Table 2

ELISA procedures for all four laboratoriesa

Assay parameter or reagentCDCVanderbilt Medical CenterSanofi PasteurGlaxoSmithKline Biologicals
Microtiter plateImmulon 2HBImmulon 2HBNunc MaxiSorpNunc MaxiSorp
Coating buffer0.05 M carbonate-bicarbonate (pH 9.6)0.05 M carbonate-bicarbonate (pH 9.6)0.05 M carbonate-bicarbonate (pH 9.6)0.05 M carbonate-bicarbonate (pH 9.6)
Coating incubation time19 h ± 5 h16–24 h18 h ± 2 hOvernight to 3 days
Coating incubation temp (°C)5 ± 328 (in a humidified chamber)21 ± 35 ± 3
Washing solutionPBS–0.05% Tween 20PBS–0.05% Tween 20PBS–0.05% Tween 200.9% NaCl–0.05% Tween 20
Washing cycle300 μl three times250 μl five times300 μl three times with soak300 μl three times
DiluentPBS–4% BSA–0.05% Tween 20PBS–0.5% BSA–0.5% Tween 20–0.005% PPG0.01 M PBS–10.0% goat serum–0.05% Tween 20PBS–0.05% Tween 20
Blocking solutionNoneNone0.01 M PBS–1.0% goat serum–0.05% Tween 201% BSA
Blocking incubation timeNoneNone45 min ± 5 min3 h
Blocking incubation temp (°C)NoneNone21 ± 337 ± 2
Serum dilutionSingle point 1:100Eight 2-fold serial dilutionsEight 2-fold serial dilutionsEight 2-fold serial dilutions
Serum incubation time2 h ± 5 min2 h ± 5 min1 h ± 5 min30 min
Serum incubation temp (°C)22 ± 428 (in a humidified chamber)37 ± 2RT with shaking
AntibodyMouse anti-human IgG FcGoat anti-human IgG FcγGoat anti-human IgG FcγGoat anti-human IgG Fc
ConjugatePeroxidaseAlkaline phosphatasePeroxidasePeroxidase
Conjugate incubation time2 h ± 5 min16–24 h1 h ± 5 min30 min
Conjugate incubation temp (°C)22 ± 428 (in a humidified chamber)37 ± 2RT with shaking
SubstrateTMBpNPPTMBOPD
Substrate incubation time10 min1 h35 min ± 5 min20 min
Substrate incubation temp (°C)22 ± 428 (in a humidified chamber)21 ± 3RT
Wavelength read (nm)450405/590450/540490/620
Reference standardIn-house pooled seraIn-house pooled seraIn-house pooled seraIn-house pooled sera
Reference standard calibrationWHO international referenceCBER lot 3CBER lot 3Japanese reference Yoken-H-3
ControlsNegative, medium, high positiveLow, high positiveNegative, high positiveMedium positive
Lower limit of quantitation (LOQ)15 IU/ml10 EU/ml4 EU/ml5 EU/ml
SoftwareGen5/Bio-Tek Instruments, Inc.SoftMax Pro/Molecular DevicesSoftMax Pro/Molecular DevicesSoftMax Pro/Molecular Devices
Analysis (curve fit)4PL4PLParallel line analysis4PL
  • a PBS, phosphate-buffered saline; BSA, bovine serum albumin; PPG, polypropylene glycol; RT, room temperature; TMB, 3,3′,5,5′-tetramethylbenzidine; pNPP, para-nitrophenylphosphate; OPD, ortho-phenylenediamine dihydrochloride; WHO, World Health Organization; CBER, Center for Biologics Research and Review; IU, international units; EU, ELISA units; 4PL, four-parameter logistic function.