Note: Descriptions are shown in the official language in which they were submitted.
2142~72
Wo 94/04922 Pcr/US93/07287
METHODS FOR MONITO~?ING EFFECTIVENESS OF INTERFER(;)N
THERAPY IN INDIYIDUALS WITH HCV INFECTIONS
Back~round of the Invention
This invention relates generally to interferon therapy, and more
particularly, relates to the monitoring of anti-HCV IgM or anti-GOR
IgG in an indindual's test sample as an indication of the clinical
ef~ectiveness and/or clinical outcome of interferon therapy used to treat
an individual with chronic HCV infection.
''''
Greater than 90% of cases of transfusion hepatitis worldwide are
attributed to non-A, non-B hepatitis (NANBH). The predominant
etiological agent of NANBH, termed Hepatitis C virus (HCV), has been ~-
cloned. An immunodominant region designated as c-1û0, encoded by
1~ the putative nonstructural (NS)-4 genomic region, has been expressed,
purified, and incorporated into immunoassays which are useful in the
detection of antibody to HCV in infected test samples. See, for e~ample,
Q.-L. Choo et al., Science 244:359-362 (1989); H. J. Alter et al., N. ~n~
J. MedO 321:1494-1500 (1989); J. I. Esteban et al., Lancet ii:294-297 (1989);
G. Huo et al., Science 244:362-364 ~1989); T. Miyamura et al., Proc. Natl.
Acad. Sci~USA 87:983-987 (1990); and C. L. Van der Poel et al., Lancet
ii:297-298 (1989).
Recently, Mishiro et al. have reported isolation of a cDNA clone
designated as GOR4701, ~om the plasma of a chimpanzee
e~perimentally infeted ~th NANBH agent. S. Mishiro et al., ancet
336:1400-1403 (1990). the GOR~7-1 cDNA clone was shown to lack ~^
detectable sequence homology to l~nown HCV sequences. Further,
unlike HC~, GOR~gene is clod~d by a single-copy gene of host cellular ` -
30 sequence. An ELA has been developed using a 27-amino acid synthetic
peptide, spGOR2, deduced from the cDNA sequence of the GOR gene.
Using this EL~. 60% to 80% if the samples were found to be reactive,
while only two percent (2%) of the voluntary donor population had anti-
GOR2 antibodies, suggesting a st~ong association between immune
response to GOR and H~V in NANBH patients.
214287~
wo 94J04922 PCrtUS93/07287
It is known that a percentage of individuals who develop NANBH
(HCV) infections pro~Tess to the chronic state of the disease, where
liver function is impaired and death ultimately may result in ~evere
cases. Interferon therapy has been shown to improve liver function in
a proportion of patients with NANB hepatitis. About fiflGy percent (50%)
of chronic Hepatitis C cases respond to alpha interferon (IFN) therapy.
No demographic, biochemical or clinical features have been identified
to predict response to therapy. Interferon induced normalization of
serum ALT levels is so far the only endpoint indicating response to IFN
10 therapy. Biochemical relapse is predicted by increasing serum ALT
leveis. In addition to ALT elevation, recurrence of HCV viremia as
measured by PCR during or after interferon treatment is discussed as a
possible predictor for relapse. However, AI,T elevations may not occur
for several months after viremia recurrence. It is not clear at this time
1~ if changes în anti-HCV IgG levels are associaterd with IFN treatment.
It would be advantageous to provide a means for predicting the
clinical outcome of interferon therapy in an individual receiving such
therapy for NANB (HCV) infection. Such a means would be useful in -~
2~ that the response to therapy could be monitored, and the state of disease
could be assessed to dete~nine remission or relapse after therapy. ;
Summarv of the Invention
The present invention provides a means for determining the ~-~
25 effect*eness and/or possible clinical outcome of interferon therapy
when it is used in individuals diagnosed with chronicECV infection, or
when interferon therapy is used as a treatment for an HCV infection.
Briefly, test samples from an indindual are taken before the start of
interferon therapy and during the course of interferon therapy, and
30 assayed for either anti-HCV IgM and/or anti-GOR IgG antibodies. A
decrease in either a~ti-HCV IgM level or anti-COR IgG level in
indicative of a clinical response to interferon therapy. 1-
Several assay procedures disclosed herein can be used to assay
35 for anti-HCV IgM activity. HCV antigens useful in these assays
include HCV CORE, HCVp33c and HCV c-100. The most preferred
antigen is HCV CORE. These assays can include a solid phase to
21 ~28~
:- - WO 94/04922 PCI /US93/07287
which HCV antigen is attached. The solid phase selected can include
polymeric or glass beads, nitrocellulose, microparticles, wells of a
reaction tray, test tubes and magnetic beads. The signal generating
compound can include an enzyme, a luminescent compound, a
chromogen, a radioactive element and a chemilllminescent compound.
