Note: Descriptions are shown in the official language in which they were submitted.
773
NON-A, NON-B HEPATITIS ASSA~ AND VACCINE
This invention relates to the detection and prevention,
of infection by a newly discovered etiological agent for
non-A, non-B (NANB) hepatitis. In particular it is concerned
with making vaccines and conducting immunoassays for this
agent or its antibodies.
NANB hepatitis is defined as clinical hepatitis which
cannot be attributed to infection by cytomegalovirus,
Epstein-Barr ~irus, or hepatitis A or B~
NANB hepatitis was first identified in transfused
individuals. Transmission from man to chimpanzee and
serial passa~es in chimpanzees provided evidence that NANB
hepatitis is due to an infectious agent or agents. NANB
hepatitis represents up to 90% of post-transfusion hepatitis
cases since HBsAg positive blood is no longer used for
transfusion. The risk of contracting NANB hepatitis after
blood transfusion has been estimated to be close to 10% in
the U.S. Therefore, it is important to screen the blood
of potential donors to detect NANB hepatitis. Further,
use of donors vaccinated against NANB hepatitis would serve
to reduce the probability of transmitting serum hepatitis.
; NANB hepatitis has been found to be associated with a
variety of virus-like particles found in serum and tissue
extracts. Hollinger et al., in "Proc. Second Symposium
on Viral Hepatitis" (San Francisco), p. 699, (1978) report
the previous work of others in locating 20-~2 nm and
60-80 nm particles associated with NANB hepatitis. Other
particules which have been reported to be associated with
NANB hepatitis are (a) small spheres and filaments 15-25 nm
in diameter, (b) 35-40 nm diameter virions which resemble
the hepatitis B DANE partic1le, (c) a 27 nm virus-like
particle identified in lots of antihemophilic factor and
liver tissue from chimpanzees infected with antihemophilic
~actor ("Morbidity and M~rtality Weekly Report'l 27 (21)
[1978]), and (d) a 20-27 nm diameter particle identified
; 35 in hepatocytes of infected animals (Schimizu ~t al.,
~A~
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"Science" 205: 197-200 [1979]). In addition, antigen-
antibody systems not linked to a definitive particle have
been identified as associated with NANB hepatitis (Kabiri
et al., "Lancet", August 4, 1979, pp. 221-224; Vitvitski
et al., "Lancet", December 15, 1973, pp. 1263-1267;
Dienstag et al., "Lancet", ~une 16, 1979, pp. 1265-1267;
and Shirachi et al., "Lancet", October 21, 1978, pp. 853-856).
In accordance with the present invention, there is
provided a new togavirus strain having ATCC (American Type
Culture Collection) accession numbers VR-2011, V~-2012,
VR-2013 or VR-201~.
The present invention also includes:
(1) compositions comprising (a~ an antigen of
one or more of the togavirus strains which is
substantially free of primate proteins, (b~ at least
one of the togavirus strains which is inactivated or
attenuated so as to be biologically non-infectious,
(c) primate antibody which is substantially free of
other primate proteins and which is capable of binding
at least one antigen of the togavirus strains, (d) non-
primate antibody which is capable of binding at least
one antigen of one or more of the togavirus strains
and (e) any of the foregoing four compositions bound
to a water insoluble substance or to a label;
(2) methods for making the compositions ~
(3) an immunoassay comprising, detecting or
determining an antigen of at least one of the togavirus
strains;
(4) an immunoassay comprising detecting or
determining the NANB hepatitis particle core antigen
or an antibody thereto î
(5) an immunoassay, comprising detecting or
determining IgM capable of binding NANB hepatitis
particle antigen;
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(6) a method for recovering NANB hepatitis
antigens comprising separating from urine a NANB
hepatitis virus antigen;
(7) a method for detecting NANB hepatitis
which comprises assaying for a NANB hepatitis particle
antigen in urine; and
(8) a method for propagating NANB hepatitis virus
comprising culturing the virus in a primate diploid
cell line.
