[Use of pH-sensitive immobilized monoclonal antibodies for optimization of immunoenzyme sandwich technique of detection of HBsAg of hepatitis B virus].
OBJECTIVE
To develop highly sensitive sandwich technique for identification of surface hepatitis B virus antigen (HBsAg) in serum and analyse of possible improvement of solid phase for immunoenzyme sandwich technique of HBsAg identification through variation of pH-dependent sorption of monoclonal antibodies on the surface of immune plates.


METHODS
Calibration curves for identification of HBsAg in sandwich techniques using 36 possible binary combinations of monoclonal antibodies of our panel (including high affinity antibodies to HBsAg produced by 6 hybridomas) were compared. Immobilization of antibodies on solid phase (by passive sorption) was performed at different pH values (2.8, 7.5, and 9.5).


RESULTS
Analysis of panel of antibodies to HBsAg produced by 6 hybridomas revealed pH-dependent monoclonal antibodies (18C8), which immobilization at low pH values together with detecting antibodies F4F3 allowed to greatly improve sensitivity of the sandwich technique. Minimal credibly detectable concentration of HBsAg in sera of persons infected with hepatitis B virus was 0.013 - 0.017 ng/ml. Validation of sandwich technique was performed on certified panel of serum samples with various concentrations of HBsAg (different serotypes).


CONCLUSIONS
Highly sensitive sandwich technique for detection of HBsAg was developed. It was shown that analysis of panel of monoclonal antibodies on pH-dependence could be used as simple methodical approach for optimization of immunoenzyme sandwich techniques for detection of different antigens.
Iakovleva
D A
DA
Dmitriev
A D
AD
Dmitriev
D A
DA
Koliaskina
G I
GI
Massino
Iu S
IuS
Pavlova
E V
EV
Pyrenkova
I Iu
IIu
Smirnova
M B
MB
Segal
O L
OL
Tarakanova
Iu N
IuN
Ulanova
T I
TI
Fartushnaia
O N
ON
Sharipova
I N
IN
Koliaskina
G I
GI
Lavrov
V F
VF
D055910:Antibodies, Immobilized / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D006508:Hepatitis Antibodies / Q000276:immunology*; D006509:Hepatitis B / Q000097:blood / Q000175:diagnosis*; D006514:Hepatitis B Surface Antigens / Q000097:blood*; D006515:Hepatitis B virus / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007124:Immunoenzyme Techniques; D012680:Sensitivity and Specificity
D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D006508:Hepatitis Antibodies; D006514:Hepatitis B Surface Antigens