Mouse Typer
®
Sub-Isotyping Kit
Instruction Manual
Protocol for Mouse Typer Sub-Isotyping
Kit (Catalog Number 172-2051)
and Mouse Typer Sub Isotyping Panel
(Catalog Number 172-2055)
For Technical Service Call Your Local Bio-Rad Office
or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)
Table of Contents
Section 1 Introduction ..........................................................1
1.1 Background.......................................................................1
1.2 Materials Required............................................................2
1.3 Storage and Stability.........................................................2
1.4 Reagents and Equipment Not Included ............................2
Section 2 Sub-Isotyping Assay.............................................3
2.1 Solutions ...........................................................................3
2.2 General Recommendations...............................................4
2.3 Procedure ..........................................................................5
Section 3 Troubleshooting Guide ........................................8
Section 4 References ...........................................................11
Section 1
Introduction
1.1 Background
The growth and widespread use of mouse monoclonal
antibody technology have created a need for a fast, accurate, and
simple means of determining immunoglobulin class and sub-class.
Several classes of mouse monoclonal antibody, all structurally
different depending on their heavy chain composition, have been
described.
properties of the various classes are unique. They differ in their
solubility and electrophoretic properties, in their susceptibility to
cleavage enzymes, and in their reactivity with protein A.
convenient kit for identifying mouse immunoglobulin class and
sub-class in tissue culture supernatant and ascitic fluids. The
ELISA based kit uses a panel of ultra pure reagents to determine
mouse sub-isotypes: IgG
and λ chain. It comes complete with all the essential reagents for
800 extremely sensitive tests (100 typings). The included protocol
and troubleshooting guide guarantee efficient and accurate assays.
Mouse Typer kit is fast and simple. Antigen is first adsorbed to a
microplate, then treated with specific monoclonal antibody. Bound
monoclonals are separately reacted with each of the Mouse Typer
rabbit anti-mouse panel reagents. Immunoglobulin class and sub-class
are then determined with Bio-Rad’s goat anti-rabbit (H + L)
horseradish peroxidase (HRP) conjugate and peroxidase substrate
system. Color development occurs immediately, with positive wells
having absorbances 3-6 times that of corresponding negatives. Using
Bio-Rad’s Model 3550 or Model 3550-UV Microplate Reader, results
are easily quantitated in less than 60 seconds.
1
Identification is essential since chemical and biological
2,3
The Mouse Typer sub-isotyping kit is an extremely
, IgG2a, IgG2b, IgG3, IgM, IgA,
1
χ
Determining a mouse monoclonal antibody’s class with the
chain,
1
1.2 Materials Required
Kit Components:
Catalog Quantity/
Number Product Description Package
172-2055 Mouse Typer Sub-Isotyping Panel, includes 10 ml each
172-1019 EIA Grade Affinity Purified Goat Anti-Rabbit IgG 1 ml
172-1064 Peroxidase Substrate System, contains 2,2’- 200 ml
ultra pure rabbit anti-mouse subclass specific
anti-serum to mouse IgG
IgM, IgA, χchain, and λ chain.
(H + L), human adsorbed, horseradish peroxidase conjugate (GAR-HRP).
Azino-di (3-ethyl-benzthiazoline sulfonate [6])
and hydrogen peroxide.
, IgG2a, IgG2b, IgG3,
1
1.3 Storage and Stability
Temperature Shelf Life
Mouse Typer Sub-Isotyping Panel 4 °C 1 year
GAR-HRP Antibody Conjugate 4 °C 1 year
Peroxidase Substrate System 4 °C >1 year
1.4 Reagents and Equipment Not Included
1. Specific mouse monoclonal antibody, culture or ascites fluid,
and its cognate antigen.
2. Sodium chloride (NaCl), sodium phosphate dibasic hepta-
hydrate (Na
(NaH
2PO4-H2
3. Tween 20, EIA Grade (Bio-Rad catalog number 170-6531).
4. Bovine Serum Albumin BSA (Sigma catalog number A 9647).
5. Thimerosal, (Sigma catalog number T 5125).
6. Oxalic acid, dihydrate (J. T. Baker catalog number 0230-1).
HPO4-7H2O), sodium phosphate monobasic
2
O), ACS Reagent Grade.
