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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES
REFERENCE NO: AHC/1998/3/3.5/2.1
TITLE: PREPARATION OF ELISA "REAGENTS"
INTRODUCTION
This method describes how to make up the reagents used for ELISA's ( AHC/1998/3/3.5/2.2, AHC/1998/3/3.5/2.3 and AHC/1998/3/3.5/2.4).
MATERIALS
Stocks
- x20 PBS
- Fish gelatin
- Peroxidase labelled antibodies (diluted 1:1 in glycerol) e.g. Anti-mouse IgG
- Tetramethylbenzidine (TMB)
- Dimethylsulphoxide (DMSO)
- Sodium Carbonate
- Sodium Bicarbonate
- TWEEN x20
- Hydrogen Peroxide 30% solution
PROCEDURE
Coupling Buffer - Sodium Carbonate/Bicarbonate pH 9.6
- 1.59g Sodium Carbonate (Na2 CO3)
- 2.93g Sodium Bicarbonate (NaH2 CO3)
- Add 800ml of distilled water and adjust pH to 9.6
- Make up to 1000 ml with distilled water, aliquot into 100ml volumes.
- Autoclave. Label and date, store at 2 to 8°C for up to 6 months.
ELISA Wash (PBST) Concentrate - x20 PBS/TWEEN
- To 500ml of x20 PBS add 50ml TWEEN
- Mix well. Label, store at room temperature for up to 6 months
- For use - dilute 1:20 in distilled water and label ELISA Wash. Store at room temperature for up to 1 month.
Diluent - use ELISA
- Blocking agent
- 0.05% fish gelatin in PBS
- To 900ml of x1 PBS, add 1.0ml of fish gelatin (warm gelatin in waterbath before pipetting -as it is very viscous).
- Mix well and aliquot into 50ml tubes.
- Label and store at <-10°C.
Substrate Buffer- Sodium Acetate Buffer pH 6.0
- 8.25g sodium acetate
- Add 800ml of distilled water
- Adjust to pH 6.0 with 0.1M citric acid (2.1g in 100ml distilled water)
- Make up to 1000ml with distilled water
- Aliquot into 100ml volumes and autoclave.
2M H2SO4
MAKE UP IN FUME HOOD (WEAR GLOVES AND EYE PROTECTION)
- Measure out 900ml of distilled water and transfer to beaker or bottle.
- Using separate cylinder measure out 110ml of conc. H2SO4.
- With constant stirring SLOWLY add the acid to the water.
- Store in glass bottle and label as corrosive.
- Store in Acids cupboard at room temperature for up to 1 year.
Substrate 'Stock' Solution
- 58mg Tetramethylbenzidine (TMB)
- 5ml DMSO
- Dissolve TMB in DMSO - wear gloves, and use fume cupboard.
- Cover container in tin foil, label as 'toxic'.
- Store at room temperature in toxic cupboard for up to 1 month.
Substrate 'Working' Solution
- 100ml of TMB stock solution per 10ml of substrate buffer.
- 10ml of H2O2 per 10ml of substrate buffer.
Stock Reagent Antibodies
CAPTURE AGENTS
|
Assay |
Reagent |
Dilution*
(for storage) |
Dilution**
(for use) |
|
Mouse Protein A |
BDH, |
1:1 glycerol |
1/2000 |
|
Mouse Rat anti mouse kappa |
|
1:10 (PBS) |
400ml in 10 ml |
|
Rat mouse anti-rat |
GAM |
1:1 glycerol |
10ml in 10 ml |
|
Human Protein G |
|
1:100 in PBS |
10m in 10ml |
* Storage at <-10°C and expiry date as per manufacturers instructions
** Store at <-10°C or below. Keep for up to 1 month.
STANDARDS
|
Assay |
Reagent |
Dilution*
(for storage) |
Dilution**
(for use) |
|
Mouse IgG1 kappa |
(NOPC21) |
1mg/ml |
1/2500 |
|
Rat IgG1 kappa |
myeloma |
1mg/ml |
1/2500 |
|
Human Human Ig |
|
5.7mg/ml |
1/5700 |
CONJUGATES
|
Assay |
Reagent |
Dilution*
(for storage) |
Dilution**
(for use) |
|
Mouse a-mouse Ig |
HRP |
1:1 (glycerol) |
1/1000 |
|
Rat a-rat IgG |
HRP |
1:1 (glycerol) |
1/1000 |
|
Human a-human Ig |
HRP |
1:1 (glycerol) |
1/1000 |
* Storage at <-10°C and expiry date as per manufacturers instructions
** Store at <-10°C or below. Keep for up to 1 month.
Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
Page layout by CERDIC
Copyright CABRI, 1998 | 
