Human free fatty acids（FFA）ELISA Kit instruction

Kit name

Human free fatty acids（FFA）ELISA Kit

Intended use

The kit is used to assay the content of Human free fatty acids（FFA）in Human serum，blood plasma and other related tissue liquid.

Test principle

The kit uses a double-antibody sandwich enzyme-linked immunosorbent assay （ELISA） to assay the level of Human free fatty acids（FFA）in samples. Add Human free fatty acids（FFA）to pre-coated Human free fatty acids（FFA）monoclonal antibody microelisa well, incubation; washing. Add HRP tagged Human free fatty acids（FFA）antibodies. After another incubation and washing, remove the unbound enzyme, add Chromogen Solution A and B, the color of the liquid change into blue, and the color finally become yellow at the effect of acid. The depth of the color is positively correlated with concentration of the Human free fatty acids（FFA）in samples.

Materials supplied

Note: Standard was diluent with Standard diluent followed by: 32、16、8、4、2、1nmol/mL.

Materials required but not supplied

1.         37 ℃ incubator

2.         Standard microplate reader

3.         Precision pipettes and Disposable pipette tips

4.         Distilled water

5.         Disposable tubes for sample dilution

6.         Absorbent paper

Assay procedure

1.        Prepare: The kit takeing out from the environment of 2-8℃ should be balanced 30 minutes at less in the room temperature before using.

2.        Diluent: Diluent the 20×wash solution.

3.        Add standard and Sample: Set Standard wells, testing sample wells and blank wells. Add Diluted standard 50μl to standard well; Add Sample dilution 40μl to testing sample well which on Assay plate, then add testing sample 10μl （sample final dilute degree is 5 times）, blank well doesn’t add anyting.

4.        Incubation: Incubate 30 minutes at 37 ℃ in incubator.

5.        Wash: Discard Liquid, drying, filling in diluted washing liquid to each well, oscillation for 1 min, discard the washing liquid with absorbent paper Pat dry. Repeat three times, Pat dry.

6.        Add HRP-conjugate reagent: Add HRP-conjugate reagent 50μl to each well, except the blank well. Mixing gently shaking, incubated 30 minutes at 37 ℃.

7.        Repeat step4.

8.        Repeat step5

9.        Add chromogen solution A and B: Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix, incubate for 15 min at 37℃.

10.    Add Stop Solution: Add Stop Solution 50μl to each well, Stop the reaction（the blue color change to yellow immediay）.

11.    Take blank well as zero, measure the optical densit （OD） at 450 nm after adding Stop Solution and within 15 min.

12.    According to standard concentration and the corresponding OD values calculated standard curve linear regression equation, then the OD values according to the sample on the regression equation to calculate the corresponding sample concentration. It should be remembered that the sample has been diluted and its actual concentration should be multiplied by the total dilution.

Specimen requirements

1.      Can’t detect the samples which contain NaN3, because NaN3 inhibits HRP activity of the horseradish peroxidase.

2.      Extract as soon as possible after Specimen collection, Extracted according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can not be tested immediay, specimen can be kept in -20℃ to preserve, but repeated freezing and thawing should be avoided.

3.      The samples shoule be centrifugated dequay and no hemolysis or granule was allowed.

Important notes

1.         The operation should be carried out in strict accordance with instructions and test results must be based on microplate reader to determine readings shall prevail.

2.         If the microelisa stripplate has not used up after open, it should be stored in the sealed bag.

3.         Recommended that all standard materials, test samples are doing double to minish the Experimental error.

4.         Please multiply total dilution times when calculation. 5 times is the best dilute time according to this ELISA Kit design.

5.         If the testing material content in the sample is excessively high, please use Special dilution to dilute certain multiple, then assay.

6.         If the color too shallow, It may be appropriate to extend the substrate incubation time.

7.         Add Sample with sampler each step and proofread its accuracy frequently to avoid the experimental error. In order to avoid cross-contamination, avoid to reusing the suction head and closure plate membrane.

8.         Use the kit in validity, not mix the reagents of different batches.

9.         Chromogen Solution B is light-sensitive, avoid prolonged exposure to light,

Summary procedures

Preparing reagents, samples and standard

Add prepared sample and standard, incubated 30 minutes at 37 ℃

Plate washed four times, adding HRP-Conjugate Reagent incubated 30 minutes at 37 ℃

Plate washed four times, adding Chromogen Solution A and B incubated 15 minutes at 37 ℃

Add stop solution

Measure within 15min

Calculation

Assay range：1-32nmol/mL

Package size: 96 determinations

Storage：  2-8℃.

validity： six months.