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                               RayBio? ?? ? 
Human RAGE ELISA Kit 
 
 
 
 
User Manual
（Revised Mar 1, 2012）
 
                                     RayBio? ?? ? Human RAGE
ELISA Kit Protocol 
                             
                               （Cat#: ELH-RAGE-001）
 
 
 
 RayBiotech, Inc.
 
We Provide You With Excellent 
Protein Array System And Service 
 
                      
 
（Toll Free）1-888-494-8555 or 770-729-2992; 770-206-2393;  
 RayBio? Human RAGE ELISA Kit Protocol     1
 
RayBiotech, Inc.
 
 
RayBio? ?? ? Human RAGE
ELISA Kit Protocol
 
TABLE OF CONTENTS
 
 
I.  Introduction……..……………………………….2
II.  Reagents……………………………..…………..2
III.  Storage.…………………………………….…….3
IV.  Additional Materials Required…………………..3
V.  Reagent Preparation……………………………..3
VI.  Assay Procedure……………………………...... 5
VII.  Assay Procedure Summary……………………...6
VIII. Calculation of Results
A.  Typical Data…..……………………….…….…..7
B.  Sensitivity……………………………….….……8
C.  Recovery…………………………..……………..8
D.  Linearity………………………………….…........8
E.  Reproducibility…………………………………..9
IX.  Specificity………………………………………. 9
X.  Troubleshooting Guide.……………………....….10 RayBio? Human RAGE ELISA Kit Protocol     2
I.  INTRODUCTION
          The RayBio? Human RAGE ELISA （Enzyme-Linked Immunosorbent
Assay） kit is an in vitro enzyme-linked immunosorbent assay for the
quantitative measurement of human RAGE in serum, plasma, cell culture
supernatants and urine. This assay employs an antibody specific for human
RAGE coated on a 96-well plate. Standards and samples are pipetted into the
wells and RAGE present in a sample is bound to the wells by the
immobilized antibody. The wells are washed and biotinylated anti-human
RAGE antibody is added. After washing away unbound biotinylated
antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are
again washed, a TMB substrate solution is added to the wells and color
develops in proportion to the amount of RAGE bound. The Stop Solution
changes the color from blue to yellow, and the intensity of the color is
measured at 450 nm. 
 
 
II.  REAGENTS
1. RAGE Microplate （Item A）: 96 wells （12 strips x 8 wells） coated with
anti-human RAGE.
2. Wash Buffer Concentrate （20x） （Item B）: 25 ml of 20x concentrated
solution.
3. Standards （Item C）: 2 vials of recombinant human RAGE.
4. Assay Diluent A （Item D）: 30 ml diluent buffer, 0.09% sodium azide as
preservative. For Standard/Sample （serum/plasma） diluent.  
5. Assay Diluent B （Item E）: 15 ml of 5x concentrated buffer. For
Standard/Sample （cell culture medium/urine） diluent. 
6. Detection Antibody RAGE （Item F）: 2 vial of biotinylated anti-human
RAGE （each vial is enough to assay half microplate）. 
7. HRP-Streptavidin Concentrate （Item G）: 200 µl 200x concentrated
HRP-conjugated streptavidin. 
8. TMB One-Step Substrate Reagent （Item H）: 12 ml of 3,3’,5,5’-
tetramethylbenzidine （TMB） in buffer solution.
9. Stop Solution （Item I）: 8 ml of 0.2 M sulfuric acid.  RayBio? Human RAGE ELISA Kit Protocol     3
III.  STORAGE
      
         May be stored for up to 6 months at 2o
 to 8o
C from the date of
shipment. Standard （recombinant protein） should be stored at -20 o
C or -80
o
C （recommended at –80 o
C） after reconstitution. Opened Microplate Wells
or reagents may be stored for up to 1 month at 2o
 to 8o
C. Return unused wells
to the pouch containing desiccant pack, reseal along entire edge.
Note: the kit can be used within one year if the whole kit is stored at -20 o
C.
Avoid repeated freeze-thaw cycles.
 
 
IV.  ADDITIONAL MATERIALS REQUIRED
1  Microplate reader capable of measuring absorbance at 450 nm.
2  Precision pipettes to deliver 2 µl to 1 ml volumes.
3  Adjustable 1-25 ml pipettes for reagent preparation.
4  100 ml and 1 liter graduated cylinders.
5  Absorbent paper.
6  Distilled or deionized water.
7  Log-log graph paper or computer and software for ELISA data
analysis.
8  Tubes to prepare standard or sample dilutions.
 