2
7. 96-well polystyrene microplates (Bio-Rad catalog number
224-0096).
8. Pipet tips (Bio-Rad catalog number 223-9302, nonsterile).
®
9. Octapette
10. Reagent reservoirs (Bio-Rad catalog number 224-4872).
11. Automatic ELISA plate reader (Bio-Rad Model 3550 Micro-
plate Reader, catalog number 170-6601 or Model 3550-UV
Microplate Reader, catalog number 170-6638, or Model 550
Microplate Reader, catalog number 170-6750).
pipet, 100 ml (Bio-Rad catalog number 224-4800).
Section 2
Sub-Isotyping Assay
2.1 Solutions
The solution volumes that follow are recommended for an
ELISA assay typing of 10 mouse monoclonal antibodies in one
microtitration plate.
Phosphate buffered (0.01 M phosphate buffer, pH 7.2)
saline, PBS, 300 ml Add 0.105 g sodium monobasic phosphate,
0.600 g sodium dibasic phosphate, and
2.550 g sodium chloride to 250 ml distilled,
deionized water. Adjust to pH 7.2 with HCl
and bring to 300 ml with water.
Phosphate buffered Divide the above solution into 100 and
saline wash solution, 200 ml fractions. Add 0.10 ml Tween 20
PBS-Tween, 200 ml to the 200 ml fraction.
Antigen solution, Dissolve or dilute antigen preparation in
10 ml 10 ml PBS to a final concentration of 1-
10 µg/ml.
3
Blocking solution, 1% BSA-PBS.
30 ml Add 0.3 g BSA to 30 ml PBS. Adjust pH
to 7.2.
Antibody conjugate Dilute GAR-HRP conjugate 1:3,000 by
solution, 10 ml adding 3.3 µl to 10 ml of PBS-Tween.
Peroxidase substrate Mix 9 ml solution A with 1 ml solution B.
solution, 10 ml Prepare fresh prior to use and use
immediately.
Color stopping (2% oxalic acid)
solution, 50 ml Add 1 g oxalic acid dihydrate to 50 ml
distilled, deionized water.
Note: If stock solutions of PBS, PBS-tween, and blocking solution
are made, include the bacteriostat thimerosal at a concentration of
0.01%. Avoid the use of sodium azide, as it is an inhibitor of peroxidase activity.
2.2 General Recommendations
1. Assay Incubation Temperature: All incubation steps are
performed at room temperature (23-25 °C), with the
microplate covered to prevent evaporation. For convenience,
any step may be carried out overnight at 4 °C. Incubation
times can be decreased to 0.5 hr if done at 37 °C.
2. Reagent Purity: All reagents should be ACS or EIA Grade.
Chemical impurities, as well as poor water quality, can cause
enzyme inhibition and/or increased backgrounds.
3. Antigen Adsorption: Coat the immunoassay microtitration
plates with 0.1 to 1.0 mg antigen per well. The optimal concentration should be determined empirically prior to subtyping. Antigen adsorption is a function of concentration,
diluent, type of assay plate, and purity of sample.
antigens also can be used for sub-isotyping.
4
4
Whole cell
4
4. Monoclonal Antibody Sample: To insure proper typing of
culture media samples, do not dilute when applying to
microplates. When using ascites fluid, dilute at least 1:1,000 with
PBS-tween before testing. Serial dilutions may be necessary to
establish the working dilution of ascites fluid required for the
best signal-to-noise ratio in the typing immunoassay.
5. GAR-HRP Antibody Conjugate: Bio-Rad’s affinity purified
antibodies should be used at recommended dilutions. These
products give excellent signal-to-noise ratios while using less
reagent. More antibody may be used, but this could result in
higher backgrounds with minimal increase in detection
sensitivity.
6. Background: Nonspecific background reactions are usually
the result of low-purity second antibody and/or using
excessive conjugate antibody concentrations. Always wash
plates thoroughly, especially after the conjugate incubation.
