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PRINCIPLE OF THE ASSAY

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

goat-anti-rabbit antibody. Standards or samples are then added to

the appropriate microtiter plate wells with a HRP-conjugated IL-17

and   antibody   preparation   specific   for   IL-17   and   incubated.   Then

substrate   solution   is   added   to   each   well.   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-17 in the

samples is then determined by comparing the O.D. of the samples

to the standard curve.

DETECTION RANGE

120 pg/ml-6000 pg/ml. The standard curve concentrations used for

the ELISA’s were 6000 pg/ml, 3000 pg/ml, 1200 pg/ml, 360 pg/ml,

120 pg/ml.

SPECIFICITY

This assay recognizes recombinant and natural chicken IL-17. No

significant cross-reactivity or interference was observed.

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SENSITIVITY

The minimum detectable dose of chicken IL-17 is typically less than

50 pg/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  （96T）

              Standards (S1-S5)                          5

              HRP-conjugate                          1 x 6ml

              Antibody                                1 x 6 ml

                                                    1 x 15 ml

              Wash Buffer

                                                 (20×concentrate)

              Substrate A                            1 x 7ml

              Substrate B                            1 x 7 ml

              Stop Solution                          1 x 7 ml

   Standard             S1           S2          S3          S4          S5

Concentration

                      120           360         1200        3000        6000

    (pg/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.

1.   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.

TECHNICAL HINTS

1.   Bring all reagents and plate to room temperature for at least 30

    minutes before use. Unused wells need store at 2-8 ℃and avoid

   sunlight.

2.   Centrifuge vials before opening to collect contents.

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3. Wash Buffer          If crystals have formed in the concentrate, warm

    to   room    temperature      and   mix   gently   until  the   crystals   have

    completely dissolved. Dilute 15 ml of Wash Buffer Concentrate

    into   deionized   or   distilled   water   to   prepare   300   ml   of   Wash

    Buffer.

4.   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.

5.   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.

6.   To   ensure   accurate   results,   proper   adhesion   of   plate   sealers

    during     incubation    steps    is  necessary.      Sealers    can    not   be

    reused.

7.   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.

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8.   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.

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 samples

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    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.

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 50μl of Standard or

     Sample per well. Standard need test in duplicate.

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

    then 50μl Antibody to each well. Mix well and then incubate for

    2 hours at 37°C.

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3.   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  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,     Blank,   and

sample and subtract the optical density of Blank. Create a standard

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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-17 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

    Quantikine Immunoassay, the possibility of interference cannot

    be excluded.

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Notes：

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