（IL-6）大鼠白介素6（IL-6）ELISA試劑盒

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                     Rat Interleukin 6 （IL-6）   

                                              ELISA Kit 

                                     Catalog No. CSB-E04640r 

                                                           （96 T） 

       This immunoassay kit allows for the in vitro quantitative determination of rat IL-6 

       concentrations in serum, plasma and other biological fluids. 

       Expiration date      six months from the date of manufacture 

       FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. 

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INTRODUCTION 

Interleukin-6   （IL-6）   is   an   interleukin   that   acts   as   both   a 

pro-inflammatory  and  anti-inflammatory  cytokine.  It  is  secreted 

by  T  cells  and  macrophages  to  stimulate  immune  response  to 

trauma,  especially  burns  or  other  tissue  damage  leading  to 

inflammation. In terms of host response to a foreign pathogen, 

IL-6  has  been  shown,  in  mice,  to  be  required  for  resistance 

against the bacterium, Streptococcus pneumoniae. I                     -6 is also a 

"myokine," a cytokine produced from muscle, and is elevated in 

response to muscle contraction. It is significantly elevated with 

exercise, and precedes the appearance of other cytokines in the 

circulation. During exercise, it is thought to act in a hormone-like 

manner  to  mobilize  extracellular  substrates  and/or  augment 

substrate   delivery.   Additionally,   osteoblasts   secrete   IL-6   to 

stimulate osteoclast formation. Smooth muscle cells in the tunica 

media      of   many      blood     vessels      also    produce       IL-6    as    a 

pro-inflammatory  cytokine.  IL-6's  role  as  an  anti-inflammatory 

cytokine is mediated through its inhibitory effects on TNF-alpha 

and IL-1,  and  activation  of  IL-1ra  and  IL-10.  IL-6  is  one of the 

most  important  mediators  of  fever  and  of  the  acute  phase 

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response. In the muscle and fatty tissue IL-6 stimulates energy 

mobilization  which  leads  to  increased  body  temperature.  IL-6 

can   be   secreted   by   macrophages   in   response   to   specific 

microbial      molecules,       referred     to    as    pathogen       associated 

molecular   patterns   （PAMPs）.   These   PAMPs   bind   to   highly 

important  detection  molecules  of  the  innate  immune  system, 

called  Toll-like  receptors  （TLRs）,  that  are  present  on  the  cell 

surface      （or    in   intracellular     compartments）          which      induce 

intracellular  signaling  cascades  that  give  rise  to  inflammatory 

cytokine production. IL-6 is also essential for hybridoma growth 

and  is  found  in  many  supplemental  cloning  media  such  as 

briclone. Inhibitors of IL-6 （including estrogen） are used to treat 

postmenopausal osteoporosis.   

PRINCIPLE OF THE ASSAY 

The microtiter plate provided in this kit has been pre-coated with 

an  antibody  specific  to  IL-6.  Standards  or  samples  are  then 

added      to    the    appropriate       microtiter     plate     wells    with     a 

biotin-conjugated  antibody   preparation   specific   for  IL-6   and 

Avidin conjugated to Horseradish Peroxidase （HRP） is added to 

each  microplate  well  and  incubated.  Then  a  TMB  （3,3',5,5' 

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tetramethyl-benzidine） substrate solution is added to each well. 

Only  those  wells  that  contain  IL-6,  biotin-conjugated  antibody 

and enzyme-conjugated Avidin will exhibit a change in color. The 

enzyme-substrate  reaction  is  terminated  by  the  addition  of  a 

sulphuric   acid  solution  and  the   color   change   is   measured 

spectrophotometrically at a wavelength of 450 nm ± 2 nm. The 

concentration  of  IL-6  in  the  samples  is  then  determined  by 

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

DETECTION RANGE 

0.312 pg/ml-20 pg/ml. The standard curve concentrations used 

for the ELISA’s were 20 pg/ml, 10 pg/ml, 5 pg/ml, 2.5 pg/ml, 1.25 

pg/ml, 0.625 pg/ml, 0.312 pg/ml. 

