Table 4.

Analysis of requirements for a set of regular and avidity mouse DTP MIAs and ELISAs

RequirementLevela for:
MIAELISA
96-well plates3 low-protein-binding filter platesc60 high-protein-binding platesh
Microbeads1.2 × 106 beads/regiondNone
Antigen1 μg/pertussis antigen; 5 μg/diphtheria or tetanus antigene200 μg/antigeni
Serum per mouse (μl)0.3 μlf24 μlj
Conjugate75 μl anti-IgG-PEg125 μl anti-IgG-HRPk
Human laborb0.5 day coupling + 1 day assay3 days
  • a Calculations are made for an experiment in which levels and avidity of IgG antibodies to 6 DTP antigens are determined in 75 mouse sera.

  • b Time needed for one operator with a maximum of handling 20 plates per day.

  • c Based on accommodating 75 sera (and controls) per plate and testing under 3 MIA conditions (0, 6, and 9 M urea).

  • d Based on the use for each bead region of 4,000 microbeads per well in 3 plates.

  • e Based on the use of approximately 20% of a standard bead stock (i.e., 6.25 × 106 beads) requiring 5 μg of pertussis antigens or 25 μg of diphtheria or tetanus antigens for conjugation.

  • f The minimal serum volume that can be accurately pipetted. This makes up 375 μl of a 1:1,250 serum dilution, which is largely sufficient to be analyzed in MIA in 3 wells (50 μl/well, 3 conditions; 0, 6, and 9 M urea).

  • g Used as a 1:200 dilution at 50 μl/well, in 3 plates.

  • h Based on accommodating 15 sera and controls (in six dilutions) and one antigen per plate and testing under 2 ELISA conditions (without and with optimized [NaSCN]).

  • i Amount per antigen needed to coat 10 ELISA plates at 2 μg/ml, 100 μl/well.

  • j Based on using 100 μl of serum dilution per well and making 200 μl of a 1:100 starting dilution per antigen (n = 6) per ELISA condition (n = 2).

  • k Used as a 1:5,000 dilution at 100 μl/well, in 60 plates.