趋化因子C-C-基元配体3样蛋白1(CCL3L1)重组蛋白

趋化因子C-C-基元配体3样蛋白1(CCL3L1)重组蛋白

实验方法双抗夹心法

反应时长3h

检测范围0.625-10ng/mL

灵敏度最小可检测剂量小于等于0.231ng/mL.

样本类型serum, plasma, tissue homogenates, cell lysates, cell culture supernates 趋化因子C-C-基元配体3样蛋白1(CCL3L1)重组蛋白and other biological fluids

Enzyme-linked Immunosorbent Assay Kit

For Chemokine C-C-Motif Ligand 3 Like Protein 1 (CCL3L1)

Organism Species: Homo sapiens (Human)

Instruction manual

FOR IN VITRO AND RESEARCH USE ONLY

NOT FOR USE IN CLINICAL DIAGNOSTIC PROCEDURES

11th Edition (Revised in July, 2013)

[ INTENDED USE ]

The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of CCL3L1 in human serum,

plasma, tissue homogenates, cell lysates, cell culture supernates and other biological fluids.

[ REAGENTS AND MATERIALS PROVIDED ]

Reagents Quantity Reagents Quantity

Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4

Standard 2 Standard Diluent 1×20mL

Detection Reagent A 1×120μL Assay Diluent A 1×12mL

Detection Reagent B 1×120μL Assay Diluent B 1×12mL

TMB Substrate 1×9mL Stop Solution 1×6mL

Wash Buffer (30 × concentrate) 1×20mL Instruction manual 1

趋化因子C-C-基元配体3样蛋白1(CCL3L1)重组蛋白[ MATERIALS REQUIRED BUT NOT SUPPLIED ]

1. Microplate reader with 450 ± 10nm filter.

2. Precision single or multi-channel pipettes and disposable tips.

3. Eppendorf Tubes for diluting samples.

4. Deionized or distilled water.

5. Absorbent paper for blotting the microtiter plate.

6. Container for Wash Solution

[ STORAGE OF THE KITS ]

1. For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection

Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20oC upon receipt while

the others should be at 4

oC.

2. For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the

above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant

pack, and reseal along entire edge of zip-seal.

Note:

It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date

of the kit. For the expiration date of the kit, please refer to the label on the kit box. All components are stable until

this expiration date. [ SAMPLE COLLECTION AND STORAGE ]

Serum - Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at

4

oC before centrifugation for 20 minutes at approximately 1000×g. Assay freshly prepared serum

immediately or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles.

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at

1000×g at 2 - 8

oC within 30 minutes of collection. Remove plasma and assay immediately or store samples

in aliquot at -20oC or -80oC for later use. Avoid repeated freeze/thaw cycles.

Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this

assay, tissues were rinsed in ice-cold PBS(0.01mol/L,pH 7.0-7.2) to remove excess blood thoroughly and

weighed before homogenization. Minced the tissues to small pieces and homogenized them in 5-10mL of

PBS with a glass homogenizer on ice(Micro Tissue Grinders woks, too). The resulting suspension was

sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell

membranes. After that, the homogenates were centrifugated for 5 minutes at 5000×g. Remove the supernate

and assay immediately or aliquot and store at ≤-20oC. Cell Lysates - Cells must be lysed before assaying according to the following directions.

1. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can

be collected by centrifugation directly).

2. Wash cells three times in cold PBS.

3. Resuspend cells in PBS (1×) and the cells was subject to ultrasonication for 4 times (or Freeze cells at ≤

-20oC. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.)

4. Centrifuge at 1500×g for 10 minutes at 2 - 8

oC to remove cellular debris.

Cell culture supernates and other biological fluids - Centrifuge samples for 20 minutes at 1000×g. Remove

particulates and assay immediately or store samples in aliquot at -20oC or -80oC for later use. Avoid repeated

freeze/thaw cycles.

Note:

1. Samples to be used within 5 days may be stored at 4

oC, otherwise samples must be stored at -20oC (≤1

month) or -80oC (≤2 months) to avoid loss of bioactivity and contamination.

2. Sample hemolysis will influence the result, so hemolytic specimen should not be detected.

3. When performing the assay, bring samples to room temperature.

趋化因子C-C-基元配体3样蛋白1(CCL3L1)重组蛋白[ REAGENT PREPARATION ]

1. Bring all kit components and samples to room temperature (18-25oC) before use.

2. Standard - Reconstitute the Standard with 0.5mL of Standard Diluent, kept for 10 minutes at room

temperature, shake gently(not to foam). The concentration of the standard in the stock solution is 10ng/mL. Please prepare 5 tubes containing 0.25mL Standard Diluent and produce a double dilution series according

to the picture shown below. Mix each tube thoroughly before the next transfer. Set up 5 points of diluted

standard such as 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL, and the last EP tubes with

Standard Diluent is the blank as 0ng/mL.

Tube 1 2 3 4 5 6

ng/mL 10 5 2.5 1.25 0.625 0

3. Detection Reagent A and Detection Reagent B - Briefly spin or centrifuge the stock Detection A and

Detection B before use. Dilute to the working concentration with Assay Diluent A and B, respectively

(1:100).

