Human Dopamine （DA） ELISA Kit  
 
 
Catalog No. CSB-E06888h
（96 tests） 
 
 
  This  immunoassay  kit  allows  for  the  in  vitro  quantitative  determination  of  DA
concentrations in serum, plasma and other biological fluids.
  Expiration date      six months from the date of manufacture
  FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. 
 
INTRODUCTION
Dopamine  is  a  neurotransmitter  occurring  in  a  wide  variety  of  animals,  including  both
vertebrates and invertebrates. In the brain, this phenethylamine functions as a neurotransmitter,
activating  the  five  types of dopamine  receptors  - D1, D2, D3, D4 and D5, and  their variants.
Dopamine  is  produced  in  several  areas  of  the  brain,  including  the  substantia  nigra  and  the
ventral  tegmental  area. Dopamine  is  also  a  neurohormone  released  by  the  hypothalamus.  Its
main function as a hormone  is  to  inhibit  the release of prolactin from  the anterior  lobe of  the
pituitary.
Dopamine  can  be  supplied  as  a  medication  that  acts  on  the  sympathetic  nervous  system,
producing effects such as increased heart rate and blood pressure. However, because dopamine
cannot  cross  the  blood-brain  barrier,  dopamine  given  as  a  drug  does  not  directly  affect  the
central  nervous  system.  To  increase  the  amount  of  dopamine  in  the  brains  of  patients with
diseases such as Parkinson's disease and dopa-responsive dystonia, L-DOPA （levodopa）, which
is the precursor of dopamine, can be given because it can cross the blood-brain barrier.
Dopamine has many functions in the brain, including important roles in behavior and cognition,
motor  activity,  motivation  and  reward,  inhibition  of  prolactin  production,  sleep,  mood,
attention, and learning. Dopaminergic neurons are present chiefly in the ventral tegmental area  
of the midbrain, substantia nigra pars compacta, and arcuate nucleus of the hypothalamus.
PRINCIPLE OF THE ASSAY
This assay employs  the competitive  inhibition enzyme  immunoassay  technique. A polyclonal
antibody  specific  for  DA  has  been  pre-coated  onto  a  microplate.  A  competitive  inhibition
reaction is launched between HRP labeled DA and unlabeled DA （Standards or samples） with
the pre-coated antibody specific for DA. The more  the amount of DA  in samples,  the  less  the
HRP labeled DA bound by pre-coated antibody. The substrate solutions are added to the wells,
respectively. And the color develops in opposite to the amount of DA bound in the initial step.
The color development is stopped and the intensity of the color is measured.
DETECTION RANGE
0.156 ng/ml-10 ng/ml. The standard curve concentrations used for the ELISA’s were 10 ng/ml,
5 ng/ml, 2.5 ng/ml, 1.25 ng/ml, 0.625 ng/m1, 0.312 ng/ml, 0.156 ng/ml .
SPECIFICITY
This assay recognizes human DA. No significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of DA is typically less than 0.039 ng/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 （96 tests）  1
Standard  2
Sample Diluent      2 x 20 ml
HRP-DA    1 x 60µl
Wash Buffer
1 x 20 ml  
（25 x concentrate）
TMB Substrate  1 x 10 ml
Stop Solution
1 x 10 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 with desiccants 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.
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.
2.  Standard      The  Standard  concentration  is  10  ng/ml.  Allow  the  standard  to  sit  for  a
minimum of 15 minutes with gentle agitation prior to making serial dilutions. The Sample
Diluent serves as the zero standard （0 ng/ml）.
3.  HRP-DA    Dilute  to  the working concentration specified on  the vial  label using Sample
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.
  Pipettes and pipette tips.
  Deionized or distilled water.
  Squirt bottle, manifold dispenser, or automated microplate washer.
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 100µl of Sample Diluent to Blank well, 50ul Standard or Sample to other wells.  
2.  Add 50µl of HRP-DA working solution to Standard and Sample well.（Don’t to Blank
well?。?
3.  Cover with the adhesive strip. Shake the plate gently for 60 seconds and incubate for 60
minutes at 37° C.  
4.  Aspirate each well and wash, repeating the process five times for a total of five washes.
Wash by filling each well with Wash Buffer （350µl） using a squirt bottle, multi-channel
pipette, manifold  dispenser  or  autowasher. 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.
5.  Add 90µl of TMB Substrate to each well. Incubate for 30 minutes at 37°C. Keeping the
plate away from drafts and other temperature fluctuations in the dark.
6.  Add 50µl of Stop Solution to each well. If color change does not appear uniform, gently
tap the plate to ensure thorough mixing.
7.  Determine the optical density of each well within 30 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 DA 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
  FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
  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.
  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
  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.