Examples of enzymes include alkaline phosphatase, horseradish
pero~idase and beta-galactosidase. Examples of enhancer compounds
include biotin, anti-biotin and avidin. Examples of enhancer compound
binding members include biotin, anti-biotin and avidin. In order to
10 block the effects of rheumatoid factor-like substances, the test sample is
subjected to conditions sufficient to block the effect of rheumatoid factor-
like substance~. These conditions comprise contacting the test sample
with a quantity of anti-human IgG to form a mi~ture, and incubating
the mi~ture for a time and under conditions sufficient to form a
15 reaction mixture product substantially free of rheumatoid factor-like
substance.
Several assay procedures disclosed herein also can be used to ;~
assay for anti-GOR IgG activity. The GOR antigen most preferred in ;~
~) this assay is GOR2. These assays can include a solid phase to which
C~OR antigen is attached. The solid phase selected can include
polymeric or glass beads, nitrocellulose, microparticles, wells of a
- reaction tray, test tubes and magnetic beads. The signal generating
compound can include an enzyme, a luminescent compound, a
25 chromogen, a radioactive element and a chemiluminescent compound.
Examples of enzymes include alkaline phosphat~se, horseradish
peroxidase and beta-galactosidase. Examples of enhancer compounds
include biotin, anti-biotin and avidin. E~amples of enhancer compound
bindi~g members include biotin, anti-biotin and avi~in. In order to
30 block the effects of rheumatoid factor-like substances? the test sample is
subjected to conditions sufficient to block the effect of rheumatoid factor-
like substances. These conditions comprise contacting the test sample
with a quantity of anti-human IgG to form a mi~ture, and incubating
the mi~ture for a time and under conditions sufficient to form a
35 reaction mi~ture product substantially ~ree of rheumatoid factor-like
substance.
wo 94/04~22 ?, 1 ~ ~ 8 7 ~ Pcr/US93/07287 ~-
Detailed Description of the Invention
The present invention provides a means for determining the
effectiveness of interferon therapy for individuals receiving su~h
- therapy for HCV infections. The inventions compnses assaying for the
5 presence of anti-GOR IgG and/or anti-HCV IgM and using these
determinations to determine clinical effectiveness and/or clinical
outcome of an individual.
The present invention employs an immunoassay which utilizes
10 specific binding members. A "specific binding membert" as used
herein, is a mem~er of a specific binding pair. That is, two different
molecules where one of the molecules through chemical or physical
means specifically binds to the second molecule. Therefore, in addition
to antigen and antibody specific binding pairs of common
~5 immunoassays, other specific binding pairs can include biotin and
avidin, carbohydrates and lectins, complementary nucleotide
sequences, effector and receptor molecules, co~actors a~d enzymes,
enzyme inhibitors and enzymes, and the like. Furthermore, specific
binding pairs can include members that are analogs of the original
a~ specific binding members, for e~ample, an analyte-analog.
Immunoreact*e specific binding members include antigens, antigen
fragments, antibodies and antibody fragments, both monoclonal and
polyclonal, and complexes thereof, including those formed by
recombinant DNA molecules. The term "hapten", as used herein,
25 refers to a par~al antigen or non-protein binLing member wh~ch is
capable of bir}ding to an antibody, but which is not capable of eliciting
antibody formatio~ unless coupled to a carTier protein.
A "capture reagent", as used herein, refers to an unlabeled
30 specific binding member which is specific either for ~he analyte as in a
sandwich assay, for the indicator reagent or analyte as in a competitive
assay, or ~or an ancillary specific binding member, which itself is
specific for the analyte, as in an indirect assay. The capture reagent
can be directly or indirectly bound to a solid phase material before the
35 per~ormance of the assay or during the performance of the assay,
thereby enabling the separa~on of immobilized complexes from the test
sample.
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. WC) 94/04922 PCr/US93/07287
Test samples which can be tested by the methods of the present
inven~on described herein include human and animal body fluids such ;
- as whole blood, serum, plasma, cerebrospinal fluid, urine, biological
5 fluids such as cell culture supernatants, tissue specimens and cell
specimens.
