大鼠瘦素(LEP)ELISA试剂盒使用说明

 大鼠瘦素(LEP)ELISA试剂盒使用说明

 

Rat Leptin (LEP) ELISA Kit

 

For  the quantitative  determination of rat leptin (LEP) concentrations in 

serum, plasma, cell culture supernates, tissue homogenates.

This package insert must be read in its entirety before using this product.

 

In order to obtain higher efficiency service, please ready to supply the lot number 

of the kit to us (found on the outside of the box).2

PRINCIPLE OF THE ASSAY

This assay employs the quantitative sandwich enzyme immunoassay technique. 

Antibody specific for LEP has been pre-coated onto a microplate. Standards and 

samples are pipetted into the wells  and  any  LEP present is bound by the 

immobilized antibody. After  removing  any unbound substances, a 

biotin-conjugated antibody specific for LEP is added to the wells. After washing,

avidin conjugated Horseradish Peroxidase (HRP) is added to the wells.

Following a wash to remove any unbound avidin-enzyme reagent, a substrate 

solution is added to the wells and color develops in proportion to the amount of 

LEP bound in the initial step. The color development is stopped and the intensity 

of the color is measured.

DETECTION RANGE

0.312 ng/ml-20 ng/ml.

SENSITIVITY

The minimum detectable dose of rat LEP is typically less than 0.078 ng/ml.

The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as 

the lowest protein concentration that could be differentiated from zero. It was 

determined the mean O.D value of 20 replicates of the zero standard added by 

their three standard deviations.

SPECIFICITY

This assay has high sensitivity and excellent specificity for detection of rat LEP. 

No significant cross-reactivity or interference  between rat LEP and analogues 

was observed.

Note: Limited by current skills and knowledge, it is impossible for us to complete 

the cross-reactivity detection between rat LEP and all the analogues, therefore,

cross reaction may still exist.3

PRECISION

Intra-assay Precision (Precision within an assay): CV%<8%

Three samples of known concentration were tested twenty times on one plate to 

assess.

Inter-assay Precision (Precision between assays): CV%<10%

Three samples of known concentration were tested in twenty assays to assess.

LIMITATIONS OF THE PROCEDURE

? FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC 

PROCEDURES.

? The kit should not be used beyond the expiration date on the kit label.

? Do not mix or substitute reagents with those from other lots or sources.

? If samples generate values higher than the highest standard, dilute the 

samples with Sample Diluent and repeat the assay.

? Any variation in  Sample Diluent, operator, pipetting technique, washing 

technique, incubation time or temperature, and kit age can cause variation 

in binding.

? This assay is designed to eliminate interference by soluble receptors, 

binding proteins, and other factors present in biological samples. Until all 

factors have been tested in the Immunoassay, the possibility of 

interference cannot be excluded.4

MATERIALS PROVIDED

Reagents Quantity

Assay plate (12 x 8 coated Microwells) 1(96 wells)

Standard (Freeze dried) 2

Biotin-antibody (100 x concentrate) 1 x 120 μl

HRP-avidin (100 x concentrate) 1 x 120 μl

Biotin-antibody Diluent   1 x 15 ml

HRP-avidin Diluent    1 x 15 ml

Sample Diluent   1 x 50 ml

Wash Buffer (25 x concentrate) 1 x 20 ml

TMB Substrate  1 x 10 ml

Stop Solution   1 x 10 ml

Adhesive Strip (For 96 wells) 4

Instruction manual 1

STORAGE

Unopened kit Store at 2 - 8°C. Do not use the kit beyond the expiration date

Opened kit

Coated assay 

plate

May be stored for up to 1 month at 2 - 8°C. 

Try to keep it in a sealed aluminum foil bag,

and avoid the damp.

Standard May be stored for up to 1 month at 2 - 8° C. If 

don’t make recent use, better keep it store at 

-20°C.

Biotin-antibody 

HRP-avidin

Biotin-antibody 

Diluent

May be stored for up to 1 month at 2 - 8°C.

HRP-avidin 

Diluent

Sample

Diluent

Wash Buffer

TMB 

Substrate

Stop Solution

*Provided this is within the expiration date of the kit.5

OTHER SUPPLIES REQUIRED

? Microplate reader capable of measuring absorbance at 450 nm, with the 

correction wavelength set at 540 nm or 570 nm.

? An incubator which can provide stable incubation conditions up to 

37°C±0.5°C.

? Squirt bottle, manifold dispenser, or automated microplate washer.

? Absorbent paper for blotting the microtiter plate.

? 100ml and 500ml graduated cylinders.

? Deionized or distilled water.

? Pipettes and pipette tips.

? Test tubes for dilution.

PRECAUTIONS

The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, 

and clothing protection when using this material.6

SAMPLE COLLECTION AND STORAGE

? Serum   Use a serum separator tube (SST) and allow samples to clot for 

two hours at room temperature or overnight at 4°C before centrifugation 

for 15 minutes at 1000 ×g. Remove serum and assay immediately or 

aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw 

cycles.

? Plasma   Collect plasma using EDTA, or heparin as an anticoagulant. 