The inventors have located a particle resembling a
togavirus in the body fluids of NANB hepatitis patients. The
. particle ranges from about 50 to 60 nm in diameter and has
a core of about 40 nm. It is rendered noninfective for
tissue culture by exposure to ether or upon heating at
: 15 25C in aqueous suspension. The particle was unexpectedly
found in urine with greater consistency than in serum.
Further, we found that the particle can be cultured ln
vitro
Four examples of the particle have been accessioned
in the American Type Culture Collection (ATCC) under
numbers VR-2011, VR-2012, VR-2013 or VR 2014.
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~ he ter~ "particle" henreforth refers to these ATCC de-
posits, collectively and indiv;d~ally. These four samples of
the particle were discovered by electronmicroscopy in the urine
of four patients in the acute state of NANB hepatitis during a
hepatitis epidemic in Algeria. The particle was also detected
by electronmicroscopy in the urine of five other NANB hepatitis
patients out of a total of ten patients whose urines were in-
spected for the particle. Additionally, three of the five urine
samples were in~ective for tissue culture. Paradoxically, none
of the particles could be detected by electronmicroscopy in the
sera of ten acute NANB hepatitis patients. Nonetheless, the
partic1e has been detected in the sera of NANB hepatitis-infected
patients. Out of eight hemodialysis patients whose serum
glutamate-pyruvate transaminose activity ~SGPT) was elevated
above normal, the serum of one patient ~aken at the peak of
SGPT activity contained the particle as revealed by electron-
microscopy. Finally, the particle has been detected in a
sample of commercial blood protein fraction concentrate which
had been linked to serum hepatitis infection in recipients of
the concentrate.
The particle may be cultured in vltro using a cell line
susceptible to infection by the particles, usually human or
- primate diploid lines, preferably of fetal origin. Examples
are the MRC-5 and Wl-38 lines. They may be obtained commercially
or similar cell lines prepared in the laboratory using conven-
tional techniques.
Any culture medium conventionally used to grow and maintain
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~6~773
the susceptible cell line is satisfactory for initially cultur-
ing and then sustaining the cells during viral replication. The
preferred medium for use with the MRC-5 and WI-38 lines is
disclosed by ~organ et al., 'IProc. Soc. Biol. Med." 73:1 (1950).
It is supplemented with antibiotics and a small proportion,
usually about from 5~ to 15~ by volume, of human or animal serum.
If human serum is used it should obviously be free of antibodies
to the particles.
The particles are replicated In vitro by culturing the host
cells to confluence, inoculating with pathologic sample ~prefer-
ably urine) having about from 1 x 106 to 1 x lOa particles/ml
and incubating at about from 30 - 40 C for about from 2 to 7
days while maintaining the culture medium pH at about neutrality.
Air is saeisfactory for the viral cultivation, i.e., no special
atmosphere was needed. The culture medium should be exchanged
every 2 days during viral replication. The first subculture
will produce about from 1 x 104 to 1 x 105 particles/ml. These
particles are not infective for further culture and are there-
fore believed to have become attenuated as a result of replica-
tion under the conditions described. As will be described below,such particles may be useful as vaccines.
The particles are recovered from the culture by simply
decanting and combining the spent media from the culture con-
tainer and purified using one or more of the procedures de-
scribed below. The purified particles ar-e preferably frozen in
a medium cons~sting of 50~ of the growth medium without serum
and 5~% glycerol by volume.
It may be desirable for a variaty of reasons to p~rify the
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6~73
particles present in pathological material such as tissues,
urine and blood fractions, or which are obtalned through tissue
culture. For example, if the particle is to be treated and
employed as a vaccine or in an immunoassay, there ordinarily
5 should be as little in the way of extraneous protein contamination
as possible. Thus, the particle or its antigens should be
substantially free of primate proteins. The term substantially
free does not mean that the particle antigen is devoid of pro-
teins present in its natural environment which are noncovalently
bound or absorbed thereto, e.g., antibodies. However, the
term does mean that all but trace contaminant concentrations
of the proteins associated with the particle or particle antigen
have been removed, e.g., normal human serum proteins, urinary
albumin and cellular proteins.
The particle antigen is defined as a substance containing
at least one epitopic site of one of the deposited particles.
