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Human Aldosterone(ALD)
ELISA Kit 
 
 
Catalog No. CSB-E07026h
(96 tests)
 
 
 
 
 

  This  immunoassay  kit  allows  for  the  in  vitro  rapid  detection  of  human  ALD
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.
 
 
 
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INTRODUCTION
Aldosterone  is  a  hormone  that  increases  the  reabsorption  of
sodium and water and the release (secretion) of potassium in the
kidneys. This  increases blood volume and,  therefore,  increases
blood pressure. Drugs that  interfere with the secretion or action
of aldosterone are in use as antihypertensives. One example  is
spironolactone,  which  lowers  blood  pressure  by  blocking  the
aldosterone  receptor.  Aldosterone  is  part  of  the
renin-angiotensin  system.  A  measurement  of  aldosterone  in
blood may be termed a plasma aldosterone concentration (PAC),
which  may  be  compared  to  plasma  renin  activity  (PRA)  as  a
PAC/PRA ratio.
Aldosterone  is  a  steroid  hormone  (mineralocorticoid  family)
produced by the outer-section (zona glomerulosa) of the adrenal
cortex  in  the adrenal gland, and acts on  the distal  tubules and
collecting ducts of the nephron, the functioning unit of the kidney
to  cause  the  conservation  of  sodium,  secretion  of  potassium,
increased  water  retention,  and  increased  blood  pressure.  The
overall effect of aldosterone  is  to  increase  reabsorption of  ions
and water in the kidney.
Its  activity  is  reduced  in  Addison’s  disease  and  increased  in
Conn syndrome. 
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PRINCIPLE OF THE ASSAY
This  assay  employs  the  competitive  inhibition  enzyme
immunoassay  technique. An antibody specific  to ALD has been
pre-coated onto a microplate. Standards or samples are added
to  the  appropriate  microtiter  plate  wells  with  HRP-conjugated
ALD and incubated. A competitive inhibition reaction is launched
between ALD (Standards or samples) and HRP-conjugated ALD
with the pre-coated antibody specific for ALD. The more amount
of ALD in samples, the less antibody bound by HRP-conjugated
ALD.  Then  the  substrate  solutions  are  added  to  the  wells,
respectively. And the color develops in opposite to the amount of
ALD  in  the sample. The color development  is stopped and  the
intensity of the color is measured.
DETECTION RANGE
The  standard  curve  concentrations  used  for  the  ELISA’s  were
1000pg/ml, 266.6 pg/ml, 66.6 pg/ml, 26.6 pg/ml, 13.3pg/ml.
SPECIFICITY
This  assay  recognizes  human  ALD.  No  significant
cross-reactivity or interference was observed. 
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SENSITIVITY
The minimum  detectable  dose  of  human  ALD  is  typically  less
than 0.66 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
Standards (S1-S5)  5  
HRP-conjugate  1 x 6 ml
Wash Buffer      
1 x 15 ml
(20×concentrate)
Substrate A  1 x 6 ml
Substrate B  1 x 6 ml
Stop Solution      1 x 6 ml
 
Standard  S1  S2  S3  S4  S5
Concentration
(pg/ml)
13.3  26.6  66.6  266.6  1000 
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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.
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. Wash  Buffer    If  crystals  have  formed  in  the  concentrate,
warm  to  room  temperature  and mix  gently  until  the  crystals 
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have  completely  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
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. 
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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 immediately
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  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.  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),
Mix well and then incubate for 1 hour at 37°C.  
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 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.   
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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
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 ALD 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. 
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  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
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.