LIT78D 8/7/98 04:08 PM Page A 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.
LIT78D 8/7/98 04:08 PM Page B Table of Contents Section 1 Introduction ..........................................................1 1.1 1.2 1.3 1.4 Background .......................................................................1 Materials Required............................................................2 Storage and Stability .........................................................2 Reagents and Equipment Not Included ............................2 Section 2 Sub-Isotyping Assay ..................
LIT78D 8/7/98 04:08 PM Page 1 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.1 Identification is essential since chemical and biological properties of the various classes are unique.
LIT78D 8/7/98 04:08 PM Page 2 1.2 Materials Required Kit Components: Catalog Number Quantity/ Package Product Description 172-2055 Mouse Typer Sub-Isotyping Panel, includes 10 ml each ultra pure rabbit anti-mouse subclass specific anti-serum to mouse IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, χ chain, and λ chain. 172-1019 EIA Grade Affinity Purified Goat Anti-Rabbit IgG 1 ml (H + L), human adsorbed, horseradish peroxidase conjugate (GAR-HRP).
LIT78D 8/7/98 04:08 PM Page 3 7. 96-well polystyrene microplates (Bio-Rad catalog number 224-0096). 8. Pipet tips (Bio-Rad catalog number 223-9302, nonsterile). 9. Octapette® pipet, 100 ml (Bio-Rad catalog number 224-4800). 10. Reagent reservoirs (Bio-Rad catalog number 224-4872). 11. Automatic ELISA plate reader (Bio-Rad Model 3550 Microplate Reader, catalog number 170-6601 or Model 3550-UV Microplate Reader, catalog number 170-6638, or Model 550 Microplate Reader, catalog number 170-6750).
LIT78D 8/7/98 04:08 PM Page 4 Blocking solution, 30 ml 1% BSA-PBS. Add 0.3 g BSA to 30 ml PBS. Adjust pH to 7.2. Antibody conjugate solution, 10 ml Dilute GAR-HRP conjugate 1:3,000 by 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 solution, 50 ml (2% oxalic acid) Add 1 g oxalic acid dihydrate to 50 ml distilled, deionized water.
LIT78D 8/7/98 04:08 PM Page 5 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.
LIT78D 8/7/98 04:08 PM Page 6 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.
LIT78D 8/7/98 04:08 PM Page 7 Samples 1-10 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F + C O N T R O L B L A N K G H Fig. 1. Suggested format for sub-isotyping 10 samples 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.
LIT78D 8/7/98 04:08 PM Page 8 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 A. High background. 1. Insufficient washing 1. Wash each well 6-7x and after conjugate antiincrease soak cycles to 30 body incubation. seconds. 2. Insufficient blocking 2.
LIT78D 8/7/98 04:08 PM Page 9 Problem Probable Cause Recommended Solution B. No reaction or weak color development. 1. Horseradish a. Improper storage of a. Store peroxidase substrate peroxidase subreagents. 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.
LIT78D 8/7/98 04:08 PM Page 10 Problem Probable Cause C. Monoclonal 1. Impure sample. exhibits multiple sub-isotype (i.e. assay shows one 2. Sample too conclone to be both centrated an IgG1 and 3. Sample is polyclonal. IgM). Recommended Solution 1. Use fresh source of culture supernatant. Purify media or ascites fluid.5-7 2. Dilute supernanants and ascites fluid, repeat test. 3. Re-clone hybridoma cells by limited dilution,8 and repeat assay. Notes to Troubleshooting Guide 1.
LIT78D 8/7/98 04:08 PM Page 11 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.
LIT78D 8/7/98 04:08 PM Page BC1 Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547 LIT78 Rev D
