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       Porcine Interleukin 6 （IL-6）

                            ELISA Kit

                      Catalog No. CSB-E06786p

                                    （96T）

?   This immunoassay kit allows for the in vitro quantitative determination of porcine

    IL-6 concentrations in serum, plasma.

?   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 a protein that in humans is encoded by the

IL6     gene.    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. IL-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 （Petersen, J Appl Physiol 2005）. 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.

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IL-6 is one of the most important mediators of fever and of the

acute phase response. It is capable of crossing the blood brain

barrier   and   initiating   synthesis   of   PGE2   in   the   hypothalamus,

thereby changing the body's temperature setpoint. 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）.

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. Il-6 is also produced by adipocytes and is thought

to be a reason why obese individuals have higher endogeneous

levels   of   CRP.   In   a   2009   study,   intranasally   administered   IL-6

was     shown      to   improve     sleep-associated       consolidation      of

emotional memories.

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

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added to the appropriate microtiter plate wells with a Horseradish

Peroxidase （HRP）-conjugated monoclonal antibody preparation

specific for IL-6 and incubated. Then substrate solution A and B

are    added    to  each   well.  Only   those   wells   that  contain   IL-6,

HRP-conjugated   antibody   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

The standard curve concentrations used for the ELISA’s were 80

pg/ml, 40 pg/ml, 20 pg/ml, 10 pg/ml, 4 pg/ml, 2pg/ml.

SPECIFICITY

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

significant cross-reactivity or interference was observed.

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SENSITIVITY

The   minimum   detectable   dose   of  porcine   IL-6   is   typically   less

than   1   pg/ml.   The   sensitivity   of  this   assay,   or   Lower   Limit   of

Detection   （LLD）   was   defined   as   the   lowest   concentration   that

could be differentiated from zero.

MATERIALS PROVIDED

              Reagent                          Quantity

              Assay plate                           1

              Standard（S  -S  ）                    6

                            1   6

              HRP-conjugate                     1 x 6 ml

                                               1 x 15 ml

              Wash Buffer

                                           （20×concentrate）

              Substrate A                       1 x 6 ml

              Substrate B                       1 x 6 ml

              Stop Solution                     1x 6 ml

         Standard               S1      S2      S3       S4      S5      S6

 Concentration （pg/ml）          2        4      10       20      40      80

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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

   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

1.  Bring all reagents and plate to room temperature for at least

    30 minutes before use. Unused wells need store at 2-8°C and

    avoid sunlight.

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

   warm   to   room   temperature   and   mix   gently   until   the   crystals

    have    compley     dissolved.    Dilute   15  ml   of  Wash     Buffer

    Concentrate into deionized or distilled water to prepare 300 ml

    of Wash Buffer.

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.

?  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

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     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

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.   Set a Blank well without any solution. Add 100μl of Standard

     or Sample per well. Standard need test in duplicate.

2.   Add 50μl of HRP-conjugate to each well （not to Blank well）.

     Mix well and then incubate for 2 hours at 37°C.

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3.   Complete   remove   the   liquid.   Then   fill   each   well   with  Wash

    Buffer   （about      200μl）,   stay   for  10   seconds     and   Spinning.

    Repeat   the   process   for   a   total  of   three   washes.   Complete

    removal       of   liquid   at  each     step    is  essential     to   good

    performance.        After  the   last  wash,    remove     any   remaining

    Wash Buffer by aspirating or decanting. Invert the plate and

    blot it against clean paper towels.

4.   Add 50μl of Substrate A and 50μl of Substrate B to each

    well, mix well. Incubate for 15 minutes at 37°C. Keeping the

    plate away from drafts and other temperature fluctuations in

    the dark.

5.   Add 50μl of Stop Solution to each well. If color change does

    not appear uniform, gently tap the plate to ensure thorough

    mixing.

6.   Determine the optical density of each well within 10 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

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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   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.

?  If samples generate values higher than the highest standard,

    dilute the samples with the appropriate Diluent and repeat the

    assay.

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?  Any      variation    in  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.

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.

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?  To ensure accurate results, proper adhesion of plate sealers

    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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