 
V.  REAGENT PREPARATION
1. Bring all reagents and samples to room temperature （18 - 25°C） before   
use.
 
2. Sample dilution: Assay Diluent A （Item D） should be used for dilution of
serum/plasma samples. 1x Assay Diluent B （Item E） should be used for
dilution of cell culture supernates/urine.
 
       Suggested dilution for normal serum/plasma: 2-20 fold*.
 
    * Please note that levels of the target protein may vary between different RayBio? Human RAGE ELISA Kit Protocol     4
        specimens.  Optimal dilution factors for each sample must be
        determined by the investigator. 
 
3. Assay Diluent B should be diluted 5-fold with deionized or distilled
     water before use.
 
4. Preparation of standard: Briefly spin the vial of Item C and then add
400 µl Assay Diluent A （for serum/plasma samples） or 1x Assay Diluent
B （for cell culture supernates/urine） into Item C vial to prepare a 50
ng/ml standard.  Dissolve the powder thoroughly by a gentle mix. 
Add 15 µl RAGE standard （50 ng/ml） from the vial of Item C, into a
tube with 485 µl Assay Diluent A or 1x Assay Diluent B to prepare a
1,500 pg/ml standard solution.  Pipette 400 µl Assay Diluent A or 1x
Assay Diluent B into each tube. Use the 1,500 pg/ml standard solution to
produce a dilution series （shown below）. Mix each tube thoroughly
before the next transfer.  Assay Diluent A or 1x Assay Diluent B serves
as the zero standard （0 pg/ml）.  
 
 
5.  If the Wash Concentrate （20x） （Item B） contains visible crystals, warm
to room temperature and mix gently until dissolved. Dilute 20 ml of
Wash Buffer Concentrate into deionized or distilled water to yield 400
ml of 1x Wash Buffer.
 
 200 µl
 1,500             500            166.7            55.56           18.52            6.17             2.06                   0          
 pg/ml            pg/ml          pg/ml           pg/ml           pg/ml           pg/ml           pg/ml               pg/ml
 
15 µl standard
+ 485 µl
200µl  200 µl  200 µl  200 µl  200 µl RayBio? Human RAGE ELISA Kit Protocol     5
6. Briefly spin the Detection Antibody vial （Item F） before use. Add 100 µl
of 1x Assay Diluent B into the vial to prepare a detection antibody
concentrate. Pipette up and down to mix gently （the concentrate can be
stored at 4°C for 5 days）.  The detection antibody concentrate should be
diluted 80-fold with 1x Assay Diluent B and used in step 4 of Part VI
Assay Procedure.
 
7. Briefly spin the HRP-Streptavidin concentrate vial （Item G） and pipette
up and down to mix gently before use.  HRP-Streptavidin concentrate
should be diluted 200-fold with 1x Assay Diluent B.
 
     For example: Briefly spin the vial （Item G） and pipette up and down
     to mix gently . Add 50 µl of HRP-Streptavidin concentrate into a tube  
              with 10 ml 1x Assay Diluent B  to prepare a 200-fold diluted HRP-  
              Streptavidin solution （don’t store the diluted solution for next day use）.
              Mix well.  
 
 
VI.  ASSAY PROCEDURE: 
 
1. Bring all reagents and samples to room temperature （18 - 25°C） before
use. It is recommended that all standards and samples be run at least in
duplicate.
 
2. Add 100 µl of each standard （see Reagent Preparation step 2） and
sample into appropriate wells. Cover well and incubate for 2.5 hours at
room temperature or over night at 4°C with gentle shaking.
 
3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by
filling each well with Wash Buffer （300 µl） using a multi-channel
         Pipette 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. RayBio? Human RAGE ELISA Kit Protocol     6
4. Add 100 µl of 1x prepared biotinylated antibody （Reagent Preparation
step 6） to each well. Incubate for 1 hour at room temperature with
gentle shaking.
 
5. Discard the solution. Repeat the wash as in step 3.
 
6. Add 100 µl of prepared Streptavidin solution （see Reagent Preparation
step 7） to each well. Incubate for 45 minutes at room temperature with
gentle shaking.
 
7. Discard the solution. Repeat the wash as in step 3.
 
8. Add 100 µl of TMB One-Step Substrate Reagent （Item H） to each well.
Incubate for 30 minutes at room temperature in the dark with gentle
shaking.
 
