ab190514
Isotyping Mouse Kit
Instructions for Use
Lateral-flow Assays for Rapid Identification of Mouse Immunoglobulin
Isotypes, Subtypes, and Light Chains.
This product is for research use only and is not intended for diagnostic
use.
Version 2 Last Updated 21 October 2014
Table of Contents
INTRODUCTION
1.
BACKGROUND
2.
ASSAY SUMMARY
GENERAL INFORMATION
3.
PRECAUTIONS
4.
STORAGE AND STABILITY
5.
MATERIALS SUPPLIED
6.
MATERIALS REQUIRED, NOT SUPPLIED
7.
LIMITATIONS
8.
TECHNICAL HINTS
2
3
4
4
4
5
5
5
ASSAY PROCEDURE
9.
ASSAY PROCEDURE
6
DATA ANALYSIS
10. INTERPRETATION OF RESULTS
8
RESOURCES
11. TROUBLESHOOTING
12. NOTES
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1
INTRODUCTION
1. BACKGROUND
Abcam’s one-step immunochromatographic mouse isotyping kit allows
for quick and easy identification of mouse isotypes, subtypes and light
chains from a single sample. The sample used can be purified
antibody, cell culture supernatant or ascites fluid. For purified antibody
and cell culture supernatant the test can also be used to determine the
relative purity of a hybridoma cell line.
Lateral-flow membranes are impregnated with separate lines
containing antibodies specific for unique amino acid sequences
contained within IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, kappa light
chains and lambda light chains. When the isotyping strips are added
to a test tube containing a diluted mouse immunoglobulin sample,
colloidal gold nanoparticles conjugated with antibody form a soluble
immune-complex. The complex is apparent in the form of a red band
which allows users to make a qualitative determination for their specific
sample.
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INTRODUCTION
2. ASSAY SUMMARY
2.1 Remove appropriate number of Red and Blue strips.
Equilibrate all reagents to room temperature.
2.2 Add Sample Buffer to glass test tube.
2.3 Add Sample to test tube containing Sample Buffer. Vortex to
mix.
2.4 Add to the test tube one Red and one Blue strip (blank sides
touching).
2.5 Incubate in test tube for 10 minutes prior to reading.
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GENERAL INFORMATION
3. PRECAUTIONS
Please read these instructions carefully prior to beginning the
assay.
All kit components have been formulated and quality control tested to
function successfully as a kit. Modifications to the kit components or
procedures may result in loss of performance.
4. STORAGE AND STABILITY
Store kit at Room Temperature immediately upon receipt.
Do not freeze any kit components.
Kit components should not be exposed to UV light or prolonged heat.
Store all components in their original packaging.
Stable for one year from the date of shipment.
5. MATERIALS SUPPLIED
Item
Amount
Storage
Condition
(Before
Preparation)
Red strips to identify – IgG1, IgG2a, IgG2b and
IgG3 subtypes
20
RT
Blue strips to identify - kappa light chain,
lambda light chain, IgA and IgM.
20
RT
15 mL
RT
1
RT
Sample buffer
Desiccant pouch
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GENERAL INFORMATION
6. MATERIALS REQUIRED, NOT SUPPLIED
These materials are not included in the kit, but will be required to
successfully utilize this assay:

Adjustable pipettes for reagent preparation.

Absorbent paper.

Distilled or deionized water.

Tubes to prepare standard or sample dilutions.
7. LIMITATIONS

Do not mix or substitute reagents or materials from other kit lots or
vendors. Kits are QC tested as a set of components and
performance cannot be guaranteed if utilized separately or
substituted.

Bacterial or fungal contamination of either samples or reagents or
cross-contamination between reagents may cause erroneous
results.

Disposable pipette tips, flasks or glassware are preferred, reusable
glassware must be washed and thoroughly rinsed of all detergents
before use.
8. TECHNICAL HINTS

Kit components should be stored as indicated. All the reagents
should be equilibrated to room temperature before use.
Reconstituted standards should be discarded after use.

