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PBL Interferon Source
131 Ethel Rd West Suite 6
Piscataway, NJ 08854
Toll-free 877-PBL-8881
 
 
 Verikine ™ Mouse Interferon Alpha
（Mu-IFN- ） ELISA Kit
Rev. 01
 
Product #42100-1
Single Plate （96 Wells*）
 
Lot Number:      
 
Expiration Date:      
 
Sensitivity: 12.5– 500 pg/ml
 
 
*Due to edge effect we recommend not using top and bottom rows （72 wells）
 
 
For research use only.  Not for use in human diagnostic or therapeutic procedures. ©
Copyright 2000 PBL Biomedical Laboratories.  All rights reserved
 
Please review the protocol in its entirety prior to use to insure proper kit
performance.  Please note that the concentrations of the Detecting Antibody
and HRP differ from lot to lot as a result of calibrating each kit for optimal
sensitivity.
 
Speed: Incubation time, 26 hr 15 min
 
Specificity: Mouse IFN-α .   No cross-reactivity with rat IFN-α,  mouse IFN-β,
mouse IFN-γ; or human IFN-α.
 
Specifications:  This kit quantitates mouse interferon alpha in media using a
sandwich immunoassay.
1,2
 The kit is based on an  ELISA with anti-secondary
antibody conjugated to horseradish peroxidase （HRP）.  Tetramethyl-benzidine
（TMB） is the substrate.  All reagents are supplied.  One pre-coated microtiter plate
（96 wells） is included.  The assay is based on the international reference standard
for mouse interferon alpha （Mu-IFN-α） provided by the National Institutes of
Health.
3
 Typical standard curves for each lot are included with the procedure. 
 Special Conditions/Comments: For retention of activity, all reagents should be
kept at 2-6
o
C in the dark.  Deionized or distilled water should be used for
preparation of all reagents.  All dilutions should be made with polypropylene tubes
and pipette tips.  Pipette tips should be changed between each dilution tube.  All
measurements for standards and samples should be performed in duplicate.  At least two control wells （wells with Dilution Buffer only） should be used for
each assay; these control values should be subtracted  from all readings
prior to any calculations or plots of the data. 
 
Please note that the blanks are higher on the top and bottom edges of
the plate. For higher sensitivity samples （<100pg/mL） avoid using
those wells.
 
**Caution** Solutions SMP003-60, SMP038-1, SMP010-60, SMP039-1 and
SMP011-15 contain 0.1 g/L thimerosal as a preservative; they should be
handled with appropriate safety precautions and discarded properly.  Since
thimerosal is highly toxic through skin contact, inhalation or ingestion,
suitable protective wear and care  should be used in handling these
solutions.  For further information, consult the material safety data sheets
for thimerosal （CAS #54-64-8）. 
 Materials Supplied:
 
Kit Components  Part Number  #42100-1  
Size: single plate    Lot#  Qty
Plate （s）  SMP037         1
Plate Sealers  N/A n/a 4
Wash Solution Concentrate  SMP003-60         50 ml
Mouse IFN Alpha Solution
10,000pg/mL
SMP038-1         0.2 ml
Dilution Buffer  SMP010-60         50 ml
Antibody Concentrate  SMP039-1         1 vial
HRP Conjugate Concentrate  SMP050-1350          1 vial
HRP Conjugate Diluent  SMP011-15         15 ml
TMB Substrate Solution  NGE-15         15 ml
Stop Solution   SCY-15         15 ml
 
Procedure:
 
1.  Before starting assays, prepare Final Wash Solution as follows.  Dilute
50 ml of the Wash Solution Concentrate to 1000 ml with distilled
water.  The Final Wash Solution should be stored in the refrigerator
and mixed thoroughly before use.   All the wash steps should be
preformed at room temperature （24
o
C） with ice cold
            （2 – 6
o
 C） wash solution. 
 2.  Construct a standard curve from 0 – 500 pg/ml by serial dilutions of
the Mouse Interferon Alpha Solution Standard in the Dilution Buffer. 
Please note that the sample curves provided are for reference
only.
Label seven polypropylene tubes and serially dilute the Mouse Interferon
Alpha Solution as shown below.   All dilutions should be made with
polypropylene tubes and pipette tips.   Pipette tips should be changed
between each dilution.  Samples of unknown interferon concentration to be
tested should also be diluted in the Dilution Buffer as required.
 
Tube No.  Vial B  S6 S5 S4 S3 S2 S1 B
Amount
Taken from
Tube to Left
（ml）
---  0.05 0.4 0.5 0.5 0.5  0.5  ---
Dilution
Buffer 
（ml）
---  0.95 0.6 0.5 0.5 0.5  0.5  1.0
Final Conc.
（pg/ml）  10,000 500 200 100  50  25  12.5  0
 
3.  Determine the number of microplate wells required to test the desired
number of samples.  We recommend the use of 14 wells for blanks （BK）
and standards （S1-S6）.  Remove extra microtiter strips from the frame, seal
in the foil bag provided and store at 2 – 6
o
C.  Unused
             strips can be used in later assays. 
 
Please note that the blanks are higher on the top and bottom edges of
the plate. For higher  sensitivity samples （<100pg/mL） avoid using
those wells.
 
4.  Place precisely 100  µl of the interferon samples prepared in Step 2 in
individual wells of the microtiter plate, at least in duplicate. individual wells of the microtiter plate, at least in duplicate.
 
  1  2  3  4  5  6  7  8  9  10  11  12
A                        
B S1 S1  BK
            
C  S2 S2  BK
            
D S3 S3
              
E  S4 S4
              
F  S5 S5
              
G  S6 S6
              
H                       
 
BK: Blank – buffer only                 S1 – S6: serially diluted standards
                          
                                      Wells should be used only for samples predicted to be 
                                       > 100pg/mL
 A recommended microplate sample layout  for blanks and standards is shown in
the diagram above.  Samples of unknown interferon concentration should be
tested as required.