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
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