SPECIFICITY 

This  assay  recognizes  recombinant  and  natural  rat  IL-6.  No 

significant cross-reactivity or interference was observed. 

SENSITIVITY 

The  minimum  detectable  dose  of  rat  IL-6  is  typically  less  than 

0.078 pg/ml. 

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The sensitivity of this assay, or Lower Limit of Detection （                     D） 

was  defined  as  the  lowest  protein  concentration  that  could  be 

differentiated from zero. 

MATERIALS PROVIDED 

               Reagent                                    Quantity 

              Assay plate                                      1 

               Standard                                        2 

               Sample Diluent                             1 x 20 ml 

               Biotin-antibody Diluent                    1 x 10 ml 

               HRP-avidin Diluent                         1 x 10 ml 

               Biotin-antibody                            1 x 120μl 

               HRP-avidin                                 1 x 120μl 

                                                          1 x 20 ml 

              Wash Buffer       

                                                      （25×concentrate） 

              TMB Substrate                               1 x 10 ml 

               Stop Solution                              1 x 10 ml 

STORAGE 

1.  Unopened  test  kits  should  be  stored  at  2-8°C  upon  receipt 

    and the microtiter plate should be kept in a sealed bag. The 

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    test kit may be used throughout the expiration date of the kit, 

    provided  it  is  stored  as  prescribed  above.  Refer  to  the 

    package label for the expiration date. 

2.  Opened test plate should be stored at 2-8°C in the aluminum 

    foil bag with desiccants to minimize exposure to damp air. The 

    kits will remain stable until the expiring date shown, provided it 

    is stored as prescribed above.     

3.  A  microtiter  plate  reader  with  a  bandwidth  of  10  nm  or less 

    and an optical density range of 0-3 OD or greater at 450nm 

    wavelength         is    acceptable        for    use      in    absorbance 

    measurement. 

REAGENT PREPARATION 

Bring all reagents to room temperature before use.   

1.  Wash  Buffer    If  crystals  have  formed  in  the  concentrate, 

    warm  up  to  room  temperature  and  mix  gently  until  the 

    crystals  have  compley  dissolved.  Dilute  20  ml  of  Wash 

     Buffer Concentrate into deionized or distilled water to prepare 

     500 ml of Wash Buffer. 

2.  Standard    Centrifuge  the  standard  vial  at  6000-10000rpm 

    for  30s.  Reconstitute  the  Standard  with  1.0  ml  of  Sample 

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     Diluent. This reconstitution produces a stock solution of 20 

     pg/ml. Allow the standard to sit for a minimum of 15 minutes 

    with  gentle  agitation  prior  to  making  serial  dilutions.  The 

     undiluted  standard  serves  as  the  high  standard  （20  pg/ml）. 

     The Sample Diluent serves as the zero standard （0 pg/ml）. 

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

     after use. 

3.   Biotin-antibody    Centrifuge the vial before opening. Dilute 

     to   the     working       concentration        using      Biotin-antibody 

     Diluent（1:100）, respectively. 

4.   HRP-avidin    Centrifuge the vial before opening. Dilute to the 

    working   concentration   using   HRP-avidin   Diluent（1:100）, 

     respectively. 

Precaution: The Stop Solution provided with this kit is an acid solution. Wear 

            eye, hand, face, and clothing protection when using this material. 

OTHER SUPPLIES REQUIRED 

    Microplate reader capable of measuring absorbance  at 450 

     nm, with the correction wavelength set at 540 nm or 570 nm. 

    Pipettes and pipette tips. 

    Deionized or distilled water. 

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    Squirt  bottle,  manifold  dispenser,  or  automated  microplate 

    washer. 

    An incubator which can provide stable incubation conditions 

     up to 37°C±0.5°C. 

