Human IgG-Fc ELISA Quantitation Kit
Manual
Catalog number: 40-288-20083F
Kit Contents:
(Enough for ten 96-well plates)
Coating Antibody (Catalog # 15-288-20083F)
Affinity-purified Chicken anti-Human IgG-Fc
Quantity: 200 pg
Calibrator (Catalog # 10-288-20083F)
Pure Human IgG-Fc Antigen
Quantity: 3 pg
HRP Detection Antibody (Catalog # 27-288-20083F)
Affinity-purified Chicken anti-Human IgG-Fc – HRP Conjugate
Quantity: 5 pg
Buffers, Substrate and Plates not included.
Notes:
Range of Detection: 300 – 0.412 ng/ml
Shelf life: One year from date of receipt.
Storage: -20°C.
Assay Condition: The kit performance has been optimized for the stated protocol using the
materials listed and standard dilutions from 300 – 0.412 ng/ml of human IgG-Fc. For alternative
assay conditions, the operator must determine appropriate dilutions of reagents. ELISA assay
reactivity is sensitive to any variation in operator, pipetting and washing techniques, incubation
time or temperature, composition or reagents, and kit age. Adjustments may be required to
position the standard curve and/or samples in the desired detection range.
Specificity: By immunoelectrophoresis and ELISA the antibodies in this kit react specifically with
human IgG-Fc, not with other human serum proteins. The following factors prepared in the
detection range of this kit, 300 – 0.412 ng/ml were assayed and exhibited no cross-reactivity or
interference.
Human Serum Albumin
Human Transferrin
Human IgM
Human IgA
Country of 0rigin: United States of America
Assay Use: For in vitro laboratory use only. Not for any clinical, therapeutic, or diagnostic use in
humans or animals. Not for animal or human consumption.
For research use only; not for diagnostic or therapeutic use.
Page 1
Human IgG-Fc Quantitative ELISA Protocol
Standard Curve
Buffer
Preparation
3
2.5
1.
Prepare the
A.
o
a
t
i
n
g
2
1.5
1
B
u
f
f
e
r
,
0.5
0.1
1
10
100
1000
Concentration
y = ( (A - D)/(1 + (x/C)^B ) ) + D:
Std (Standards: Concentration vs MeanValue)
A
0.417
B
1.002
C
10.594
D
2.998
R^2
1
0
.05 M Carbonate-Bicarbonate, pH 9.6
B.
Wash Solution, 0.05% Tween 20 in PBS, pH 7.4
C.
Blocking Solution, 50 mM Tris, 0.14 M NaCl, 1% BSA, pH 8.0
D.
Dilution buffer, 50 mM Tris, 0.14 M NaCl, 1% BSA, 0.05% Tween 20, pH 8.0
E.
Enzyme Substrate, TMB (KPL, Cat # 50-76-00)
F.
Stopping Solution, 2 M H2SO4 or other appropriate solution
Step-by-Step Method (Perform all steps at room temperature, 25 o C
1.
Coat with Capture Antibody
A.
B.
C.
D.
E.
.
Blocking (Postcoat)
A.
B.
C.
3.
Determine the number of single wells needed. Standards, samples, blanks and/or
controls should be analyzed in duplicate. Insert the required number of microtiter well
strips into a holder.
Dilute the capture antibody with dilution buffer to a concentration of 2 Hg/ml. Coat
each well with 100 Hl of capture antibody dilution.
Incubate coated plate for 60 minutes.
After incubation, aspirate the Capture Antibody solution from each well.
Wash each well with W ash Solution as follows:
1.
Fill each well with Wash Solution
2.
Remove Wash Solution by aspiration
3.
Repeat for a total of 3 washes.
Add 200 Hl of Blocking Solution to each well.
Inc u ba te 30 m in ut es .
After incubation, remove the Blocking Solution and wash each
well three times as in Step 1.E.
Standards and Samples
For research use only; not for diagnostic or therapeutic use.
Page 2
C
A.
Dilute the calibrator to a concentration of 300 ng/ml. Do 1:3 serial dilutions (1 volume
sample into 2 volumes diluent) down to a concentration of 0.412 ng/ml.
Dilute the samples, based on the expected concentration of the analyte, to fall within
the concentration range of the standards.
Transfer 100 Hl of standard and sample to assigned wells.
Inc u ba te pl a te 6 0 m in ut es .
After incubation, remove samples and standards and wash each well 5 times as in
Step 1.E.
B.
C.
D.
E.
4.
HRP Detection Antibody
A.
Dilute the HRP conjugate in dilution buffer to a concentration of 50 ng/ml. (Note:
Adjustments in dilution may be needed depending on substrate used, incubation time,
and age of kit).
B.
Tr ansfer 100 Hl to each well.
C.
Inc u ba te 60 m in ut es .
D.
5.