Tween 20 is essential in all wash steps after blocking. At a
0.05% concentration, it will not disrupt antigen-antibody
interactions.
For further assistance in determining sub-isotyping of
monoclonal antibodies, contact Bio-Rad Technical Services (in the
USA 1-800-424-6723) or your local technical representative.
2.3 Procedure
Before starting the assay, read through the entire protocol.
1. Adsorb antigen (Ag) to the microplate by adding 100 µl Ag
solution to all wells (0.1-1.0 µg Ag/well). Cover the plate and
incubate at room temperature for at least 1 hour.
Note: If the Ag coated plates are not used immediately, cover
and store at 4 °C. Antigen solution should contain 0.01%
thimerosal. Plates can be stored for up to 1 month.
5
2. Remove any unbound Ag by flooding the wells of the assay
plates with PBS. Use a plastic wash bottle or an automatic
plate washer. When using a wash bottle, fill each well, soak
for 15 seconds, then vigorously shake off solution in a sink
(flick-washing). Repeat 2 times.
3. To prevent nonspecific binding, fill all the wells with 300 µl
blocking solution(1% BSA-PBS). Let stand at room temperature
for 30 minutes, then flick-wash the plate 3x with PBS-tween.
4. Add hybridoma culture fluid (undiluted) or ascites (diluted)
samples, 100 µl/well, using the suggested format outlined in
Figure 1. Six wells of columns 2-11 are used (1 sample per
column). Column 1 is reserved for substrate blank and column
12 for positive control (
i.e., mouse serum). Cover and incubate
the plate for 1 hour at room temperature.
5. Empty the plates of hybridoma supernatant or diluted ascites
fluid. Flick-wash the plate 3x with PBS-Tween.
6. Add appropriate rabbit anti-mouse panel reagents using the
format in Figure 1. All of rows A-H are filled with respective
panel reagent, 100 µl/well. Incubate the covered microplate for
1 hour at room temperature.
6
Fig. 1. Suggested format for sub-isotyping 10 samples
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
+
C
O
N
T
R
O
L
B
L
A
N
K
Samples 1-10
on 1 microplate.
7. Empty the plates of panel reagents and flick-wash 5x with
PBS-tween.
8. With the exception of column 1, fill the wells with diluted goat
anti-rabbit horseradish peroxidase conjugate, 100 µl/well. Cover
and incubate for 1 hour.
9. During the incubation with conjugate, prepare peroxidase
substrate solution (see Section 2.1).
10. Wash off unreacted conjugate solution by flick-washing the
plate 4x with PBS-tween. Wash an additional time with PBS.
Invert and tap the plate over paper toweling to rid the wells of
excess wash solution. Add 100 µl peroxidase substrate
solution. Positive reactions will appear immediately. Identification of mouse sub-isotype can be assessed after a 10 to 30
minute room temperature incubation.
11. Stop color development by adding 100 µl/well 2% oxalic acid
color stopping solution. Results can be documented using the
Model 3550, 3550-UV, or Model 550 Microplate Reader at
415 nm.
7
Note: When assessing color development using Model 3550,
Model 550, or the Model 3550-UV Microplate Reader, wash
the bottom of the assay plate thoroughly with distilled water
and wipe dry with non-lint toweling.
Section 3
Troubleshooting Guide
Problem Probable Cause Recommended Solution
A. High back- 1.Insufficient washing 1. Wash each well 6-7x and
ground. after conjugate anti- increase soak cycles to 30
body incubation. seconds.
2.Insufficient blocking 2.Increase blocking step to
after antigen adsorption. 60 minutes.
3. Tween 20 absent from 3. Include Tween 20 in all
washes. washes and solutions after
blocking.
4.GAR-HRP conjugate 4. Use recommended
concentration too high. dilutions. Generally, the
less dilute, the higher the
background.
5.Color developed too 5. Decrease color developlong. ment time by one-half.
6.Substrate too old (high 6. Use fresh solution A and
green color at working solution B
dilution).