9. Add 50 µl of Stop Solution （Item I） to each well. Read at 450 nm
immediay.
 
 
VII.  ASSAY PROCEDURE SUMMARY
1. Prepare all reagents, samples and standards as instructed.
 
 
2. Add 100 µl standard or sample to each well.
    Incubate 2.5 hours at room temperature or over night at 4o
C.
 
 
3. Add 100 µl prepared biotin antibody to each well.
    Incubate 1 hour at room temperature.
 
 
4. Add 100 µl prepared Streptavidin solution.
    Incubate 45 minutes at room temperature.
 RayBio? Human RAGE ELISA Kit Protocol     7
 
5. Add 100 µl TMB One-Step Substrate Reagent to each well.
    Incubate 30 minutes at room temperature.
 
 
6. Add 50 µl Stop Solution to each well.
    Read at 450 nm immediay.
 
 
VIII. CALCULATION OF RESULTS
 
Calculate the mean absorbance for each set of duplicate standards, controls
and samples, and subtract the average zero standard optical density. Plot the
standard curve on log-log graph paper or using Sigma plot software, with
standard concentration on the x-axis and absorbance on the y-axis. Draw the
best-fit straight line through the standard points.
 
A. TYPICAL DATA 
 
These standard curves are for demonstration only. A standard curve must be
run with each assay.
 
    
Assay Diluent A
 Human RAGE concentration （pg/ml）
1 10 100 1000 10000
OD=450 nm 0.01
0.1
1
10
              
Assay Diluent B
 Human RAGE concentration （pg/ml）
1 10 100 1000 10000
OD=450 nm 0.001
0.01
0.1
1
10
        
                    RayBio? Human RAGE ELISA Kit Protocol     8
        
B.  SENSITIVITY
 
The minimum detectable dose of RAGE is typically less than 3 pg/ml. 
 
C.  RECOVERY
Recovery was determined by spiking various levels human RAGE into
human serum, plasma and cell culture media. Mean recoveries are as
follows:
 
Sample Type                     Average % Recovery         Range （%）
 
Serum                                 89.25                                  79-98
 
Plasma                                78.58                                  67-93
 
Cell culture media              103.4                                  94-112
 
 
 
D.  LINEARITY
Sample Type                                   Serum        Plasma    Cell Culture Media
 
1:2       Average % of Expected        115.5          116.2           100.2
            Range （%）                           104-123      105-125      91-109
 
1:4       Average % of Expected        122.5          122.5            101.0
            Range （%）                           113-131      113-132       91-110 
 
 
 
 
 RayBio? Human RAGE ELISA Kit Protocol     9
E.  REPRODUCIBILITY
 
Intra-Assay:  CV<10%
Inter-Assay:  CV<12%
 
 
IX.  SPECIFICITY
Cross Reactivity:  This ELISA kit shows no cross-reactivity with the
following cytokines tested: human Angiogenin, BDNF, BLC, ENA-78, FGF-
4, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12
p70, IL-12 p40, IL-13, IL-15, IL-309, IP-10, G-CSF, GM-CSF, IFN-γ,
Leptin （OB）, MCP-1, MCP-2, MCP-3, MDC, MIP-1α, MIP-1 β, MIP-1δ,
PARC, PDGF, RANTES, SCF, TARC, TGF-β, TIMP-1, TIMP-2, TNF-α,
TNF-β, TPO, VEGF.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 RayBio? Human RAGE ELISA Kit Protocol     10
X.  TROUBLESHOOTING GUIDE 
 
Problem     Cause     Solution
1. Poor standard
    curve
   1. Inaccurate pipetting              1. Check pipettes   
      2. Improper standard
    dilution
   2. Ensure a brief spin
    of Item C
    and dissolve the
    powder thoroughly 
    by a gentle mix.
2. Low signal     1.Too brief incubation
   times
   1. Ensure sufficient
    incubation time; 
    assay procedure
    step 2 may change
to over night
      2. Inadequate reagent
    volumes or improper
    dilution
  2. Check pipettes and
    ensure correct
    preparation
3. Large CV     1. Inaccurate pipetting      1. Check pipettes
4. High background    1. Plate is insufficiently
     washed
  1. Review the manual
     for proper wash. If
     using  a plate
     washer, check that 
     all ports are
     unobstructed.
      2. Contaminated wash 
     buffer
   2.  Make fresh wash 
     buffer
5. Low sensitivity    1. Improper storage of the
    ELISA kit                                
  1. Store your standard
    at<-20o
C after
    reconstitution, 
    others at 4
o
C. Keep
    substrate solution
    protected from light 
    2. Stop solution     2. Stop solution should
    be added to each
    well before measure
 
 
 RayBio? Human RAGE ELISA Kit Protocol     11
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 RayBio? Human RAGE ELISA Kit Protocol     12
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 RayBio? Human RAGE ELISA Kit Protocol     13
 
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