Thus, a "particle antigen" could be found on a particle which is
otherwise immunologically distinct from deposited particles. A
"particle antigen" may also be present on a fragment or in
solution which is free of any other antigenic sites present on
the deposited particles. Particle antibody is defined as an
antibody capable of binding a particle antigen as described.
It should be noted that the term refers not to the water
solubility of the antigen or antibody but rather to their origin
or specificity.
-- The particles may also be reproduced in vivo by injecting
particles (usually or preferably lO9 - lûl particles) from
pathological material into a chimpanzee not having an antibody
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titer for the partlcles, followed by daily inspection of serum
and aliquots for the first appearance of the particle. Plasma-
pheresis can then be used to recover large quantities of con-
taminated plasma. Alternatively, the animal may be sacrificed
and the liver excised and h~mogenized as a source of particles.
Particle antigens, whether soluble or in the form of
intact particles, are first separated.from water insoluble
contaminants having greater dimensions or different density
than the intact particles. Such contaminants may include
animal cell debris, e.g., from ~issue culture, cellular
microorganisms, e.g., bacteria in urine, or chylomicra in
sera. This gross separation is generally accomplished by
low speed centrifugation or filtration, the parameters of
which will obviously vary dependlng upon the nature and degree
of contamination and can be determined by routine experimenta-
tion. Either technique may be supplemented by sedimentation
with a sucrose density gradient. However, filtration is pre-
ferred for large volumes of particle suspension. Ordinarily,
filters having an average pore diameter of 0.8 micron are useful
in retaining gross contamination and passing the particles.
The particle may be separated from undesired water soluble
materials after gross contamination is removed. Where it is
desired to only recover intact part7c!es or their water in-
soluble fragments, e.g., discrete core or envelope fragments,
it is convenient to simply remove all water soluble constituents
from the sample. This is preferably accompanied by replacement
of the contaminating solutes with one or more protein stabili-
zers such as hydroxyl compounds, e.g., sugars, glycerol or
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carbohydrates, sulfhydryl compounds such as cysteine, and pro-
teins such as serum albumin. Buffers may also be included.
The foregoing objectives may be accomplished by ultra-
filtering the sample through a membrane having a molecular
wçight cut-off intermediate the approximately 50 million
of the particle and the nearly 1 million of most commonly
encountered protein contaminants. The ultrafiltrate is
then washed with a buffered solution of the stabilizer,
the ultrafilter back-washed with the solution and the washed
ultrafiltrate then recovered.
It may be more convenient to precipitate the particles
or their large molecular weight fragments using one or more
of various well-known flocculating or protein precipitating
agents, particularly salting out agents or flocculating
polymers. Examples include polyethylene gllycol and ammon;um
sulfate. The precipitating agent may be readily removed from
the precipitate by dialysis and the particles resuspended
for further purificat;on or use.
The concentration of polyethylene glycol to be used
will depend upon whether or not the particles are intact,
the electrolyte constituents of the sample, the temperature,
the molecular weight of the polyethylene glycol and the degree
of contamination by the particles. Polyethylene glycol of
average molecular 6000 may be added to serum to a concentra-
tion of about from 10 to 15~ by weight at a temperature ofaboue from 1 to 5 C to precipitate the particles. Precipi-
tation from urine may not require as much polethylene glycol
as with serum.
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77
The ammonium sulfate concentration to be used is de-
pendent upon the factors noted above for polyethylene glycol,
except that the precipitant molecular weight is not a variable.
Generally, ammonium sulfate at about from 12 to 18~ of satura-
; 5 tion is satisfactory to precipitate the particles.
Certain other methods, while ordinarily only useful on asmall scale, are well suited for obtaining particles of a high
degree of purity. Such high purity particles are useful in
immunoassays, especially as immunogens or labelled reagents.
Suitable methods include ultracentrifugation, e.g., sedimenta-
tion with a sucrose gradient or isopycnic centrifugation with
cesium chloride, zone electrophoresis, and chromatography with
ion exchange resins, gels or insolubilized specific binding
proteins.
Chromatography is the most versatile method since it may
be readily scaled up for commercial manufacture of the particles.