An enhancer can be used to detect the generated signal in the
assay. By "enhancer" is meant a moiety which can bolster a signal
10 generated in an immunoassay, thereby amplifying the generated
signal. Several me~ods of enhancing and amplifying a signal
generated in an immunoassay are known in the artO Also, the use of a
signal enhancer such as the use of avidin-biot;in also is known. For
example, U. S. Patent No. 4,228,237 to Hevey et al. describes the use of a
15 biotin labelled specific binding substance for a ligand used in a method
which also employs an enzyme labelled with avidin. The use of a biotin-
anti-biotin system is described in European Patent Application No.
160,900, published on November I3, 1985.
ao The term "probe," as used herein, means a member of the
specific binding pair attached to an "enhancer" compound. An
"enhancer" compound can be any compound used in the assay which
can enhance the signal generated by the ~ignal generating compound.
Thus, enhancer compounds include haptens such as biotin, and also
include fluorescein, di-nitrophenol, and the like.
The indicator reagent comprises a signal generating compound
(label) which is capable of generating a measurable signal detec~able by
e~ternal means conjugated (attached) to a specific ~inding member.
"Specific binding member,~ as used herein, means ~l member of a
- ~pecific binding pair. That is, two different molecules where one of the
- molecules through chemical or physical means specifically binds to the
second molecule. In addition to being an antibody member of a specific
binding pair for HCV, the indicator reagent also ca~ be a member of
35 any specif ic binding pair, including either hapten-anti-hapten systems
such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin,
a complementary nucleotide sequence, an effector or a receptor
2142~72
WO ~4/04922 PCI /US~3/072~7 '
molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or
an enzyme, and the like. An immunoreactive specific binding member
can be an antibody, an antigen, or an antibody/antigen comple~ that is
capable of binding either to HCV as in a sandwich assay, to the capture
5 reagent as in a competitive assay, or to the ancillary specific binding
member as in an indirect assay. Thus, if an enhancer is utilized in the
assay, the indicator reagent comprises a signal generating compound
conjugated to an enhancer-specific compound (enhancer compound
binding mem~er), such as biotin or anti-biotin, avidin or biotin, and
10 others known to those skilled in the art. For example, if the enhancer
compound utilized is biotin, then anti-biotin, or avidin, can be used as
the enhancer-specific compound.
The various signal generating compounds (labels) contemplated
~5 include a chromogen such as bromo-chloro-indole-phosphate (BCIP),
catalysts such as enzymes, luminescent compounds such as
fluorescein and rhodamine, chemiluminescent compounds such as
acridinium, phenanthridinium or 1,2-dio~etane compounds,
radioactive elements, and direct visual labels. Egamples of enzymes
2~ include alkaline phosphatase, horseradish pero~idase, beta-
galactosidase, and the like. The selection of a particular label is not
critical, but it will be capable of producing a signal either by itself or in
conjunction with one or more additional substances, such as the use of
enzyme substrates when enzymes are employed as the signal
25 generating compound.
It is contemplated that the reagent employed for the assay can be
provided in the form of a kit with one or more containers such as vials
or bottles, with each container containing a separate reagent such as a
30 monoclonal antibody, or a cocktail of monoclonal antibodies, employed
- in the assay.
The assay co~figuration may involve the use of a solid phase in
performance of the present invention. A "solid phase", as used herein,
35 refers to any material which is insoluble, or can be made insoluble by a
subsequent reaction. The solid phase can be chosen for its intrinsic
ability to attract and immobilize the capture reagent. Alternatively, the
2~ ~2C7,~ ~
: wo 94/04922 Pcr/US93/07287
solid phase can retain an additional receptor which has the ability to
attract and immobilize the capture reagent. The additional receptor
can include a charged substance that is oppositely charged wit~h respect
- to the capture reagent itself or to a charged substance conjugated to the
6 capture reagent. As yet another alternative, the receptor molecule can
be any specific binding member which is immobilized upon the solid
phase and which has the ability to immobilize the capture reagent
through a specific binding reaction. The receptor molecule enables the
indirect binding of the capture reagent to a solid phase material before -
the performance of the assay or during the performance of the assay. If
an assay device is utilized to perform the assays of the present
invention, it can have many configurations, several of which are
dependent upon the material chosen as the solid phase. For example,
the solid phase can include any suitable porous material. By "porous
~5 is meant that the material is one through which the test sample caneasily pass and includes both bibulous and non-bibulous solid phase
materials. In the present invention, the solid phase can include a
fiberglass, cellulose, or nylon pad for use in a pour and flow-through
assay device having one or more layers containing one or more of the
2D assay reagents; a dipstick for a dip and read assay; a test strip for
wicking (e.g., paper) or thin layer chromatographic or capillary action
(e.g., Mtrocellulose) techniques; or other porous or open pore matenals
well known to those skilled in the art (e.g., polyethylene sheet material).
