Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1998-03-09
2001-04-10
Nutter, Nathan M. (Department: 1711)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007100, C435S239000, C436S518000, C436S524000, C436S527000, C436S528000, C436S531000, C436S532000, C436S815000, C530S826000
Reexamination Certificate
active
06214541
ABSTRACT:
This invention relates to a method of detecting specific antibodies directed against HPV proteins in body fluids. Furthermore, this invention concerns a kit usable for this purpose. Moreover, the invention deals with native HPV proteins suitable for carrying out the method according to the invention.
As is known, many people suffer from persistent infections caused by human papilloma viruses (hereinafter referred to as HPVs) . As is also known, over 95% of all anogenital carcinomas, particularly of cervical carcinoma, and a considerable percentage of the carcinomas in the mouth/pharyngeal space are associated with persistent infections caused by HPVs.
In addition, there are indications that an uncontrolled expression of HPV genes, particularly genes of E6 and/or E7, is necessary for the formation of carcinomas in cells having a persistent infection caused by HPVs.
Therefore, the detection of such an expression could be a possibility serving for the early detection of HPV-associated carcinomas.
Thus, it is the object of the present invention to provide a method by which the uncontrolled expression of HPV genes, particularly of genes E6 and/or E7, can be detected.
According to the invention this is achieved by the subject matters defined in the claims.
Thus, the subject matter of the invention relates to a method of detecting specific antibodies directed against HPV proteins, which comprises the following steps:
(I) incubating a native, carrier material-bound HPV protein with body fluids, and
(II) reacting specific antibodies (a) bound to the HPV protein
with labeled antibodies (b) directed against antibodies (a), or
with unlabeled antibodies (b) and the latter with labeled antibodies (c) directed against antibodies (b).
The method according to the invention is based on the applicant's finding that antibodies often exist in human beings who show uncontrolled expression of HPV genes, particularly of genes E6 and/or E7, which antibodies are directed against the expression products of these genes.
The above expression “HPV protein” comprises any protein of any HPV type. In particular, this expression relates to HPV-E6 and HPV-E7 proteins, more particularly of HPV 16 and HPV 18. The HPV protein may include a wild-type sequence. It may also have a sequence deviating therefrom, and the deviations may manifest themselves in the form of additions, deletions and/or substitutions of one or more amino acids. Furthermore, the HPV protein may be part of a fusion protein.
Common methods can be employed for the preparation of an HPV protein. It is favorable to insert a nucleic acid coding for an HPV protein, particularly a DNA, in an expression vector and employ the latter for the transfection of host cells and transformation of host cells, respectively.
A person skilled in the art knows a nucleic acid coding for an HPV protein (cf. Schwarz, E., 443-466 in “Papilloma Viruses and Human Diseases”, (1987), Springer-Verlag). As regards HPV-E6 and HPV-E7 proteins, particularly of HPV 16 and HPV 18, he is also familiar with the following fact: EMBL data bank, AC K02718 for HPV 16; Swiss protein data bank P03126 for HPV 16-E6; Swiss protein data bank P03129 and Dürst, M. et al., Proc. Natl. Acad. Sci USA 60, (1983), 3812-3815 for HPV 16-E7; EMBL data bank, AC M20325 for HPV 18; Swiss protein data bank, P06463 for HPV 18-E6; Swiss protein data bank, P06788 for HPV 18-E7.
Furthermore, the person skilled in the art is familiar with expression vectors. In the case of an expression vector for
E. coli
these are e.g. pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8, the latter being preferred. For the expression in yeast these are e.g. pY100, Ycapdl and pREP-L20, the latter being preferred. For the expression in animal cells, e.g. PKCR, PEFBOS, cDM8 and pCEV4 have to be indicated, while particularly the baculovirus expression vector pAcSGHisNT-A is suitable for the expression in insect cells.
Moreover, the person skilled in the art knows host cells. Examples of such host cells comprise the
E. coli
strains HB101, DH1, x1776, JM101, JM109, BL21 and SG13009, the latter being preferred, the yeast strains saccharomyces cerevisiae and saccharomyces pombe, the latter strain being preferred, and the animal cells L, NIH 3T3, FM3A, CHO, COS, Vero and HeLa as well as the insect cells sf9.