Centrifuge for 15 minutes at 1000 x g, 2  - 8°C within 30 minutes of 

collection. Assay immediately or aliquot and store samples at  -20°C or 

-80°C. Avoid repeated  freeze-thaw cycles. Centrifuge the sample again 

after thawing before the assay.

? Cell Culture Supernates   Remove particulates by centrifugation for 15 

minutes at 1000 x g, 2 - 8°C and assay immediately or aliquot and store 

samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.

? Tissue Homogenates   100mg tissue was rinsed with 1X PBS, 

homogenized in 1 ml of 1X PBS and stored overnight at -20°C. After two 

freeze-thaw cycles were performed to break the cell membranes, the 

homogenates were centrifuged for 5 minutes at 5000 x g, 2 - 8°C. The 

supernate was removed and assayed immediately. Alternatively, aliquot 

and store samples at -20°C or -80°C. Centrifuge the sample again after 

thawing before the assay. Avoid repeated freeze-thaw cycles.7

SAMPLE PREPARATION

Serum and plasma samples require a 5-fold dilution into Sample Diluent. The 

recommended dilution factor is for reference only. The optimal dilution factor 

should be determined by users according to their particular experiments.

Note:

1. CUSABIO is only  responsible for the kit itself, but not for the samples 

consumed during the assay. The user should calculate the possible 

amount of the samples used in the whole test. Please reserve sufficient 

samples in advance.

2. Samples to be used within 5 days may be stored at 2-8°C, otherwise 

samples must be stored at -20°C (≤1month) or -80°C (≤2month) to avoid 

loss of bioactivity and contamination.

3. Grossly hemolyzed samples are not suitable for use in this assay.

4. If the samples are not indicated in the manual, a preliminary experiment to 

determine the validity of the kit is necessary. 

5. Please predict the concentration before assaying. If values for these are 

not within the range of the standard curve, users must determine the 

optimal sample dilutions for their particular experiments.

6. Tissue or cell extraction samples prepared by chemical lysis buffer may 

cause unexpected ELISA results due to the impacts of certain chemicals.

7. Owing to the possibility of mismatching between antigen from other 

resource and antibody used in  our kits (e.g., antibody targets 

conformational epitope rather than linear epitope), some native or 

recombinant proteins from other manufacturers may not be recognized by 

our products.

8. Influenced by the factors including cell viability, cell number and also 

sampling time, samples from cell culture supernatant may not be detected 

by the kit.

9. Fresh samples without long time storage are recommended for the test. 

Otherwise, protein degradation and denaturalization may occur in those 

samples and finally lead to wrong results.8

REAGENT PREPARATION

Note: 

? Kindly use graduated containers to prepare the reagent. Please don’t 

prepare the reagent directly in the Diluent vials provided in the kit.

? Bring all reagents to room temperature (18-25°C) before use for 30min.

? Prepare fresh standard for each assay. Use within 4 hours and discard 

after use.

? Making serial dilution in the wells directly is not permitted. 

? Please carefully reconstitute Standards according to the instruction, and 

avoid foaming and mix gently until the crystals have completely dissolved. 

To minimize imprecision caused by pipetting, use small volumes and 

ensure that pipettors are calibrated. It is recommended to suck more than 

10μl for once pipetting.

? Distilled water is recommended to be used to make the  preparation for 

reagents or samples. Contaminated water or container for reagent 

preparation will influence the detection result.

1. Biotin-antibody (1x) - Centrifuge the vial before opening. 

Biotin-antibody requires a 100-fold dilution. A suggested 100-fold dilution 

is 10 μl of Biotin-antibody + 990 μl of Biotin-antibody Diluent.

2. HRP-avidin (1x) - Centrifuge the vial before opening.

HRP-avidin requires a 100-fold dilution. A suggested 100-fold dilution is 10 

μl of HRP-avidin + 990 μl of HRP-avidin Diluent.

3. Wash Buffer(1x)- If crystals have formed in the concentrate, warm up to    

room temperature and mix gently until the crystals have completely 

dissolved. Dilute 20 ml of Wash Buffer Concentrate (25 x) into deionized or 

distilled water to prepare 500 ml of Wash Buffer (1 x).9

4. Standard 

Centrifuge the standard vial at 6000-10000rpm for 30s. 

Reconstitute the  Standard with 1.0 ml of  Sample Diluent. Do not 

substitute other diluents. This reconstitution produces a stock solution of 

20 ng/ml. Mix the standard to ensure complete reconstitution and allow the 

standard to sit for a minimum of 15 minutes with gentle agitation prior to 

making dilutions. 

Pipette 250 μl of Sample Diluent into each tube (S0-S6). Use the stock 

solution to produce a 2-fold dilution series (below). Mix each tube 

thoroughly before the next transfer. The undiluted Standard serves as the 

high standard (20 ng/ml). Sample Diluent serves as the zero standard (0 

ng/ml).

Tube S7 S6 S5 S4 S3 S2 S1 S0

ng/ml 20 10 5 2.5 1.25 0.625 0.312 010

ASSAY PROCEDURE

Bring all reagents and samples to room temperature before use. Centrifuge 

the sample again after thawing before the assay. It is recommended that all 

samples and standards be assayed in duplicate. 