The solid phase, however, is not limited to porous materials. The solid
phase can also comprise polymeric or glass beads, microparticles,
tubes, sheets, plates, slides, wells, tapes, test tubes, or the like, or any
other material which has an intrinsic charge or which can retain a
charged substance.
.
Natural, synthetic, or naturally occurring materials that are
synthetically modified, can be used as a solid phase includi~g
polysaccharides, e.g., cellulose materials such as paper and cellulose
derivatives such as cellulose acetate and nitrocellulose; silica;
inorganic materials such as dea~tivated alumina, diatomaceous earth,
MgSO4, or other inorganic finely divided mate~ial uniformly dispersed
in a porous polymer matri~, with polymers such as vinyl chloride, vinyl
chloride-propylene copolymer, and vinyl chloride-vinyl acetate
21~287 ?
WO 94/04922 ; ~ PCr/VS93/07287~ -
copolymer; cloth, both naturally occurring (e.g., cotton) and synthetic
(e.g., nylon); porous gels such as silica gel, agarose, dextran, and
gelatin; polymeric films such as polyacrilamide; and the like. ~The solid
phase should have reasonable strength or strength can be provided by
5 means of a support, and it should not interfere with the production of a
detectable signal.
Preferred solid phase materials for flow-through assay devices
include filter paper such as a porous fiberglass material or other fiber
10 matri~ materials. The thickness of such material is not critical, and
will be a matter of choice, largely based upon the properties of the
s~mple or analyte being assayed, such as the fluidity of the test sample.
To change or enhance the intrinsic charge of the solid phase, a
L~ charged substance can be coated directly to the mateI~al or onto
microparticles which are then retained by a solid phase support
material. Alternatively, microparticles can serve as the solid phase, by
being retained in a column or being suspended in the mixture of soluble
reagents and test s~mple, or the particles themselves can be retained
ao and immobilized by a solid phase support material. By "retained and
immobilized" is meant that the particles on or in the support material
are not capable of substantial movement to positions elsewhere within
~e support material. The particles can be selected by one skilled in the
art from a~y suitable type of particulate material and include those
25 composed of polystyrene, polym~thylacrylate, polypropylene, late~,
polytetrafluoroethylene, polyacrylonitrile, polycarbonate, or similar
materials. The size of the particles is not critical, although it is
preferred that the average diameter of the particles lbe smaller than the
average pore size of the support material being used.
Solid supports are known to those in the art and include the walls
of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads,
nitrocellulose strips, membranes, microparticles such as late~
par~cles, chips of glass, plastic, derivatized plastic, metal and silicon,
35 and others.
21~2Q7~
WO 94/04922 PCI /US93/07287
9 ,;
Accordingly, a test sample which may contain HCV IgM is
contacted with a solid support to which HCV antigen has been
attached, to form a mixture. This mixture is incubated for a time and
under conditions sufficient to form HCV antigen/antibody complexes.
5 Then, a probe comprising a mammalian anti-human IgM to which an
enhancer has b~en attached is contacted with the HCV
antigen/antibody complexes, to form a second mixture. This second
mi~ture is incubated for a time and under conditions sufficient to form
a second mixture reaction product. Ne~t, an indicator reagent which
10 comprises an enhancer compound binding member and a signal
generating compound capable of generating a measurable signal is
contacted with the second mixture reaction product . This third
mi~ture is incubated for a time and under conditions sufficient to form ~ ?
indicator reagent reaction products. The presence and/or amount ~of
15 HCV IgM is determined by detecting the signal generated. The amount
of HCV IgM present in the test sample is proportional to the signal
generated.
Another assay comprises an assay wherein a test sample which
ao may contain HCV IgM is contacted with a solid support to which HCV
an~igen has been attached, to form a mixture. This mi~ture is ~-
incubated for a time and under conditions sufficient to form HCV
antigen/antibody complexes. Then, an indicator reagent which
comprises a signal generating compound capable of generating a ;
2B measurable signal attached to a specific binding member for HCV IgM
is contacted with the complexes, to form a second mixture. This second
m~ture is incubated for a time and under conditions sufficient to form
a reaction. The presence and/or amount of HCV IgM present in the
t~st sample is deter~mined by detecting the signal genl~rated. The
30 amount of HCV IgM present in the test sample is proportional to the
signal generated.