Gel chromatography systems using cross-linked dextran beads are
preferred due to the comparatively low cost. A column of suit-
I able gel can be selected which will permit diffusion of pro-
teins and low molecular weight substances into the void volume
of the gel beads, thereby retarding the progress of these con-
taminants through the column, while allowing the particles to
pass through virtually unimpeded. The gel which is selected
will thus be a matter of routine experimen~ation.
Any of the above methods may be combined as desired. For
example, yel chromatography of contaminated serum on Biogel A5M
and subsequent isopycnic centrifugation in cesium chloride is
very effective at removing human serum proteins.
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7~73
lf a large proportion of the particle antigens present in
a sample are particle fragments or water soluble protein anti^
gens, then the most expeditious technique for separating them
may be affinity chromatography. Antibodies capable of binding
the particle antigens are covalently linked or absorbed to an
insoluble support using conventional procedures. The insoluble
antibody is placed in a column and the sample is passed through.
Immunologically--bound antigen is washed with buffer and then
released by changing the ionic strength or pH of the wash
buffer. Generally, acid pH is effective at releasing the
bound antigen. This technique is highly effective in separating
closely related proteins from the particle antigens.
The substances which may be removed from urine upon
purification of the particles include one or more of urea,
bilirubin, uric acid, albumin, mucins, and electrolytes com-
prising calcium, iron, sodium, potassium, magnesium, chloride,
phosphate, ammonia and sulfate.
All of the foregoing materials may also be separated
from particl~-bearing serum or plasma. In addition, one or
more of the following lipids or proteins may also be removed
from serum in purifying the particles: Cholesterol; phos-
phatides, fatty acids, albumin, ~l-globulins, ~2-globulins,
B-globulines, fibrinogen, gamma globulins and lipoprotein
members of the ~1 and B globulins.
The particles obtained from pathological sources such
as serum or urine may be complexed with antibody. Particle
antigens free of adherent antibody are desirable for use as
reagents for immunoassays. They may be obtained by gr~wing the
1`
773
12
particle in tissue culture, followed by passing the spent
culture through a column of insolubilized staphylococcal
Protein A made in known fashion. The Protein A will specifi-
cally absorb most residual immune complexes of particla antigen
and antibody.
Purified antibody to particle antigen is also useful
in immunoassays. The impure anti-serum source may be the
sera of patients convalescing from a NAN8 hepatitis infection
or an animal which has been immunized against the par~icle
antigens. The immunized animals may be nonprimates, generally
guinea pigs, rabbits, goats or horses, which are not susceptible
to hepatitis and thus can be injected with particles which
would otherwise be infective and potentially fatal. The
antibody-containing gamma globulin fraction may be purified by
any conventional protein fractionation procedures previously
employed to separate gamma globulin from plasma, e.g., alcohol,
polyethylene glycol or ammonium sulfate precipitation.
The degree of antibody purity desired will depend upon
the source and intended use. Gamma globulin for therapeutic
or prophylactic administration in the treatment or prevention
of NANB hepatitis should be of human origin and should be of
as high degree of purity as commercially ~easible. Usually,
this at least entails removing the other desirable fractions
of human plasma, e.g., antihemophilic factor or prothrombin
complex, from the donor plasma. On the other hand, it is
usually sufficient to employ unfractionated serum from non-
primates in immunoassays, either as insoluble or labelled
reagents as will be described below.
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~2~77~3
Purified particle antigens are further treated for use
as vaccines. It ;s preferred to use as a starting material for
preparation of vaccine the particle antigens produced by tissue
culture of the infectious virus. The antigens are preferably
recovered as intact particles by purifying them as described
above. However, it is also possible to prepare a suitable
vaccine from particles purified from the serum or urine of
acute-phase human patients, or from fragments of the particles
from any source, including water soluble antigenic proteins.
Such proteins native to the viral envelope are preferred.
These proteins may be purified by affinity chromatography,
also as described above. It is desirable but not necessary
to purify the particle antigens substantially free of human
proteins, particularly any adherent gamma globulins in the
event a circulatory fluid of an acute NANB hepatitis patient
is used as the starting material. However, it is more important
that the antigens be free of proteins not of human origin which
may be introduced by way oF the nutrient medium, cell lines
; or tissues in which the virus is cultured.
