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 Porcine Immunoglobulin G（IgG）

                            ELISA Kit

                      Catalog No. CSB-E06804p

                                    （96T）

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

    IgG concentrations in serum .

?   Expiration date   six months from the date of manufacture

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

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PRINCIPLE OF THE ASSAY

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

IgG.   Standards   or   samples   are   then   added   to   the   appropriate   microtiter

plate   wells   with  Horseradish     Peroxidase    （HRP）    -conjugated    antibody

preparation specific for porcine IgG， mix well and incubated. The more the

amount     of  porcine  IgG   in  samples,   the  less  HRP-conjugated      antibody

preparation    specific  for  porcine   IgG  bound    by  pre-coated   porcine   IgG.

Then a TMB （3,3',5,5' tetramethyl-benzidine） substrate solution is added to

each well. And the color develops in opposite to the amount of porcine IgG

in the sample. The color development is stopped and the intensity of the

color is measured.

DETECTION RANGE

4.69μg/ml-300     μg/ml.   The   standard    curve   concentrations    used   for  the

ELISA’s were 300 μg/ml, 150μg/ml, 37.5 μg/ml, 9.375 μg/ml, 4.69 μg/ml.

SPECIFICITY

This    assay   recognizes    porcine   IgG.   No   significant  cross-reactivity   or

interference was observed.

SENSITIVITY

The    minimum     detectable    dose   of  porcine   IgG   is  typically  less  than

1.17μg/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.

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MATERIALS PROVIDED

               Reagent                                      Quantity

              Assay plate                                        1

               Standard                                      6×0.5ml

               Sample Diluent                               2 x 20 ml

               HRP -conjugate                               1 x 6 ml

                                                            1 x 20 ml

              Wash Buffer

                                                         （25×concentrate）

               TMB Substrate                                1 x 10 ml

               Stop Solution                                1 x 10 ml

    Standard            S1        S 2         S3         S4         S5         S6

 Concentration

                         0        4.69      9.375       37.5        150       300

     （μg/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 to minimize exposure to

    damp air. The test kit may be used throughout the expiration date of the

    kit. Refer to the package label for the expiration date.

2.   Opened    test  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.

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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.

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.

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.

    Avoid repeated freeze-thaw cycles.

?    Recommend to dilute the serum samples with Sample Diluent（1:2000）

     before   test.  The   suggested    2000-fold    dilution  can  be  achieved    by

     adding 5μl sample to 195μl of Sample Diluent. Complete the 2000-fold

     dilution by adding 5μl of this solution to 245μl of Sample Diluent. The

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     recommended dilution factor is for reference only. The optimal dilution

     factor   should    be   determined     by   users   according     to  their  particular

     experiments.

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.

1.    Add 50μl Standard or Sample to per well. Add 50ul HRP-conjugate to

     each   well   immediay.   Mix   well   with   the   pipette   or   shake   the   plate

     gently for 60 seconds.

2.    Then incubate for 30 minutes at 37° C.

3.    Aspirate   each   well   and   wash,  Wash   by   filling   each   well   with   Wash

     Buffer    （200μl）   using   a  squirt   bottle,  multi-channel     pipette,   manifold

     dispenser or autowasher. Repeating the process for a total of five time

     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 90μl of TMB Substrate to each well. Incubate for 20 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  when    the   last  four  wells

     containing the lowest concentration of standards develop obvious blue

     color. If color change does not appear uniform, gently tap the plate to

     ensure thorough mixing.

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6.    Determine the optical density of  each well within 15 minutes, using a

     microplate reader set to 450 nm.

CALCULATION OF RESULTS

Average the duplicate readings for each standard, control, and sample and

divide the average zero standard optical density. Create a standard curve

by reducing the data using computer software. As an alternative, construct

a standard curve by plotting the absorbance ratio 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

porcine   IgG   concentrations   versus   the   ratio   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 Calibrator 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 Calibrator 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   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   during

     incubation steps is necessary.

?  Substrate   Solution   should   remain   colorless   until   added   to   the   plate.

     Keep Substrate Solution protected from light. Substrate Solution should

     change from colorless 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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