In yet another assay configuration, a test sample is contacted
with mammalian anti-human IgM which is coated on a solid phase,
35 and reacted for a time and under conditions sufficient for human
IgM/a~ti-human IgM comple~es to form. These comple~es then are
contacted with a probe which comprises at least one HC~T antigen
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WO 94/04922 PCrlUS93/07287 '.
selected from HCV CORE, HCV 33c and HCV c-100 attached to an
enhancer compound. The prefelTed enhancer compound is biotin.
These are rPacted for a time and under conditions sufficient to form
antigen/antibody/antibody complexes. Ne~:t, these complexes are
5 contacted with an indicator reagent comprising a signal generating
compound conjugated to an enhancer compound bindi~g member. The
most preferred signal generating compound is the enzyme alkaline
phosphatase. The most preferred enhancer compound binding
member is anti-biotin. The resultant mixture is reacted for a time and
10 under conditions sufficient for a reaction to occur. If an enzyme is
utilized, the signal is detected and measured after addition of an
enzyme substrate. The amount of HCV IgM present in the test sample
is proportional to the signal generated.
~6 Yet other assay confi~rations can be adapted to detect HCV IgM
by practicing the teachings of this invention, and are contemplated to be
within the scope of this invention
The presence of anti-GOR IgG antibody also can be assayed and
20 monitored during and after interferon therapy for HCV infection by
using the assays disclosed hereinabove and substituting the
appropriate GOR antigens. An antibody test for GOR peptides utilizing
the synthetic spGOR2 and spGOR346 peptides is known in the art and
disclosed in S. U. Mehta et al., J. Clin. Immunolo~r 12(3):178-184 (1992).
25 Thus, a test sample which may contain GOR IgG is contacted with a
solid support to which GOR antigen has been attached, to form a
mi~ture. This mi~ture is incubated for a time and ulnder conditions
sufficient to form GOR antigen/antibody comple~es. Then, a probe
comprising a mammalian ajnti-human IgG to which an enhancer has
30 been attached is contacted with the GOR antigen/antibody comple~es, to
form a second mi~ture . This second mi~ture is incubated for a time
and under conditions sufficient to form a second miYture reaction
product. Ne~Rt, an indicator reagent wbich comprises an enhancer
compound binding member and a signal generating compound capable
35 of generating a measurable signal is contacted with the second mi~cture
reaction product . This third mi~ture is incubated for a time and under
conditions sufficient to form indicator reagent reaction products. The
2142~7~ `
`- W 0 94/04922 PC~r/US93/07287
presence and/or amount of GOR IgG is determined by detecting the
signal generated. The amount of GOR IgG present in the test sample is
proportional to the signal generated.
Another assay comprises an assay wherein a test sample which
may contain GOR IgG is contacted with a solid support to which GOR
antigen has been attached~ to form a mixture. This mi~ture is
incubated for a time and under conditions sufficient to form GOR
antigen/antibody complexes. Then, an indicator reagent which
comprises a signal generating compound capable of generating a
measurable signal attached to a specific binding member ~or GOR IgG
is contacted with the complexes, to form a second mixture. This second
mixture is incubated for a time and under conditions sufficient to form
a reaction. The presence and/or amount of GOR IgG present in the test
sample is determined by detecting the signal generated. The amount of
GOR IgG present in the test sample is proportional to the signal
generated.
In yet another assay configuration, a test sample is contacted
ao with mammalian anti-human IgG which is coated on a solid phase,and reacted for a time and under conditions sufficient for human
IgG/anti-human IgG complexes to form. These complexes then are
contacted with a probe which comprises at least one GOR antigen
selected from spGOR346 and spGOR2 attached to an enhancer
compound. The preferred enhancer compound is biotin. These are
reacted for a time and under conditions suf~cient to form
antigen/antibody/antibody comple~es. Next, these complexes are
contacted with an indicator reagent comprising a signal generating
compound conjugated to an enhancer compound bL~lding member. The
most preferred signal generating compound is the lenzyme alkaline
phosphatase. The most preferred enhancer compound binding
- member is anti-biotin. The resultant mixture is reacted for a time and
under conditions sufficient for a reaction to occur. If an enzyme is
u~ilized, the signal is detected and measured afl;er addition of an
enzyme subs~rate. The amount of GOR IgG present in the test sample
is proportional to the signal generated.
21~287 ~
W~ 94/04922 PCI /US93/07287 (: -.
Yet other assay configurations can be adapted to detect GOR IgG
by practicing the teachings of this invention, and are contemplated to be
within the scope of this invention. ~ ~
. .
