DETECTION OF ISOTYPE SPECIFIC IgG ANTIBODIES

Wednesday, May 05, 2004

Description
Response to immunization with Tetanus Toxoid Vaccine is an indicator of immune
function in infants and children which can be measured by comparing the amount of
specific antibody detected before and after immunization using an ELISA based immunoassay.

Procedure
Day One
5.1 Coat 96 well microtiterplate with 100 microliters/well Tetanus Toxoid coating antigen
at 0.4 mcg/ml in coating buffer
5.2 Cover plate with adhesive cover, incubate for 30 minutes at 37 degrees centigrade and
then overnight at 4 degrees centigrade
Day Two
5.3 Wash three times with wash solution, by hand or with automatic plate washer
5.4 Add blocking buffer, 100 microliters/well
5.5 Cover and incubate for one hour at 37 degrees centigrade
5.6 Wash three times with wash solution, by hand or with automatic plate washer
5.7 Add samples (patient and control sera, blank and non-specific antibody)
100 microliters/well in triplicate
5.8 Cover and incubate for one hour at 37 degrees centigrade
5.9 Wash three times with wash solution, by hand or with automatic plate washer
5.10 Add HRP anti-human IgG conjugate, 100 microliters/well
5.11 Cover and incubate for one hour at 37 degrees centigrade
5.12 Wash three times with wash solution, by hand or with automatic plate washer
5.13 Add OPD substrate solution, 100 microliters/well
5.14 Incubate at room temperature in the dark
5.15 Read O.D. at 450 nm, at 5, 10, 15 and 30 minutes time points
5.16 Identify linear area of standard curve
5.17 Calculate sample concentrations

Recipes
w1
3.1 Tetanus Toxoid Antigen (Connaught Labs), 12 mgs/ml
3.2 Monoclonal anti-human tetanus IgG antibody, 0.2 mcg/ml, from
human hybridoma culture supernatant. Cell Line (ATCC; HB-8177)
3.3 Samples: pre and post positive tetanus control sera, pre and post patient sera
and non-specific IgG antibody control supernatant
3.4 Carbonate-Bicarbonate Buffer Tablets (Sigma Cat #C304)
3.5 Tween 20 (Sigma or equivalent vendor)
3.6 Phosphate Buffered Saline
3.7 Bovine Serum Albumin (Sigma Cat#A-2153 or equivalent vendor)
3.8 HRP (Peroxidase) conjugated, isotype specific goat anti-human IgG antibody,
1 mg/ml; (Protos ImmunoResearch, Burlingame,CA, Cat #701)
3.9 OPD (o-phenylenediamine dihydrochloride) Peroxidase Substrate Tablet Sets
3.10 (Sigma, Cat# P9187)
3.11 Pipettes, tubes and other necessary disposables
3.12 96-well, Flat-Bottom EIA Microtiter Plates (Nunc-immuno plate maxisorp)
3.13 ELISA 450nm O.D. Reader (Molecular Devices or equivalent)
3.14 Microtiter plate washer (Denley or equivalent)
4. REAGENT PREPARATION
4.1 Coating Buffer:
?0.05 M Carbonate-B, pH 9.6
?Dissolve the contents of one Carbonate-Bicarbonate Buffer Tablet in
100 ml of distilled or deionized water
?Can be stored at 4 degrees centigrade for up to a week
4.2 Blocking/Diluent Buffer:
?0.25% BSA (Bovine Serum Albumin) in PBS (Phosphate Buffered Saline)
?0.25 gms of BSA in 100 mls PBS
?Prepare and use fresh
4.4 Wash Solution:
?0.02% Tween 20 in distilled or deionized water
?200 microliters Tween 20 in 1000 ml distilled or deionized water
?Prepare and use fresh
4.5 Tetanus Coating Antigen:
?0.4 mcg/mL Connaught Tetanus Toxoid Antigen
?Using a glass container, make a 1:30,000 dilution in coating buffer of the
stock Connaught Tetanus Toxoid antigen which is at 12 mg/ml
?Prepare and use fresh
4.6 Tetanus Toxoid IgG antibody Standards:
?Prepare the following concentrations of Human Hybridoma IgG antibody
to Tetanus Toxoid (starting concentration = 200 mcg/ml) in diluent buffer:
100, 10, 1, 0.1, 0.01, 0.001 mcg/mL
?Prepare and use fresh
4.6 Patient and Control Pre and Post Tetanus Immunization sera:
?Dilute Pre and Post patient and positive control serum 1:20,000 to 1:50,000
in diluent buffer
?Prepare and use fresh
4.7 Background and non-specific controls:
?Use diluent buffer for blank background wells
?For a control for the monoclonal antibody to tetanus, dilute non-specific
IgG-containing control supernatant at 1:2 dilution
4.8 HRP (Peroxidase) Conjugated IgG antibody:
?Dilute the HRP conjugated Goat anti-human IgG (gamma-chain specific)
antibody 1:1000 in diluent buffer
?Prepare and use fresh
4.9 OPD (o-phenylenediamine Dihydrochloride) Substrate Solution:
?Remove one OPD Tablet (silver foil package) and one Urea Hydrogen
Peroxide Tablet (gold foil package) from refrigerated storage and allow the
packages to come to room temperature.
?Open each package and without touching the tablets drop them into a 50 ml
tube containing 20 ml distilled or deionized water
?Vortex until dissolved
?Prepare and use within one hour, store in the dark
?Discard after use using proper biohazard material handling protocols.

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DETECTION OF ISOTYPE SPECIFIC IgG ANTIBODIES