7.Whole cell antigens 7a. Use extracted antigens.
have endogenous 7b. Use other enzyme conperoxidase activity. jugated antibodies.
7c. Destroy endogenous
activity by incubating
adsorbed Ag with mixture of methanol/ H2O
(99 ml methanol, 1 ml
30% H2O2), 100 µl/well
for 1 hr.
2
8
Problem Probable Cause Recommended Solution
B. No reaction or
weak color
development.
1. Horseradish a.Improper storage of a. Store peroxidase substrate
peroxidase sub- reagents. at 4 °C.
strate solution b. Substrate solutions hy- b. Use fresh peroxidase
inactive (Note 1). drolyzed due to age. substrate solutions.
2. Goat anti-rabbit a.Antibody improperly a. Store at 0-4 °C. Avoid
horseradish stored. bacterial contamination
peroxidase and repeated freeze thaw
conjugate is cycles.
inactive or non- b. Nonsaturating concen- b. If possible , increase
saturating trations of monoclonal concentration of
(Note 2). or conjugate antibody monoclonal, conjugate, or
used in incubations. both. Use caution, the less
the conjugate is diluted
the greater the nonspecific
color produced.
3. Monoclonal anti- a. Monoclonal impro- a. Avoid bacterial contami-
body solution is perly stored. nation and heat inactiva-
inactive or non- tion.
saturating b. Antibody titer too low. b. Increase amount of hybri-
(note 3). doma culture media added
to plates (i.e. 200 µl).
May also be necessary to
increase conjugate antibody concentration.
c. Tween 20 deteriorates c. Eliminate Tween 20 from
reactivity of antibodies. all solutions and buffers
except wash after
blocking. Could result in
increased backgrounds.
9
Problem Probable Cause Recommended Solution
C. Monoclonal 1.Impure sample. 1. Use fresh source of cul-
exhibits multiple ture supernatant. Purify
sub-isotype (i.e. media or ascites fluid.
assay shows one 2.Sample too con- 2. Dilute supernanants and
clone to be both centrated ascites fluid, repeat test.
an IgG1 and 3.Sample is polyclonal. 3. Re-clone hybridoma cells
IgM). by limited dilution,8and
repeat assay.
Notes to Troubleshooting Guide
1. Activity Test for Horseradish Peroxidase Substrate Solution.
Combine 1.0 ml of substrate solution with 10 ml of antibody
conjugate. If no color develops in 5 minutes, substrate solution
is at fault.
2. Activity Test for Antibody Conjugate Solution.
Combine 1.0 ml of horseradish peroxidase substrate solution
(tested above) and 1.0 ml of diluted antibody conjugate
solution. If no color develops in 5 minutes, the conjugate is
suspect. Repeat procedure using fresh conjugate antibody
dilution.
3. Activity Test for Monoclonal Antibody Solution.
Use RID, Ouchterlony precipitation, or ELISA assay to
determine reactivity. Repeat procedure with more
concentrated monoclonal antibody solution.
5-7
10
Section 4
References
1. Hybridomas, American Type Culture Collection, Rockville, MD
(1984).
2. Beyer, C. F., J. Immunol. Methods, 67, 79 (1984).
3. Langone, J. J., J. Immunol. Methods, 51, 3 (1982).
4. Clone Selector®Mouse, Human, or Rat Monoclonal Antibody
Screening Kit Instruction Mannual, Bio-Rad Laboratories, Hercules,
CA.
5. Stanker, L. H., Vanderlaan, M. and Juarez-Salinas, H., J. Immunol.
Methods, 157 (1985).
6. Bio-Radiations 49, Bio-Rad Laboratories, Hercules, CA.
7. Bio-Radiations 53, Bio-Rad Laboratories, Hercules, CA.
8. Kwan, S., Yelton, D. E. and Scharff, M. D., Genetic Engineering,
Vol. 2, (Setlow, J. K. and Hollaender, A., eds.) pp. 31-46, Plenum
Publishing Corp., New York (1980).
Octapette is a trademark of Costar.
11
Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547
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