4. Wash Solution - Dilute 20mL of Wash Solution concentrate (30×) with 580mL of deionized or distilled water

to prepare 600mL of Wash Solution (1×).

5. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual

solution into the vial again.

Note:

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

2. Prepare standard within 15 minutes before assay. Please do not dissolve the reagents at 37oC directly.

3. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction,

and avoid foaming and mix gently until the crystals are 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.

4. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once. 5. If crystals have formed in the Wash Solution concentrate (30×), warm to room temperature and mix gently

until the crystals are completely dissolved.

6. Contaminated water or container for reagent preparation will influence the detection result.

[ SAMPLE PREPARATION ]

1. We are 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. 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. Sample should be

diluted by 0.01mol/L PBS(PH=7.0-7.2).

3. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is

necessary.

4. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due

to the impacts from certain chemicals.

5. Due to the possibility of mismatching between antigen from other origin 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.

6. Influenced by the factors including cell viability, cell number or sampling time, samples from cell culture

supernatant may not be detected by the kit.

7. Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and

denaturalization may occur in those samples and finally lead to wrong results.

[ ASSAY PROCEDURE ]

1. Determine wells for diluted standard, blank and sample. Prepare 5 wells for standard, 1 well for blank.

Add 100μL each of dilutions of standard (read Reagent Preparation), blank and samples into the appropriate

wells. Cover with the Plate sealer. Incubate for 2 hours at 37oC. 2. Remove the liquid of each well, don’t wash.

3. Add 100μL of Detection Reagent A working solution to each well. Incubate for 1 hour at 37oC after covering

it with the Plate sealer.

4. Aspirate the solution and wash with 350μL of 1× Wash Solution to each well using a squirt bottle,

multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1~2 minutes. Remove the remaining

liquid from all wells completely by snapping the plate onto absorbent paper. Totally wash 3 times. After the

last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against

absorbent paper. 5. Add 100μL of Detection Reagent B working solution to each well. Incubate for 30 minutes at 37oC after

covering it with the Plate sealer.

6. Repeat the aspiration/wash process for total 5 times as conducted in step 4.

7. Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 10 - 20 minutes at

37oC (Don't exceed 30 minutes). Protect from light. The liquid will turn blue by the addition of Substrate

Solution.

8. Add 50μL of Stop Solution to each well. The liquid will turn yellow by the addition of Stop solution. Mix the

liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure

thorough mixing.

9. Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the

surface of the liquid. Then, run the microplate reader and conduct measurement at 450nm immediately.

Note:

1. Assay preparation: Keep appropriate numbers of wells for each experiment and remove extra wells from

microplate. Rest wells should be resealed and stored at -20oC. 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. 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 standards, samples, and reagents. Also, use separated 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 are added to the well strips, DO NOT let the strips DRY at any time during the assay. Incubation

time and temperature must be controlled.

4. Washing: The wash procedure is critical. Complete removal of liquid at each step is essential for 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 false elevated absorbance reading.

5. Controlling of reaction time: Observe the change of color after adding TMB Substrate (e.g. observation

once every 10 minutes), 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. Please protect it from light.

7. The environment humidity which is less than 60% might have some effects on the final performance,

therefore, a humidifier is recommended to be used at that condition.

[ TEST PRINCIPLE ]

The microtiter plate provided in this kit has been pre-coated with an antibody specific to CCL3L1. Standards or

samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to

CCL3L1. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and

incubated. After TMB substrate solution is added, only those wells that contain CCL3L1, biotin-conjugated

antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is

terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at

a wavelength of 450nm ± 10nm. The concentration of CCL3L1 in the samples is then determined by comparing

the O.D. of the samples to the standard curve.

[ CALCULATION OF RESULTS ]

Average the duplicate readings for each standard, control, and samples and subtract the average zero standard

optical density. Construct a standard curve by plotting the mean O.D. and concentration for each standard and

draw a best fit curve through the points on the graph or create a standard curve on log-log graph paper with

CCL3L1 concentration on the y-axis and absorbance on the x-axis. Using some plot software, for instance, curve

expert 1.30, is also recommended. If samples have been diluted, the concentration read from the standard curve

must be multiplied by the dilution factor.

[ TYPICAL DATA ]

In order to make the calculation easier, we plot the O.D. value of the standard (X-axis) against the known

concentration of the standard (Y-axis), although concentration is the independent variable and O.D. value is the

dependent variable. However, the O.D. values of the standard curve may vary according to the conditions of

assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), plotting log of

the data to establish standard curve for each test is recommended. Typical standard curve below is provided for

reference only.

Typical Standard Curve for CCL3L1, Human ELISA.

[ DETECTION RANGE ]

0.625-10ng/mL. The standard curve concentrations used for the ELISA’s were 10ng/mL, 5ng/mL, 2.5ng/mL,

1.25ng/mL, 0.625ng/mL.

[ SENSITIVITY ]

The minimum detectable dose of CCL3L1 is typically less than 0.231ng/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 by adding two standard deviations to the mean optical

density value of twenty zero standard replicates and calculating the corresponding concentration.

