PRINCIPLE OF THE METHOD：

The LEP kit is a solid phase phase sandwich enzyme linked immuno sorbent assay（ELISA）. Samples , including standards of  known LEP concentrations and unknowns are pipetted into these wells. During the first incubation, the LEP antigen and a biotinylated monoclonal antibody specific for LEP are simultaneously incubated.Afterwashing, the enzyme（streptavidin-peroxydase）is added. After incubation and washing to remove all the unbound enzyme, a substrate solution which is acting on the bound enzyme is added to induce a coloured reaction product. The intensity of this coloured product is directly proportional to the concentration of LEP present in the samples.

REAGENTS PROVIDED AND RECONSTTTUTION：

MATERIAL REQUIRED BUT NOT PROVIDED：

l        Distilled water

l       Pipettes:10ul、50ul、100ul、200ul、1000ul。

l        Vortex mixer and magnetic stirrer.

SAFETY：

l        For research use only

l        Avoid any skin contact with H2SO4 and TMB. In case of contact, wash thoroughly water.

l        Do not eat, drink, smoke or apply cosmetics where kit reagents are used.

l        Do not pipette by mouth.

PROCEDURAL  NOTES.  QUALITY ONTROL：

l        When not in use, kit components should be stored refrigerated or frozen as indicated on vials or bottles. All reagents should be warmed to room temperature before use. Lyophilized standards should be discarded after use.

l        Once the desired number of strips has been removed, immediay reseal the bag to protect the remaining strips from edterioration.

l        Cover or cap all reagents when not in use.

l        Do not mis or interchange reagents between different lots.

l        Do not use reagents beyond the expiration date of the kit .

l        Use a clean disposable plastic pipette tip for each reagent, standard, or specimen addition in order to avoid cross-contamination, for the dispensing of H2SO4 and substrate solution, avoid pipettes with metal parts.

l        Use a clean plastic container to prepare the washing solution.

l        Thoroughly mix the reagents and samples before use by agitation or swirling.

l        All residual washing liquid must be drained from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Never insert absorbent paper directly into the wells.

l        The TMB solution is light sensitive. Avoid prolonged exposure to light, also, avoid contact of the TMB solution with metal to prevent colour development. Warning TMB is toxic avoid direct contact with hands. Dispose off properly. If a dark blue colour develops within a few minutes after preparation, this indicates that the TMB solution has been contaminated and must be discarede. Read absorbances within 1 hour after completion of the assay.

l        When pipetting reagents, maintain a consistent order of addition from well-to-well. This will ensure equal incubation times for all wells.

l        Respect incubation times described in the assay procedure.

SPECIMEN  COLLECTION\ PROCESSING AND STORAGE：

l        Serum---Avoid any inintentional stimulation of the cells by the procedure. Use pyrogen\endotoxin free collecting tubes. Serum should be removed rapidly and carefully from the red cells after clothing. For that, after clothing, centrifuge at approximay 1000×g for 10 min and remove serum.

l        Plasma---EDTA\ citrate and heparin plasma can be assayed. Spin samples at 1000×g for 30 min remove particulates. Harvest plasma.

l        Cell culture supernatants---Remove particulates and aggregates by spinning at approximay 1000×g for 10 min.

l        tissue homogenate-----Add normal saline to tissue homogenate, 1000×g for 10 min, test supernatants

l        Storage---If not analyzed shortly after collection, samples should be aliquoted（250-500ul） to avoid freeze-thaw cycles and stored frozen at -70℃. Avoid multiple freeze-thaw cycles of frozen specimens. When possible, avoid use of badly hemolyzed or lipemic sera. If large amounts of particles are present, this should be removed prior to assay by centrifugation or filtration.

l        Recommendation---Do not thaw by heating at 37℃ or 56℃. Thaw at room temperature and make sure that sample is compley thawed and homogenous before assaying.

PREPARATION OF REAGENTS：

l        Standards: Standard have to be reconstituled with the volume of standard buffer diluent indicated on the vial. This reconstitution produces a stock solution of 50ng/ml LEP. Allow standard to stand for 5 minutes with gentle swirling prior to making dilutions. Serial dilutions of standard must be made before each assys and cannot be stored.

Washing buffer 50×concentrate:  Dilute 50 times in distilled water.

Manual Technique：

STEP 1 ： Determine the number of microwell strips required to test the desired number of samples,plus appropriate number of wells needed for running blanks standards. Each sample, standard and blank should be assayed in duplicate. Remove sufficient microwell strips from the pouch，Add 50ul of standard diluent to standard wells B1,B2,  C1,C2,  D1,D2,  E1,E2,  F1,F2. Reconstitute standard vial with the appropriate volume as described in the chapter reagents preparation. Preparation. Pipet 100ul of standard into wells A1 and A2 （see plate scheme below）. Transfer 50ul from A1 and A2 to B1 and B2 wells. Mix the contents by repeated aspirations and ejections. Take care not to scratch the inner surface of microwells. Repat this procedure from the wells B1,B2 to wells C1,C2 and from wells C1,C2 to D1,D2 and so on creating two parallel rows of LEP standard dilutions ranging，Add 50ul of standard diluent to the bland wells. Add 50ul of sample to sample wells.

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STEP2：Add 50ul of diluted biotinylated anti-LEP to all wells.

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Incubate for 45 min.at 37℃Wash 5 times

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STEP3：Distribute 100ul of streptavidin-HRP solution to all wells, including blank wells.

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Incubate for 30 min.at 37℃Wash 5 times

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STEP4：  Add 50ul Substrate A and Substrate B to each well。Incubate for 5 min at 37℃

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STEP 5 ：    Add 50μL of Stop Solution

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Read absorbance at 450nm within 30 min

CALCULATION OF RESULTS：

l        Calculate the average absorbance values （A₄₅₀） for each set of reference standards, control, and samples. Construct a standard curve by plotting the mean absorbance obtained for each reference standard against its concentration on linear graph paper, with absorbance on the vertical （y） axis and concentration on the horizontal （x） axis.Using the mean absorbance value for each sample, determine the corresponding concentration of in from the standard curve.

l         The minimum detectable concentration in this assay is estimated to be0.1 ng/ml

l         Do not extrapolate the standard curve beyond the max  standard curve point. The dose-response is non-linear in this region and good accruacy is difficult to obtain.

l         This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.