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INTRODUCTION

Insulin-like Growth Factor I (IGF-I), also known as somatomedin

C,    is  a  member       of  the   insulin    superfamily.     It  was    originally

discovered as a mediator of growth hormone actions on somatic

cell    growth,    but    has   also    been     shown     to   be   an   important

regulator of cell metabolism, differentiation and survival. IGF-I is

synthesized as a preproprotein that is proteolytically cleaved to

generate   the   mature   protein   linked   by   three   disulfide   bonds.

Mature IGF-I is highly conserved among mammals, with 100%

sequence identity between the Porcine, bovine, porcine, equine

and canine proteins. IGF-I is synthesized in the liver and multiple

other   tissues.   It   is   found   in   blood   and   other   body  fluids   as   a

complex        with    specific     high    affinity    IGF     binding     proteins

(IGFBP-1to-6).   The   IGFBPs   are   expressed   in   specific  patterns

during development. They are modulators of IGF actions, which

control IGF bioavailability to specific cell-surface receptors. Their

functions      are   further    regulated      by   IGFBP      proteases,      which

proteolytically cleave the IGFBPs to lower the affinity with which

they bind IGFs and increase IGF bioavailability. Some IGFBPs

also     have    IGF-independent          effects    on   cell   functions.     IGF-I

circulates      primarily    as    a  ternary     complex      with   IGFBP-3       or

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IGFBP-5 and the acid-labile subunit (ALS). Some IGF-I is also

present   in   binary   complexes   with   other   IGFBPs.   Whereas   the

ternary complexes are generally restricted to the vasculature, the

binary complexes freely enter the tissues.

IGF-I     actions     are   mediated       by   two    type    I  transmembrane

receptor tyrosine kinases: the IGF-I receptor (IGF-I R), and the

insulin   receptor   (INS   R)   that   exists   in   two   alternatively   spliced

isoforms   (INS   R-A   and   -B).   Both   IGF-I   R   and   INS   R   share   a

highly   homologous   structure   and   are   ubiquitously   expressed.

Functional        IGF-I      receptors       are    tetrameric       glycoproteins

composed         of  two   disulfide-linked   IGF-I      Rs    or  disulfide-linked

hybrids of one IGF-I R and one INS R. Whereas IGF-I binds with

high-affinity     to   homodimeric        IGF-I    R   and    heterdimeric      IGF-I

R:INS      R-A    or   –B   hybrids,     high-affinity    binding     of  insulin    is

observed only with dimeric INS R or IGF-I R:INS R-A hybrid but

not with IGF-I R:INS R-B hybrid. The signaling responses from

the various receptors are different depending whether insulin or

IGF-I is used as the activating ligand.

PRINCIPLE OF THE ASSAY

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

an   antibody   specific   to   IGF-1.   Standards   or   samples   are   then

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added       to   the    appropriate      microtiter    plate    wells    with    a

biotin-conjugated   antibody   preparation           specific   for   IGF-1   and

Avidin conjugated to Horseradish Peroxidase (HRP) is added to

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

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

Only those wells that contain IGF-1, 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   IGF-1   in   the   samples   is   then   determined   by

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

DETECTION RANGE

7.8 ng/ml-500 ng/ml. The standard curve concentrations used for

the ELISA’s were 500 ng/ml, 250 ng/ml, 125 ng/ml, 62.5 ng/ml,

31.2 ng/ml, 15.6 ng/ml, 7.8 ng/ml.

SPECIFICITY

This   assay   recognizes   recombinant   and   natural   human  IGF-1.

No significant cross-reactivity or interference was observed.

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SENSITIVITY

The minimum detectable dose of human IGF-1 is typically less

than 2 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.

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

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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

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   completely   dissolved.   Dilute   20   ml   of   Wash

     Buffer Concentrate into deionized or distilled water to prepare

    500 ml of Wash Buffer.

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

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

     Diluent. This reconstitution produces a stock solution of 500

     ng/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 (500 ng/ml).

    The Sample Diluent serves as the zero standard (0 ng/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.

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   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

    samples   to   clot   for   30   minutes   before   centrifugation   for   15

     minutes at 1000 g. Remove serum and assay immediately 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   immediately   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.

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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.    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.

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

    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)

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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  IGF-1   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.

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   Any       variation    in   Standard      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

    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.

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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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Notes

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