Besides, the person skilled in the art is familiar with conditions of cultivating transformed host cells and transfected host cells, respectively. He also knows methods of isolating and purifying an expressed HPV protein.
In the method according to the invention, one or more HPV proteins, particularly a HPV-E6 and/or HPV-E7 protein, more particularly of HPV 16 and HPV 18, can be used simultaneously or one after the other. They are present in native form. Thus, all natural epitopes of specific antibodies, which are disposed on the HPV proteins, are provided. This optimizes the detection of specific antibodies over HPV proteins in denatured form. For obtaining an HPV protein in native form, such a protein
can be folded back by common methods when it is present in denatured form. It is favorable to dissolve the denatured protein in a urea buffer, e.g. 10 mM Na phosphate, pH 7.4, 10% glycerin, 2 mM DTT, 8 M urea, feed it to a conventional hydroxylapatite column, e.g. HA Ultrogel, IBF Biotechnics, elute it with a conventional elution buffer of the above urea molarity, e.g. 150 mM NaPi, pH 7.8, 10% glycerin, 2 mM DTT, 1 M NaCl, 8 M urea, dialyze it against a common dialysis buffer of descending urea molarity, e.g. 50 mM Tris-HCl, pH 7.5, 0.1 mM ZnAc, 1 mM DTT, 50 mM NaCl, 4 M urea, feed it to a common anion exchanger, e.g. Q sepharose (Pharmacia), elute it with a conventional elution buffer of a urea molarity equal to the above dialysis urea buffer, e.g. 50 mM Tris-HCl, pH 7.5, 0.1 mM ZnAc, 1 mM DTT, 1 M NaCl, 4 M urea, subject it to a common collecting step, e.g. gel filtration, and dialyze it against a conventional dialysis buffer, e.g. 5 mM hepes, 5% glycerin, 1 mM DTT, 20 &mgr;M ZnAc.
As an alternative, it is favorable to dissolve the denatured protein in a urea buffer, e.g. 100 mM phosphate buffer, pH 8.0, 8 M urea, and subject it to step-wise dialysis against common dialysis buffers, e.g. 50 mM Mops/NaOH, pH 7.8, 500 mM NaCl, 20% glycerin, 5 mM DTT, 100 &mgr;M ZnCl
2
. The dialysis buffers have descending urea molarities, e.g. 8 M, 3 M, 1 M and 0 M, respectively. The dialyses are carried out as usual, e.g. within 24 hours each. After the last dialysis, a folded-back HPV protein is obtained which is collected as usual, e.g. by gel filtration, particularly by Superdex 200 (Pharmacia). An HPV protein which was folded back as described above, also belongs to the subject matter of the present invention.
According to the invention an HPV protein is bound to a carrier material. Any material suitable for binding proteins, particularly microtiter plates, small tubes, microspheres and slides, can be used as such a carrier material. The attachment between HPV protein and carrier material may be effected according to common methods. It is favorable for the HPV protein to be present together with a tag polypeptide forming the C terminus, e.g. 11 amino acids, of SV40t antigen as fusion protein, so that it can be linked with the carrier material via a generally obtainable antibody directed against the tag polypeptide.
According to the invention an HPV protein linked with a carrier material is incubated with body fluids. These body fluids comprise all fluids that can be obtained from an animal body, particularly a mammal and more particularly a human being. The fluids comprise preferably serum, lymph, saliva, sputum, urine, stool, liquor, bile and gastrointestinal secretions. Furthermore, they also comprise fluids which can be isolated from solid tissues such as lungs, brain and bone marrow, smears and biopsies as well as tumors, e.g. anogenital carcinomas. The HPV protein can be incubated with body fluids according to common methods.
The above incubation serves for binding antibodies which are specific to an HPV protein. Such antibodies
Braspenning Joris
Frey Manfred
Martens Regina
Meschede Wolfgang
Pawlita Michael
Burns Doane Swecker & Mathis L.L.P.
Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Recht
Nutter Nathan M.
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