Enzyme
A.
B.
C.
D.
6.
After incubation, remove H RP Conjugate and wash each well 5 times as in Step 1.E.
Substrate Reaction
Prepare the substrate solution according to the manufacturer’s recommendation.
Transfer 100 Hl of substrate solution to each well.
Incubate plate so that the high calibrator is at an OD450 of 2-3. (Approximately 3-10
minutes depending on age of kit.)
To stop the TMB reaction, apply 100 Hl of 2 M H2SO4to each well. If using another
substrate, use the stop solution recommended by manufacturer.
Plate Reading
Using a microtiter plate reader, read the plate at the wavelength that is appropriate for the
substrate used (450 nm for TMB).
Calculation of Results
1.
2.
3.
4.
Average the duplicate readings from each standard, control, and sample.
Subtract the zero reading from each averaged value above.
Create a standard curve by reducing the data using computer software capable of generating
a four parameter logistic (4-PL) curve-fit. Other curve fits may also be used.
A standard curve should be generated for each set of samples. See example below:
Standards (ng/ml)
Sample
St01
Concentration
300
St02
100
St03
33.333
St04
11.111
St05
3.704
St06
1.235
BackCalcConc
200.562
267.358
485.07
105.378
90.365
122.977
31.805
32.608
35.212
10.555
11.334
11.143
3.597
3.836
3.928
1.112
Wells
A1
A2
A3
B1
B2
B3
C1
C2
C3
D1
D2
D3
E1
E2
E3
F1
Values
2.869
2.9
2.943
2.763
2.728
2.794
2.354
2.366
2.402
1.705
1.751
1.74
1.07
1.102
1.114
0.661
For research use only; not for diagnostic or therapeutic use.
MeanValue Std.Dev.
2.904
0.037
CV%
1.3
2.762
0.033
1.2
2.374
0.025
1.1
1.732
0.024
1.4
1.095
0.023
2.1
0.676
0.013
1.9
Page 3
St07
0.412
1.223
1.223
0.346
0.502
0.434
F2
F3
G1
G2
G3
0.683
0.683
0.498
0.533
0.518
0.516
0.018
3.4
Smallest standard value: 0.516
Largest standard value: 2.904
Technical Hints
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
The Capture antibody should be diluted with coating buffer immediately prior to its addition to the
wells. Coated plates are stable overnight at 4ºC when covered.
Change pipette tips between each addition of standard, sample and reagents to avoid crosscontamination.
Standards and samples should be pipetted to the bottom of the wells and all other reagents should be
added to the side of the wells to avoid contamination.
Ensure that all buffers are not contaminated or expired. When troubleshooting ELISA results, it is
recommended to prepare all new buffers in new vessels.
Do not add Sodium Azide to any of the buffers.
Sample and Conjugate dilutions should be made shortly before use.
Wash buffer should be aspirated from wells, as pouring wash buffer from wells may cause crosscontamination.
When preparing dilutions, wipe excess antibody/analyte from pipette tips to ensure accurate dilutions.
Incubation time of the Enzyme Substrate will depend on the substrate used and the intensity of the
color change. The high standard should have an O.D. reading of between 2 and 3. The low standard
should have an O.D. reading above background.
The Stopping solution should be added to the wells in the same order as the Enzyme Substrate.
For research use only; not for diagnostic or therapeutic use.
Page 4
Troubleshooting
The following are some common problems encountered with the use of ELISA kits, and some of the causes of
these problems.
1.
Problem: Low absorbance
 Incorrect dilutions or pipetting errors.
 Improper incubation times
 Improper mixing of the TMB substrate. Each component is mixed in equal parts.
 Wrong filter on microtiter reader. Wavelength should be 450 nm for TMB, 490 nm for
OPD, or 405 nm for ABTS.
 Kit materials or reagents are contaminated or expired.
 Incorrect reagents used.
.
Problem: High Absorbance
 Cross contamination from other samples or positive control.
 Incorrect dilutions or pipetting errors.
 Improper washing.
 Wrong filter on microtiter reader.
 Contaminated buffers or enzyme substrate.
 Improper incubation times.
 Kit materials or reagents are contaminated or expired.
3.
Problem: Poor Duplicates
 Poor mixing of specimens.
 Incorrect dilutions or pipetting errors.
 Technical error.
 Inconsistency in following ELISA protocol.
 Inefficient washing.
4.
Problem: All wells are positive
 Contaminated buffers or enzyme substrate.
 Incorrect dilutions or pipetting errors.
 Kit materials or reagents are contaminated or expired.
 Inefficient washing.
5.
Problem: All wells are negative
 Procedure not followed correctly.
 Contaminated buffers or enzyme substrate.
 Contaminated conjugate.
 Kit materials or reagents are contaminated or expired.
For research use only; not for diagnostic or therapeutic use.
Page 5