SAMPLE COLLECTION AND STORAGE 

     Serum    Use  a  serum  separator  tube  （SST）  and  allow 

     samples  to  clot  for  30  minutes  before  centrifugation  for  15 

     minutes at 1000 g. Remove serum and assay immediay or 

     aliquot  and  store  samples  at  -20°C.  Centrifuge  the  sample 

     again   after   thawing   before   the   assay.   Avoid   repeated 

    freeze-thaw cycles. 

     Plasma    Collect plasma using citrate, EDTA, or heparin as 

     an anticoagulant. Centrifuge for 15 minutes at 1000 g within 

     30  minutes  of  collection.  Assay  immediay  or  aliquot  and 

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

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

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

ASSAY PROCEDURE 

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Bring   all   reagents   and   samples   to   room   temperature   before   use.   It   is 

recommended that all samples, standards, and controls be assayed in duplicate. 

All  the  reagents  should  be  added  directly  to  the  liquid  level  in  the  well.  The 

pipette should avoid contacting the inner wall of the well. 

1.  Add 100μl of Standard, Blank, or Sample per well. Cover with 

     the adhesive strip. Incubate for 2 hours at 37°C.   

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

3.  Add 100μl of Biotin-antibody working solution to each well. 

     Incubate   for   1   hour   at   37°C.   Biotin-antibody   working 

     solution may appear cloudy. Warm up to room temperature 

     and mix gently until solution appears uniform. 

4.  Aspirate  each  well  and  wash,  repeating  the  process  three 

     times for a total of three washes. Wash: Fill each well with 

     Wash  Buffer  （200μl）  and  let  it  stand  for  2  minutes,  then 

     remove  the  liquid  by  flicking  the  plate  over  a  sink.  The 

     remaining drops are removed by patting the plate on a paper 

     towel. Complete removal of liquid at each step is essential to 

     good performance. 

5.  Add  100μl  of  HRP-avidin  working  solution  to  each  well. 

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     Cover the microtiter plate with a new adhesive strip. Incubate 

    for 1 hour at 37°C. 

6.  Repeat the aspiration and wash five times as step 4. 

7.  Add 90μl of TMB Substrate to each well. Incubate for 10-30 

     minutes  at  37°C.  Keeping  the  plate  away  from  drafts  and 

     other temperature fluctuations in the dark.   

8.  Add 50μl of Stop Solution to each well  when the first four 

    wells   containing   the   highest   concentration   of   standards 

     develop obvious blue color. If color change does not appear 

     uniform, gently tap the plate to ensure thorough mixing. 

9.  Determine the optical density of each well within 30 minutes, 

     using a microplate reader set to 450 nm. 

CALCULATION OF RESULTS 

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

recommended, which can be downloaded from our web. 

Average the duplicate readings for each standard, control, 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 

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the  mean  absorbance  for  each  standard  on  the  y-axis  against 

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

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

the log of the IL-6 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. 

LIMITATIONS OF THE PROCEDURE 

    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. 

    It  is  important  that  the  Standard  Diluent  selected  for  the 

     standard   curve   be   consistent   with   the   samples   being 

     assayed. 

    If samples generate values higher than the highest standard, 

     dilute the samples with the appropriate Standard Diluent and 

     repeat the assay. 

    Any     variation     in   Standard       Diluent,     operator,      pipetting 

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    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 

    Quantikine   Immunoassay,   the   possibility   of   interference 

    cannot be excluded. 

TECHNICAL HINTS 

    Centrifuge vials before opening to collect contents. 

    When mixing or reconstituting protein solutions, always avoid 

    foaming. 

    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. 

    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. 

    To ensure accurate results, proper adhesion of plate sealers 

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     during incubation steps is necessary. 

     Substrate Solution should remain colorless or light blue until 

     added to the plate. Keep Substrate Solution protected from 

     light. Substrate Solution should change from colorless or light 

     blue to gradations of blue. 

     Stop Solution should be added to the plate in the same order 

     as the Substrate Solution. 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 Substrate Solution. 

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