Use a clean disposable plastic pipette tip for each reagent,
standard, or specimen addition in order to avoid crosscontamination; for the dispensing of the Stop Solution and
substrate solution, avoid pipettes with metal parts.

Thoroughly mix the reagents and samples before use by agitation
or swirling.
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ASSAY PROCEDURE
9. ASSAY PROCEDURE
9.1
For Cell Culture Fluids
9.1.1
9.1.2
9.1.3
9.1.4
9.1.5
9.1.6
Equlilbrate all reagents to room temperature.
Remove the required number of strips from each
canister. Close canister securely.
Add 375 µL of sample buffer to each 12 x 75 mm
polystyrene or glass test tube.
Add 25 µL of your sample of cell culture fluid to the
test tube containing 375 µL of sample buffer and
vortex to mix. This is a 1/16 dilution of your sample.
Add to the test tube one red strip (IgG1, IgG2a,
IgG2b and IgG3) and one blue strip (kappa, lambda,
IgA and IgM). The blank side of each strip should
be touching the other so both outward facing strips
can be read while positioned in the test tube.
Allow the strips to incubate in the test tubes for up to
10 minutes prior to reading.
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ASSAY PROCEDURE
9.2
For Purified Antibodies above 1.0 mg/mL or Ascites
Fluid
9.2.1
9.2.2
9.2.3
9.2.4
9.2.5
9.2.6
9.2.7
9.2.8
Equlilbrate all reagents to room temperature.
Remove the required number of strips from each
canister. Close canister securely.
Pipet 45 µL of sample buffer to the first 12 x 75 mm
polystyrene or glass test tube.
Add 5 µL of your purified antibody or ascites sample
to the first test tube containing 45 µL of sample
buffer and gently mix. This is a 1/10 dilution of your
sample.
Into a second test tube pipet 395 µL of sample
buffer.
Add 5 µL of the previously mentioned 1/10 sample
dilution to 395 microliters of sample buffer in second
test tube. This is a 1/800 dilution of your sample.
Add to the second test tube one red strip (IgG1,
IgG2a, IgG2b and IgG3) and one blue strip (kappa,
lambda, IgA and IgM). The blank side of each strip
should be touching the other so both outward facing
strips can be read while positioned in the test tube.
Allow the strips to incubate in the test tubes for up to
10 minutes prior to reading.
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DATA ANALYSIS
10. INTERPRETATION OF RESULTS
Interpret the results at 8-10 minutes once the positive control band
appears.
The test is valid if a positive control band appears below the “CCC”
region of the strip (see image).
A colored red band will form below the printed text indicating the
isotype, subtype and light chain of the monoclonal antibody.
Positive for IgG1 = red band below the G1 text on red strip.
Positive for IgG2a = red band below the G2a text on red strip.
Positive for IgG2b = red band below the G2b text on red strip.
Positive for IgG3 = red band below the G3 text on red strip.
Positive for IgA = red band below the Ag text on blue strip.
Positive for IgM = red band below the Mg text on blue strip.
Positive for Kappa Light Chain = red band below the k k k text on blue
strip.
Positive for Lambda Light Chain = red band below the λ λ λ text on
blue strip
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RESOURCES
11. TROUBLESHOOTING
Problem
Cause
Solution
Antibody
concentration is too
high
Dilute sample and test
again.
May be an indication
of a mixed clone in
your cell culture
supernantant
Re-cloning the hybridoma
may be necessary.
No heavy and or
light-chain appears
on either strip, but a
positive control band
is visible.
Antibody
concentration of
sample is too low or
hybridoma isn’t
secreting
immunoglobulin
Re-test the sample at a
higher concentration.
Positive result for
Kappa or Lambda,
but negative for IgG
subtype, IgA or IgM.
Secreted antibody
may be IgE, IgD or
IgG2c.
N/A
Multiple bands
appear on a single
strip.
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RESOURCES
12. NOTES
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RESOURCES
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