When assaying for HCV antl-IgM, it is preferred that the test
sample be treated such as to remove rheumatoid factor-like substances
which may be present in the test sample and which may interfere with
the per~ormance of the assay. Such treatment can be performed in a
variety of ways known to those skilled in the art and include
preadsorbing the test sample with protein A or protein G, heat
aggregated IgG, and the subjection of the test s~mple to an amount of
anti-human IgG sufficient to bind a substantial amount of the
interfering rheumatoid factor-like substances. The most preferred
me'~od for treating the test sample comprises diluting the test sample
1~ in a diluent sample buffer which contains an amount of goat anti-
human IgG suffirient to bind the rheumatoid factor-like substances
which may be present in the test sample. This dilution step preferably
is performed prior to contacting the test sample with the capture
reagent, HCV antigen. The preferred buffer is one which can remove
ao any interfering IgG which may be present in the test sample. Thus,
buffers which contain a sufficient quantity of anti-IgG can be used as
the diluent sample buffer. E2~amples Qf buffers that can b~ used in the
assay include Tris buffered saline, phosphate buffered saline, and
others known to those skilled in the art. The most preferred buffer
comprises a Tris buffered saline (pH 7.2) to which goat anti-human IgG
has been added. Fur~her, other compounds may be added to this buf~er
to block non-specific binding. The selection of these compolmds
depends upon the constituents chosen for the assay, and are within the
ordinar~ skill of the a~rtisa~.
The oIigin of the mammalian anti-human IgM or anti-IgG may
be goat, rabbit, sheep, or other mammalian anti-human IgM known in
the art. Preferably, the m~mmalian origin of the anti-human IgM and
anti-human IgG is goat.
When HCV or GOR antigen is used as a capture reagent in the
assays described herein, at least one HCV or GOR antigen is used,
2~ ~2872 :
W O 94/04922 P ~ /US93/07287
either when attached to a solid phase or in solution These HCV
antigens include HCV CORE, HCV 33c and HCV c-100. The GOR
antigens include GOR2 and GOR 346. We have determined th~t HCV
CORE is the most preferred antigen to utilize in performing the assay
- 5 for detection of anti-HCV IgM antibodies, but that HC~ 33c and HCV
c-100 also can be used alone or in any combination. Thus, HCV CORE
antigen can be combined with HCV 33c and/or c-100, or other HCV
antigens, and used as capture antigens in methods described herein.
It ~lso is contemplated that a sandwich assay can be performed
wherein a soluble capture reagent can include an analyte-specific
binding member which has been ~ound to ~ charged substance such as
an anionic substance. The present invention also can be used to
conduct a competitive assay. In a competitive configuration~ the soluble
1~ capture reagent again includes a specific binding member which has
been attached to a charged substance, such as an arlionic polymer, with
which to bind a specific binding partner.
Alterna~vely, it also is contemplated that the assay can be
performed by scanning probe microscopy, in which an analyte, analyte
analog or analyte specific substance which has been bound to a test
piece, is contacted with the test sample suspected of containing the
analyte, incubated for a time and under conditions suf~cient for a
reac~on to occur, and then the presence of analyte is determined by
using scanning probe microscopy.
The present invention will now be described by way of Examples,
which are intended to demonstrate, but not to limit, the spirit a~d
scope of the in~ention.
EXAMPLES
- Example 1
Anti-IgM Activitv
1. Patient Selection and CateFQriza~ioIl
Thirty-four (34) patients with chronic active hepatitis C at
di~erent time points during interferon therapy (1 to 5 million units of
interferon for at least si2~ (6) months) were included. Complete
responders (CR) (11/34) were defined by normaliza~on of serum
21~2S,~7 `,~
wo 94/04922 PCr/US93/07287 '
transaminases during treatment. Non-responders (NR) (23/34) were
defined as those individuals who showed transaminases levels above
two (2) times the normal values (28 IU/MI.). In total, 119 s~mples were
tested for HCV IgM antibodies. Samples were collected appro2~imately
one (1) month before, two (2) months after the beginning of and at the
completion of IFN therapy. In a number of patients, samples drawn
approximately six (6) months after therapy also were available. -
2. EICV Detection in Samples
All samples initially were tested by the Abbott HCV 2nd
10 Generation assay ELA and the Abbott HCV Supplemental EIA
(available ~om Abbott Laboratories, Abbott Park, IL and Abbott, GmbH
Diagnostika, Weisbaden, Germany). Anti-HCV IgM was determined
by utilizing a solid phase enzyme immunoassay ("IgM Combo",
available from Abbott, GmbH Diagnostika, Weisbaden, Germany). The
1~ anti-HCV IgM assay utilized recombinant antigens derived from the
structural (core) and non-structural (NS3/NS4) part o~ the virus. These
methods are detailed in (~hau et al., ~. Virol. Methods. 35:343-352, 1991).