1. Prepare all reagents, working standards, and samples as directed in the 

previous sections.

2. Refer to the Assay Layout Sheet to determine the number of wells to be 

used and put any remaining wells and the desiccant back into the pouch 

and seal the ziploc, store unused wells at 4°C.

3. Add 100μl of standard and sample per well. Cover with the adhesive strip

provided. Incubate for 2 hours at 37°C. A plate layout is provided to record 

standards and samples assayed.

4. Remove the liquid of each well, don’t wash. 

5. Add 100μl of  Biotin-antibody (1x)  to each well. Cover with a new 

adhesive strip. Incubate for 1 hour at 37°C.  (Biotin-antibody (1x) may 

appear cloudy. Warm up to room temperature and mix gently until solution 

appears uniform.)

6. Aspirate each well and wash, repeating the process two times for a total of 

three washes. Wash by filling each well with Wash Buffer (200μl) using a 

squirt bottle, multi-channel pipette, manifold dispenser, or autowasher,

and let it stand for 2 minutes, complete removal of liquid at each step is 

essential to good performance. After the last wash, remove any remaining 

wash Buffer by aspirating ordecanting. Invert the plate and blot it against 

clean paper towels.

7. Add 100μl of HRP-avidin (1x) to each well. Cover the microtiter plate with 

a new adhesive strip. Incubate for 1 hour at 37°C.

8. Repeat the aspiration/wash process for five times as in step 6.

9. Add 90μl of TMB Substrate to each well. Incubate for 15-30 minutes at 

37°C. Protect from light.

10. Add 50μl of Stop Solution to each well,  gently tap the plate to ensure 

thorough mixing.11

11. Determine the optical density of each well within  5 minutes, using a 

microplate reader set to 450 nm. If wavelength correction is available, set 

to 540 nm or 570 nm. Subtract readings at 540 nm or 570 nm from the 

readings at 450 nm. This subtraction will correct for optical imperfections 

in the plate. Readings made directly at 450 nm without correction may be 

higher and less accurate.

*Samples may require dilution. Please refer to Sample Preparation section.

Note:

1. The final experimental results will be closely related to validity of the 

products, operation skills of the end users and the experimental 

environments. 

2. Samples or reagents addition: Please use the freshly prepared Standard. 

Please carefully add samples to wells and mix gently to avoid foaming. Do 

not touch the well wall as possible. For each step in the procedure, total 

dispensing time for addition of reagents or samples to the assay plate 

should not exceed 10 minutes. This will ensure equal elapsed time for 

each pipetting step, without interruption. Duplication of all standards and 

specimens, although not required, is recommended. To avoid 

cross-contamination, change pipette tips between additions of each 

standard level, between sample additions, and between reagent additions. 

Also, use separate reservoirs for each reagent.

3. Incubation: To ensure accurate results, proper adhesion of plate sealers 

during incubation steps is necessary. Do not allow wells to sit uncovered 

for extended periods between incubation steps. Once reagents have been 

added to the well strips, DO NOT let the strips DRY at any time during the 

assay. Incubation time and temperature must be observed.

4. Washing: The wash procedure is critical. Complete removal of liquid at 

each step is essential to good performance. After the last wash, remove 

any remaining Wash Solution by aspirating or decanting and remove any 

drop of water and fingerprint on the bottom of the plate. Insufficient 

washing will result in poor precision and falsely elevated absorbance 

reading. When using an automated plate washer, adding a 30 second 

soak period following the addition of wash buffer, and/or rotating the plate 

180 degrees between wash steps may improve assay precision.12

5. Controlling of reaction time: Observe the change of color after adding TMB 

Substrate (e.g. observation once every 10 minutes), TMB Substrate

should change from colorless or light blue to gradations of blue. If the color 

is too deep, add Stop Solution in advance to avoid excessively strong 

reaction which will result in inaccurate absorbance reading.

6. TMB Substrate is easily contaminated.  TMB Substrate should remain 

colorless or light blue until added to the plate. Please protect it from light.

7. Stop Solution should be added to the plate in the same order as the TMB 

Substrate. The color developed in the wells will turn from blue to yellow 

upon addition of the Stop Solution. Wells that are green in color indicate 

that the Stop Solution has not mixed thoroughly with the TMB Substrate.13

ASSAY PROCEDURE SUMMARY

*Samples may require dilution. Please refer to Sample Preparation section.14

CALCULATION OF RESULTS

Using the professional soft "Curve Expert 1.3" to make a standard curve is 

recommended, which can be downloaded from our web.

Average the duplicate readings for each standard and sample and subtract the 

average zero standard optical density. 

Create a standard curve by reducing the data using computer software capable 

of generating a four parameter logistic (4-PL) curve-fit. As an alternative, 

construct a standard curve by plotting the mean absorbance for each standard 

on the x-axis against the concentration on the y-axis and draw a best fit curve 

through the points on the graph. The data may be linearized by plotting the log of 

the LEP concentrations versus the log of the O.D. and the best fit line can be 

determined by regression analysis. 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.

 

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