Porcine myelin basic
protein,MBP  
ELISA Kit 
 
 
 
 
  This  immunoassay  kit  allows  for  the  in  vitro  rapid  detection  of  porcine  MBP
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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PRINCIPLE OF THE ASSAY
This  assay  employs  the  competitive  inhibition  enzyme
immunoassay technique. The microtiter plate provided in this kit
has been pre-coated with goat-anti-rabbit antibody. Standards or
samples are added to the appropriate microtiter plate wells with
an antibody specific for MBP and Horseradish Peroxidase （HRP）
conjugated MBP,  then  incubated. Then  substrate  solutions are
added to the wells, respectively. 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 MBP 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 10
ng/ml, 5 ng/ml, 2 ng/ml, 1 ng/ml, 0.5 ng/ml.
SPECIFICITY
This  assay  recognizes  porcine  MBP.  No  significant 
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cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of porcine MBP  is  typically  less
than 0.25 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
Standards （S1-S5）  5 x0.5ml
Antibody  1 x 6 ml
HRP-conjugate  1 x 6 ml
Wash Buffer      
1 x 15 ml
（20×concentrate）
Substrate A  1 x 7 ml
Substrate B  1 x 7 ml
Stop Solution      1 x 7 ml
 
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Standard  S1  S2  S3  S4  S5
Concentration
（ng/ml）
0.5  1  2  5  10
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.
TECHNICAL HINTS
1.  Bring all reagents and plate to room temperature for at least 
  5
30 minutes before use. Unused wells need store at 2-8℃and
avoid sunlight.
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.
3.  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.
4. 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.
5.  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.
6.  Stop Solution should be added to the plate in the same order 
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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  to  clot  for  30 minutes  before  centrifugation  for  15
minutes at 1000 x g. Remove serum and assay immediay 
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or aliquot and store samples at -20°C. Centrifuge t he 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 x 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 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 3 hour at 37°C.  
3.  Fill  each  well  with Wash  Buffer  （about  250µl）,  stay  for  10 
  8
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 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.  
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  curve  by  reducing  the  data  using  computer  software 
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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 MBP 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.
  Any  variation  in  operator,  pipetting  technique,  washing
technique,  incubation  time  or  temperature,  and  kit  age  can 
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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.