The HCV antigens included clone c100-3 (as described by Kuo et al., `~
Science 244:362-364 ~1989]) chimeric polypeptide expressed in yeast, plus
ao recombinant HCV polypeptides expressed in E~coli that included those
from pHCV-23 (c100 fragment, lacking the lSrst 107 N-terminal amino
acids)~ pHCV-29 (CKS-33c), pHCV-34 (CKS-CORE) and pHCV-35 (~ pI.
CORE), and pHCV-45 (NS4/NS5 junction). The amino acids sequences
of these HCV polypep~des are known in the art and are described in
25 European Patent Application 0 388 232, published September 19, 1990
(see pages 32 and 34).
All HCV proteins were expressed as CMP-KDO synthetase (CKS)
fusion proteins (as taught by T. J. Bolling and W. Mandecki, "An
~s~herichia coli expression vector for high-level production of
30 heterologous proteins in fusion with CMP-KDO synthetase,"
Biotechniaues 8:488-490 [1990]), with the egception of pHCV-35, which
was egpressed in a lambda (O pL expression system.
Anti-HCV IgM res~lts were egpressed as specimen absorbance
divided by mean absorbance (both absorbance measured at 492 nm) of
35 three (3) negative controls (S/N). In 45~ healthy blood donors, the mean
S/N value was 1.0 i 0.5 (mean S/N + 1 standard deviation [SDJ).
Samples with S/N > 4 were characterized further for their IgM antigen
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specificity using single bead HCV IgM assays utilizing the HCV-Core,
HCV-NS3 and HCV-NS4 antigens.
A neutralization assay using a different recombinant copstruct
was used for confirmation testing. Rheumatoid factor interference was
6 eliminated by modifying the specimen diluent as taught hereinabove,
which allowed the separation of IgG antibodies.
3. Results
Before IFN therapy anti-HCV IgM values were d~termined to be
significantly higher in NR than in CR ~t-test). In contrast, no
significant di~erence was seen for serum AI,T levels in NR and CR
before IFN therapy (t-test). In patients with complete response to I~N
therapy, decreasses of HCV IgM and ALT levels be~ore, during and
after therapy was observed. In patients who e~ibited no response to
IFN therapy, HCV IgM and ALT levels both remained elevated.
Nineteen of twenty-three (83%) patients of the NR group had high or
increasing HCV IgM levels throughout IFN therapy. It also was
observed that in both the NR and CR anti-H(~7 IgM response was
directed almost always against the structural (Core) p~t of the virus. -~
Low level anti-C100 or anti-33c IgM was found only in 1/34 patients
ao tested. These data are summarized in the following Table 1 which
summarizes HCV IgM values (S/N) and ALT levels seen in chronic
hepatitis C Patients with no response, and in Table 2 which
summarizes HCV IgM values (S/N) and ALT levels seen in chronic
hepatitis C Patients with complete response
~
Table 1
(No Response 1;Q IFN~Q, Pa~ent~ - 23 )
Before IFN At start of IFN At enc! of IFN After IFN
Theraov Thera~v I h~ Thera~
Mean IgM 13.9 + 13.8 13.5 + 13.7 13.8 ~ 13.8 16.8 + 13.8
(SIN) +SD
No. Samples 23 ~2 ~ 13
Mean ALT 88+35 66+25 81+58 86+42
(IUI~IL) +SD
No. Samples 16 ~ ~ ao
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Table 2
(ComplQte Res~onse to EN~ No. Patients _ 11)
Before IFNAt~ tofIFN Atend of IFN After IFN
Thera~v Thera~Y TheraDv Thera~v
Mean IgM6.3 + 6.0 4.4 + 3.4 3.6 + 4.3 2.2 + 1.4
(S/N) +SD
No. Samples 10 11 11 7
Me~n ALT 130+163 18+13 15+14 15+1Q
(IU/ML) +SD
No. Sampl~s 8 11 10 8
5 4. Conclusions
From the data presented herein, anti-HCV IgM ~core) levels can
predict response to IF therapy. It also was observed that decreasing
an~-HCv IgM (core) levels can be an early indicator for relapse du~ing
and after IFN therapy. Also, it was observed that increasing anti-H(~v
10 IgM (core) levels can be an early indicator for relapse during and after
IFN therapy. Finally, anti-HCv IgM can give additional diagnostic
information in conjuction with ALT serum levels for patients with
chronic hepatitis C infections.
