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Method for detecting nanbv associated seroconversion

a seroconversion and nanbv technology, applied in the field of recombinant expression vectors, can solve the problems of increasing false positives, limited development of antibodies detection immunoassays, and reducing assay specificity and sensitivity

Inactive Publication Date: 2009-06-18
F HOFFMAN LA ROCHE LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

Enables the production of high-yield, pure HIV and HCV antigens, enhancing the accuracy and sensitivity of antibody detection in immunoassays and providing effective prognostic markers for HIV infection and HCV diagnosis.

Problems solved by technology

The development of immunoassays for the detection of antibodies has been limited by difficulties in producing sufficient quantities of specific antigens that are essentially free of immunoreactive contaminants.
The presence of contaminants that react with antibodies present in patient samples results in lower assay specificity and sensitivity and an increase in false positive results.
Furthermore, the p24 antigen reappears in the blood of infected individuals concomitant with the decline of anti-p24 antibody in patients showing the deterioration in their clinical condition that accompanies transition into full-blown AIDS.

Method used

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  • Method for detecting nanbv associated seroconversion
  • Method for detecting nanbv associated seroconversion
  • Method for detecting nanbv associated seroconversion

Examples

Experimental program
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Effect test

example 1

Isolation of the HIV p24 Gene and Construction of Expression Vector

[0110]The gag region from the pHXB2CG plasmid clone of HTLV IIIB (obtained from Dr. Robert Gallo, National Cancer Institute, Bethesda, Md.) was isolated by EcoRV restriction enzyme digestion of plasmid pHXB2CG and the resulting 2.86 kilobase fragment was isolated and inserted by ligation into the EcoRV site of a modified pUC8 vector (pUC8NR) to form plasmid PUCGAG (FIG. 1, Step 1).

[0111]The plasmid (PUCGAG) was mutagenized to generate an ATG translational initiation codon and an NdeI restriction enzyme site (CATATG) at the beginning of the p24 structural gene by the following series of manipulations (FIG. 1, Step 2). After transformation of pUCGAG into the methylation deficient dam-strain of E. coli, New England Biolabs, a gap was created in the pUCGAG DNA at the p24 amino terminus by cutting with the ClaI and PstI restriction enzymes to form gapped pUCGAG that lacks the smaller DNA segment from the p24 amino terminu...

example 2

Formation of Composite DNAs Comprising the pGEXp24 Vector with an Inserted Gene for a Conserved Envelope gp41 (Subtype 0) Antigen

[0120]The plasmid pGEXp24, was linearized by digestion with the restriction enzyme PpuMI and purified by phenol-chloroform extraction followed by precipitation with ethanol. Two complementary oligonucleotides (sequences given by nucleotides 686 to 763 and the complement of nucleotides 689 to 766 of SEQ ID NO:1) forming protruding cohesive termini when annealed, were synthesized. The synthetic oligonucleotides were allowed to form a duplex by mixing and heating to 90° C. for a approximately 3 minutes, followed by annealing at room temperature for a period of 10 minutes. The hybrid molecule represents a hybrid gene sequence encoding the p24 molecule interrupted after codon 225 by a linker amino acid (lysine), envelope sequence (amino acids 227-249) for the conserved region of HIV Subtype 0 gp41 polypeptide, strain ANT, followed by a repetition of p24 residue...

example 3

Purification of Recombinant p24-gp41 (subtype 0) Fusion Proteins

[0124]Plasmids containing the lambda promoter (pL) are normally carried in a strain of bacteria containing a lysogen of bacteriophage lambda in order to minimize the expression of the gene product of interest during the manipulation of DNAs. The pGEX7-based plasmids described in Example 1 were all carried in a lysogen of the MM294 strain of E. coli. Expression from the lambda promoter of pGEX7 can be demonstrated by transfer of the plasmid into an uninfected bacterial host (e.g., E. coli strain W3110, accession no. #27325, ATCC, Rockville, Md.) and inactivation of the cl repressor protein at 42° C. Competent E. coli (strain W3110, 100 μl bacterial suspension) were transformed with 1 μl of pGEXp24gp41-ANT, pGEXp24gp41-MVP or pGEXp24gp41-X84328. After 60 minutes on ice, the bacteria were diluted to 1 ml with LB medium and incubated for a further 60 minutes at 30° C. Aliquots of the culture were than plated on ampicillin c...

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Abstract

The present invention relates to recombinant expression vectors which express segments of deoxyribonucleic acid that encode recombinant HIV and HCV antigens. These recombinant expression vectors are transformed into host cells and used in a method to express large quantities of these antigens. The invention also provides compositions containing certain of the isolated antigens, diagnostic systems containing these antigens and methods of assaying body fluids to detect the presence of antibodies against the antigens of the invention.

Description

[0001]This is a continuation of pending application Ser. No. 12 / 077,046 filed Mar. 14, 2008 which is a continuation of pending application on appeal Ser. No. 10 / 677,956 filed Oct. 1, 2003 which is a divisional of application Ser. No. 08 / 931,855 filed Sep. 16, 1997, now U.S. Pat. No. 6,692,751 B1, which is a continuation-in-part application of Ser. No. 08 / 563,733, filed Nov. 28, 1995, now abandoned, and of Ser. No. 08 / 272,271, filed Jul. 8, 1994, which is a continuation of Ser. No. 07 / 616,369, filed Nov. 21, 1990, abandoned, which is a continuation-in-part of Ser. No. 07 / 573,643, filed Aug. 27, 1990, abandoned; the disclosures of which are incorporated herein by reference.FIELD OF THE INVENTION[0002]The present invention relates to recombinant expression vectors which have segments of deoxyribonucleic acid (DNA) that encode recombinant HIV and HCV antigens operatively linked to the sequence AGGAGGGTTTTTCAT (nucleotides 1 to 15 of SEQ ID NO:1) to control expression of the antigens. Th...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/70A61K38/00C07K14/16
CPCA61K38/00C12Q1/703C12N2740/16222C07K14/005Y10S436/82
Inventor ZEBEDEE, SUZANNEINCHAUSPE, GENEVIEVENASOFF, MARC S.PRINCE, ALFRED M.
Owner F HOFFMAN LA ROCHE LTD
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