[ SPECIFICITY ]

This assay has high sensitivity and excellent specificity for detection of CCL3L1. No significant cross-reactivity or interference between CCL3L1 and analogues was observed.

Note:

Limited by current skills and knowledge, it is impossible for us to complete the cross- reactivity detection between

CCL3L1 and all the analogues, therefore, cross reaction may still exist.

[ RECOVERY ]

Matrices listed below were spiked with certain level of recombinant CCL3L1 and the recovery rates were

calculated by comparing the measured value to the expected amount of CCL3L1 in samples.

Matrix Recovery range (%) Average(%)

serum(n=5) 84-98 93

EDTA plasma(n=5) 81-95 87

heparin plasma(n=5) 93-105 101

[ LINEARITY ]

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of CCL3L1 and their

serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1：2 1：4 1：8 1：16

serum(n=5) 87-94% 79-89% 96-104% 82-93%

EDTA plasma(n=5) 80-92% 89-103% 83-101% 88-102%

heparin plasma(n=5) 85-95% 81-96% 99-105% 78-90%

[ PRECISION ]

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level CCL3L1 were tested

20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level CCL3L1 were tested

on 3 different plates, 8 replicates in each plate. CV(%) = SD/meanX100

Intra-Assay: CV<10%

Inter-Assay: CV<12%

[ STABILITY ]

The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within

the expiration date under appropriate storage condition.

To minimize extra influence on the performance, operation procedures and lab conditions, especially room

temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the

whole assay is performed by the same operator from the beginning to the end.

[ ASSAY PROCEDURE SUMMARY ]

1. Prepare all reagents, samples and standards;

2. Add 100μL standard or sample to each well. Incubate 2 hours at 37oC;

3. Aspirate and add 100μL prepared Detection Reagent A. Incubate 1 hour at 37oC;

4. Aspirate and wash 3 times;

5. Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37oC;

6. Aspirate and wash 5 times;

7. Add 90μL Substrate Solution. Incubate 10-20 minutes at 37oC;

8. Add 50μL Stop Solution. Read at 450nm immediately.

[ IMPORTANT NOTE ]

1. Limited by the current condition and scientific technology, we can't completely conduct the comprehensive

identification and analysis on the raw material provided by suppliers. So there might be some qualitative and

technical risks to use the kit.

2. The final experimental results will be closely related to validity of the products, operation skills of the end

users and the experimental environments. Please make sure that sufficient samples are available.

3. Kits from different batches may be a little different in detection range, sensitivity and color developing time.

Please perform the experiment exactly according to the instruction attached in kit while electronic ones from

our website is only for information.

4. Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.

5. Protect all reagents from strong light during storage and incubation. All the bottle caps of reagents should be

covered tightly to prevent the evaporation and contamination of microorganism.

6. There may be some foggy substance in the wells when the plate is opened at the first time. It will not have

any effect on the final assay results. Do not remove microtiter plate from the storage bag until needed.

7. Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting for the

plate reader may lead to incorrect results. A microplate plate reader with a bandwidth of 10nm or less and an

optical density range of 0-3 O.D. or greater at 450 ± 10nm wavelength is acceptable for use in absorbance

measurement. Please read the instruction carefully and adjust the instrument prior to the experiment.

8. Even the same operator might get different results in two separate experiments. In order to get better

reproducible results, the operation of every step in the assay should be controlled. Furthermore, a

preliminary experiment before assay for each batch is recommended.

9. Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our

in-house data due to some unexpected transportation conditions or different lab equipments. Intra-assay

variance among kits from different batches might arise from above factors, too.

10. Kits from different manufacturers with the same item might produce different results, since we haven’t

compared our products with other manufacturers.

11. The instruction manual also suits for the kit of 48T, but all reagents of 48T kit are reduced by half.

[ PRECAUTION ]

The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection

when using this material.

[ TROUBLE SHOOTING ]

Problem Possible Source Correction Action

Poor

Standard

Curve

Improper standard curve preparation Ensure accurate operation of the dilution

Incomplete washing and aspiration Adequate washing and adequate aspiration

Inaccurate Pipetting Check and Calibrate pipettes

Poor

Precision

Incomplete washing of wells Ensure sufficient washing

Inadequate mixing and aspiration reagents Adequate aspiration and mixing reagents

Reused pipette tips, containers and sealers Change and use new pipette tips, containers and sealers

Inaccurate Pipetting Check and Calibrate pipettes

Low

O.D

Values

Inadequate reagent volumes added to wells Calibrate pipettes and Add adequate reagents

Incorrect incubation times Ensure sufficient incubation times

Incorrect incubation temperature Reagents balanced to room temperature

Conjugate or substrate reagent failure Mix conjugate &substrate,color should develop immediately

No stop solution added Follow the assay protocol in the kit manual

Read beyond suggested reading time Read within the time recommended in the manual

Sample

Values

Improper Sample Storage Store the sample properly and use the fresh sample

Improper sample collection and preparation Take proper sample collection and preparation method

Low quantity of analyte in samples Use new sample and repeat assay