Biotinylated anti mouse igg

ELISA plates (Nunclon Delta Surface; Nalge Nunc International, Rochester, NY, USA) were coated with MP4 at 3 μg/ml in PBS at 4 °C overnight. Plates were washed with PBS + 0.05% Tween and blocked with 1% milk powder (MP) in 0.05% PBS + 0.05% Tween at room temperature for 2 h. Serum samples were plated at 1:1000 dilution in 1% MP and incubated at 4 °C overnight. Negative controls contained 1% MP only. After renewed washing, biotinylated anti-mouse IgG (diluted 1:800 in 0.1% MP; eBioscience, San Diego, CA, USA) was added to the plates for overnight incubation at 4 °C. After incubation with avidin-horseradish peroxidase (diluted 1:1000 in 0.1% MP; BD Biosciences, San Jose, CA, USA) at room temperature for 2 h, plates were developed with tetramethylbenzidine (eBioscience). The reaction was stopped with 0.16 M sulfuric acid and read at 450 nm using a Perkin Elmer (Waltham, MA, USA) Victor 3 1420 Multilabel Counter with Wallac 1420 software version 3.00 revision 5.

ELISA plates (Nunclon™ Delta Surface™ 96 MicroWell, Nalge Nunc International, Rochester, NY, USA) were coated overnight at 4 °C with 100 μl of 3 μg/ml MP4 in PBS. The plates were blocked with 1% milk powder (MP; Herler Bio Magermilchpulver, Heirler-Cenovis GmBH) in PBS/0.05% Tween (Biochemica, Billingham, UK) at room temperature for 2 h. The serum samples were diluted 1:1000 in 1% MP/0.05% Tween in PBS and incubated at 4 °C overnight. Negative control wells contained 1%MP/0.05% Tween in PBS. Biotinylated anti-mouse IgG (eBioscience, Waltham, MA, USA) was diluted 1:800 in 0.1% MP/PBS and incubated overnight at 4 °C. Strepavidin-horseradish peroxidase (BD Biosciences) was diluted 1:1000 in 0.1% MP/PBS and added for 2 h before 100 μl tetrametyhylbenzidin substrate (eBioscience) was used for development. The reaction was stopped with 0.16 M sulfuric acid and analyzed at 450 nm using a Perkin Elmer (Waltham, MA, USA) Victor³ 1420 Multilabel Counter with Wallac 1420 software version 3.00 revision 5.

We purchased anti-CD3-coated 96-well plates from BD Biosciences. Roswell Park Memorial Institute (RPMI)-1640 culture medium was obtained from GibcoBRL Corp. Rabbit anti-human CD4 and CD8 were purchased from Dako. Biotinylated anti-human CD25 was obtained from Sigma. The following antibodies and QDs were purchased from Invitrogen: biotinylated anti-mouse IgG, anti-rabbit IgG (H + L)-conjugated QD 655, and streptavidin-conjugated QD 605. Additionally, before the antibody labeling, all QDs were centrifuged to remove aggregates of QDs.