buffer (see Subheading 2.3, item 3) and plate washes (see Subheading 3) (see Note 2).
3. Blocking Buffer: Dilute fetal bovine serum (FBS) solution
tenfold in Dulbecco’s phosphate buffered saline (without calcium and magnesium) to achieve a 10% FBS solution.
4. Diluent buffer: Dilute FBS solution tenfold in Wash buffer to
achieve a 10% FBS solution.
5. Citrate buffer: Prepare 0.1 M citric acid as follows: Dissolve
21.01 g of citric acid, monohydrate (C 6 H 8 O 7 ∙H 2 O) in 1 L
water. Separately, prepare 0.1 M sodium citrate solution as
follows: Dissolve 29.41 g of sodium citrate tribasic dehydrate
(C 6 H 5 Na 3 O 7 ∙2H 2 O) in 1 L water. Autoclave both solutions at
~110
C for 15 min. Prepare citrate buffer solution by combining 205 mL 0.1 M citric acid, 0.1 M 295 mL sodium citrate
and 500 mL water. Store the citrate buffer solution, 0.1 M
citric acid and 0.1 M sodium citrate at 4
C.
6. Stop solution (4 N H 2 SO 4 ): Slowly dilute 50 mL of 36 N
H 2 SO 4 in 400 mL of water (see Note 3).
7. O-phenylenediamine dihydrochloride (OPD) substrate tablets
(5 or 30 mg/tablet).
8. Hydrogen peroxide solution (30%w/w).
2.3 Equipment
and Plasticware
1. Serum separator Microtainer.
2. Microcentrifuge tubes, 1.5 mL.
3. 1.2 mL Cluster tubes, 8-tube strip, racked.
4. Sealing mats, round cap for 1.2 mL tubes.
Fig. 1 Schematic overview of experimental procedures
Determination of Antigen-Specific IgG Antibody Titers
539
3. Blocking Buffer: Dilute fetal bovine serum (FBS) solution
tenfold in Dulbecco’s phosphate buffered saline (without calcium and magnesium) to achieve a 10% FBS solution.
4. Diluent buffer: Dilute FBS solution tenfold in Wash buffer to
achieve a 10% FBS solution.
5. Citrate buffer: Prepare 0.1 M citric acid as follows: Dissolve
21.01 g of citric acid, monohydrate (C 6 H 8 O 7 ∙H 2 O) in 1 L
water. Separately, prepare 0.1 M sodium citrate solution as
follows: Dissolve 29.41 g of sodium citrate tribasic dehydrate
(C 6 H 5 Na 3 O 7 ∙2H 2 O) in 1 L water. Autoclave both solutions at
~110
C for 15 min. Prepare citrate buffer solution by combining 205 mL 0.1 M citric acid, 0.1 M 295 mL sodium citrate
and 500 mL water. Store the citrate buffer solution, 0.1 M
citric acid and 0.1 M sodium citrate at 4
C.
6. Stop solution (4 N H 2 SO 4 ): Slowly dilute 50 mL of 36 N
H 2 SO 4 in 400 mL of water (see Note 3).
7. O-phenylenediamine dihydrochloride (OPD) substrate tablets
(5 or 30 mg/tablet).
8. Hydrogen peroxide solution (30%w/w).
2.3 Equipment
and Plasticware
1. Serum separator Microtainer.
2. Microcentrifuge tubes, 1.5 mL.
3. 1.2 mL Cluster tubes, 8-tube strip, racked.
4. Sealing mats, round cap for 1.2 mL tubes.
Fig. 1 Schematic overview of experimental procedures
Determination of Antigen-Specific IgG Antibody Titers
539