L~ E}~ampl~ 2
Anti-GOR I~G ~c~i~tv
1. Patient Selection~.nd (~at~ori~ion
Thirty-three (33) patients with chronic active hepatitis C at
different time points dunng interferon therapy (1 to 5 million units of
ao interferon alpha-2B, three times a week, for at least six (6) months)
were included. Complete responders (CR) (11/33) were defined by
normalization of serum transaminases during treatment. Non-
responders (NR) (22/33) were defiIled as those individuals who showed
transaminases levels above two (2) times the normal values (28 IU/M~).
25 SamI)les were tested for GOR IgG and GOR IgM antibodies. Samples
we~e collected appro2~mately one (1) month before, two (2) months after
the beginning of and at the completion of IFN therapy. In a number of
patients, samples drawn approgimately six (6) months after therapy
also were available.
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2. GOR Detection in Samvles
All samples initially were te~ted by the Abbott ~CV 2nd
~-eneration assay ELA and the Abbott HCV Supplemental ELA
(available from Abbott Laboratories, Abbott Park, IL and Abbott, GmbH
Diagnostika, Weisbaden, Germany). Anti-HCV IgM was determined
by utilizing a solid phase enzyme immunoassay ("IgM Combo",
available from Abbott, GmbH Diagnostika, Weisbaden, Germany). The
anti-HCV IgM assay utilized recombinant antigens derived from the
structural (core) and non-structural (NS3/NS4) part of the virus. These
methods are detailed in Chau et al., J. Virol. Methods. 35:343-352, 1991).
The HCV antigens included clone c100-3 (as described by Kuo et al.,
Science 244:362-364 [1989]) chimeric polypeptide expressed in yeast, plus
recombinant HCV polypeptides expressed in E. coli that included those
from pHC~1-23 (c100 fragment, lacking the first 107 N^terminal amino
L5 acids), pHCV-29 (CKS-33c), pHCV-34 (CKS-CORE) and pHCV-35 (~ pL
CORE), and pHCV-46 (NS4~NS5 junction). The amino acids sequences
of these HCV polypeptides are known in the art and are described in
European Patent Application 0 388 232, published September 19, 1990
(see pages 32 and 34).
All HCV proteins were expressed as CMP-KDO synthetase (CKS)
fusion proteins (as taught by T. J. Bolling and W. Mandecki, "An
Escherichia coli expression vector for high-level production of
heterologous proteins in fusion with CMP-KDO synthetase,"
BiotechIi~ues 8:488-490 [1990]), with the exception of pHCV-35, which
was expressed in a lambda (O pL expression system.
Anti-GOR IgG and IgM levels were determied by the assay
method for IgG described hereinabove and disclosed in S. U. Mehta et
al., J. Clin. Immunol. 12 ~3):178-183 (1992). VVhen assaying for aIlti-
GOR IgM levels, t~e appro~riate rare reagents were those as descriibed
for the HCV IGM assay, but utilizing the synthetic peptides spGOR346
and spGOR2.
3. Results
It was found that 11 patients had normalized ALT levels at the
end of ~erapy (CR). Non-responders (NR) numbered 22. It was
35 observed that eight of eleven (73%) of CR showed decrease of anti-GOR
IgG. Further, anti-GOR IgM was higher in NR than CR before
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18
therapy, while there were no significant changes in this value during
therapy.
It thus is possible to monitor the ef~ectivness of interferon
5 therapy during its use in individuals infected with HCV, especially
those who have been diagnosed with chronic acute HCV infection. The
~mount of anti-HCV IgM pre-interferon therapy and/or anti-GOR IgG
pre-interferon therapy is compared to the amount of of anti-HCV IgM
pre-interferon therapy and/or anti-GOR IgG pre-interferon therapy
10 during therapy. A decrease in the level of HCY IgM antibodies or anti-
GOR IgG antibodies during therapy is an indication that the patient is
responding to the interferon therapy. Also, it is possible that dosages
may be varied depending upon the results obtained by this monitoring.
The embodiments described and presented herein are intended as
~5 examples rather than as limitations. Thus, the descr~ption of the
invention is not intended to limit the invention to the par~cular
embodiments disclosed, but it is intended to encompass all equivalents
and subject matter within the spirit and scope of the invention as
described and contemplated above, and as set forth in the following
a~ claims.
