe standards. It is recommended that each standard solution and each blank well bearranged with multiple wells as much as possible.
• c) Sample injection: 1) Blank well: no sample or anti LIP antibody labeled with biotinor Streptavidin-HRP is added to comparison blank well; add chromogen solution A & B andstop solution, each other step operation is the same. 2) Standard solution well: Add 50μlstandard and Streptavidin-HRP 50μl (biotin antibodies have united in advance in thestandard so no biotin antibodies are added). 3) Sample well to be tested: Add 40μl sampleand then 10μl LIP antibodies, 50μl Streptavidin-HRP. Then cover it with seal platemembrane. Shake gently to mix. Incubate at 37℃ for 60 minutes.
• d) Preparation of washing solution: Dilute the washing concentration (30X) withdistilled water for later use.
• e) Washing: carefully remove the seal plate membrane, drain liquid and shake off theremainder. Fill each well with washing solution, let stand for 30 seconds, then drain. Repeatthis procedure five times then blot the plate.
• f) Color development: First add 50μl chromogen solution A to each well, and then add 50μl chromogen solution B to each well. Shake gently to mix. Incubate for 10 minutes at37℃ away from light for color development.
• g) Stop: Add 50μl Stop Solution to each well to stop the reaction (color changes fromblue to yellow immediately at that moment).
• h) Assay: Take blank well as zero, measure the absorbance (OD) of each well one byone under 450nm wavelength, which should be conducted within10 minutes after havingadded stop solution.
• i) According to standards concentrations and corresponding OD values, calculate thelinear regression equation of the standard curve. Then according to the OD value ofsamples, calculate the concentration of the corresponding sample. Statistical software couldalso be employed.

• a) Hold kit at room temperature for at least 30 minutes once removed from 2-8 ℃
• environment. Coated ELISA plates should be stored in sealed bag if not used after opening.
• b) When adding samples, sample injector must be used each time and should also be
• frequently checked for precision to avoid individual error.
• c) The operating instructions must be strictly followed and the reading of ELISA
• reader must be set as the standard for determining the experiment result.
• d) Pipette tips and seal plate membrane in hand should not be used more than once inorder to avoid cross contamination.
• e) All samples, washing concentration, and all waste should be disposed as infectiveagents.
• f) Other reagents not needed shall be packed or covered. Reagents of different batchesshall not be mixed and should be used before their respective validity dates.
• g) Substrate B is sensitive to light and therefore should not be over-exposed to light.

• ① 37℃ incubator
• ② Standard ELISA reader
• ③ Precision pipettes and disposable pipette tips
• ④ Distilled water
• ⑤ Disposable tubes
• ⑥ Absorbent paper

Intra-assay Precision (Precision within an assay): 3 samples with low, middle andhigh level Human LIP were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, middle andhigh level Human LIP were tested on 3 different plates, 8 replicates in each plate.

• a) Samples containing NaN3 shall not be tested as they inhibit the activity of HorseRadish Peroxidase (HRP).
• b) Upon sample collection, extraction should be carried out as soon as possible inaccordance with related documents. After extraction, the experiment should be conductedimmediately as well. Otherwise, the sample should be preserved at -20 ℃ . Repeatedfreeze-thaw cycles should be avoided.
• c) Serum: Allow the serum to clot for 10-20 minutes at room temperature, then placein centrifuge (at 2000-3000 RPM) for approximately 20 minutes. Collect the supernatants carefully. If sediments have occurred during storage, centrifugation should be repeated.
• d) Blood Plasma: In accordance with sample collection requirements, EDTA orsodium citrate should be used for anticoagulation. Add EDTA or sodium citrate and mixthem for 10-20 minutes, then place in centrifuge(at 2000-3000 RPM) for approximately 20minutes. Collect supernatants carefully. If sediments have occurred during storage,centrifugation should be repeated.
• e) Urine: Collect with sterile tube. Place in centrifuge (at 2000-3000 RPM) forapproximately 20 minutes. Collect the supernatants carefully. If sediments have occurredduring storage, centrifugation should be repeated.When collecting pleuroperitoneal fluid and cerebrospinal fluid, please follow theprocedures mentioned above.
• f) Cell Culture Supernatant: Collect with sterile tubes when examining secretecomponents. Place in centrifuge (at 2000-3000 RPM) for approximately 20 minutes.Collect the supernatants carefully. When examining the components within the cell, usePBS (PH 7.2-7.4) to dilute cell suspension to cell concentration of approximately 1million/ml. Degrade cells through repeated freeze-thaw cycles to release interiorcomponents. Place in centrifuge (at 2000-3000 RPM) for approximately 20 minutes.Collect the supernatants carefully. If sediments have occurred during storage, centrifugationshould be repeated.
• g) Tissue Sample: Incise sample and weigh. Add a given amount of PBS (PH 7.4).Immediately freeze with liquid nitrogen for later use. Thaw sample and hold at 2-8℃. Adda given amount of PBS (PH 7.4), then homogenize the sample thoroughly by hand or withhomogenizer. Place in centrifuge (at 2000-3000 RPM) for approximately 20 minutes.Collect the supernatants carefully. Aliquot and keep one for examination and freeze theothers for later use.

• a) Washing by Hand: Shake off liquids in the wells of the ELISA plate. Lay severalbibulous papers on the test bed and pat the ELISA plate hard several times downward. Theninject at least 0.35ml of diluted washing concentrate and soak for 1-2 minutes. Repeat asneeded.
• b) Washing by Automatic Plate Washer: If an automatic plate washer is available, itshould only be used in the test by those proficient with its function.