Polysiloxane-poly(propylene oxide) hybrid discs as solid phase in anti-HCV detection using a recombinant core protein.
In this work, siloxane-poly(propylene oxide) discs (PPO disc) prepared using the sol-gel process were used as solid phase in enzyme-linked immunosorbent assays (ELISA) for the detection of anti-hepatitis C virus (HCV) antibodies. The HCV RNA from serum (genotype 1b) was submitted to the RT-PCR technique and subsequent amplification of the HCV core 408 pb. This fragment was cloned into expression vector pET42a and expressed in Escherichia coli as recombinant protein with glutathione S-transferase (GST). Cell cultures were grown and induced having a final concentration of 0.4 x 10(-3) mol L(-1) of IPTG. After induction, the cells were harvested and the soluble fraction was analyzed using polyacrilamide gel 15% showing a band with an approximate molecular weight of 44 kDa, the expected size for this GST-fused recombinant protein. The recombinant protein was purified and confirmed by immunological detection using HCV-positive serum and showed no cross-reactivity with positive samples for other infectious diseases. An ELISA was established using 1.25 ng of recombinant protein per PPO disc, a dilution of 1:10,000 and 1:40 for a peroxidase conjugate and serum, respectively, and solutions of hydrogen peroxide and 3,3',5,5'-tetra-methylbenzidine in a ratio of 1:1. The proposed methodology was compared with the ELISA conventional polystyrene-plate procedure and the performance of the PPO discs as a matrix for immunodetection gave an easy synthesis, good performance and reproducibility for commercial application.
Tagliavini
S A
SA
Unesp-Universidade Estadual Paulista, Instituto de Química, R. Prof. Francisco Degni s/n, 14800-900 Araraquara, Brazil. tagliavinisa@yahoo.com.br
Mikawa
A Y
AY
Yamanaka
H
H
Henrique-Silva
F
F
Costa
P I
PI
D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D018937:Hepatitis C Antibodies / Q000097:blood*; D011108:Polymers / Q000737:chemistry*; D011409:Propylene Glycols / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D012833:Siloxanes / Q000737:chemistry*
D018937:Hepatitis C Antibodies; D011108:Polymers; D011409:Propylene Glycols; D011994:Recombinant Proteins; D012833:Siloxanes; C012504:polypropylene glycol