While it is conceivable that noninfectious particle antigen
can be isolated free of infective particles without further
treatment it is desirable to process the particle antigen-
containing suspension to attenuate or inactivate any potentially
infective virions. This may be accomplished in a fashion that
will not significantly affect the immunogenic or antigen charac-
teristics of the preparation by adding an inactivating agent.
Formalin is effective for this purpose. A satisfactory procedure
entails adding formalin to the antigen-containing solution or
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suspension and incubating under conditions which will destroy
the infectivity of the composition, usually incubation at
about from 25 to 40 C for about from 4 to 5 days. The
formalin is then removed or neutralized, although a bacterio-
statically effective amount of formalin, e.g., 10 ug/ml, maybe left active. The viral particle count is adjusted to about
from 1 x 107 to 1 x lo~l particles by the addition of a
physiologically acceptable carrier such as saline or by removal
of excess diluent through ultrafiltration, or lyophilization
followed by reconstitution with an aqueous solutton of the
carrier.
Vaccination may be conducted in conventional fashion, for
example by subcutaneous administration of 1 ml amounts of the
vaccine at biweekly ineervals for 14 weeks.
Any of the immunoassay methods heretofore disclosed in
the art may be used to determine particle antigens or anti-
bodies. The following disclosure is genera~ly expressed in
terms of determining antigens. However, antibodies may be
determined using the same methods.
The analyt;cal methods described herein for assaying
the particle antigens or antibodies should not be construed
as requiring the determination of all of the particle antigens
; or epitopic sites. Similarly, it is not necessary to assay
for all of the antibodies which can bind the population of
epitopic sites presented by the particle. The reason for
this is that mutations and varieties o~ viruses are frequently
found in nature. Thus, some an~ibodies originally obtained
by immunizing an animal against the particle may not bind, or
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773
may bind very weakly to related NANB hepatltis strains. For-
tunatel~;, the ineact particle described herein is so large
that a considerable number of epitopic sites on or within the
particle can bind an equal number of discrete, specific anti-
5 bodies. Thus, changes in one or more of the epitopic sitesby mutation or degradation of the particle structure due to
environmental stress will not adversely affect an immunoassay
using antibodies raised aaainst the entire population of
epitopic sites since a proportion of the antibody population
will remain capable of binding an equivalent proportion of the
antigens. Similarly, antibody produced against even a single
antigen common to the particle and the sample antigen can be
effectively employed in an assay for either the intact particle
or any fragment thereof which contains the antigen. This is
the case (a) whether or not the antibody used to detect the
antigen was in fact raised by immunization against applicants'
deposited particle, against a cross-reacting strain isolated
de novo from nature, or against a fragment of said strain or
new isolate, and (b) whether or not the antigen used to detect
the antibody was derived from the deposited particle, a new
tsolate of a cross-reacting strain or a fragment of either
the deposited particle or new isolate. Cross-reactivity is
deemed to exist if an antibody raised against an antigen of
the deposited particle can bind with the antigen in question
with at least about 40~ of the avidity as the antibody will bind
the antigen against which it was originally raised.
Particle antigens may be divided into two groups~ surface
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and core antigens. Surface antigens are those which are ex-
pressed by the intact particle. These antigens are generally
those viral proteins which are exposed to the environment
through the lipid-containing envelope. They are the antigens
of greatest interest in diagnosis because the intact particle
is most likely to be infective and is generally found in early
states of the disease.
Incomplete particles may be found or manufactured which
consist of the 40 nm diameter core only. Stress which destroys
the envelope or incomplete synthesis of the particle can expose
the core surface antigens. These core antigens may be deter-
mined in the same fashion as the surface antigens. Core
antigen may be prepared from intact particles by contacting
the particles with a detergent such as sodium dodecyl sulfonate
which ruptures the envelope by acting on its lipid content.
The core may be readily separated from the residual disrupted
envelope by conventional procedures such as ultracentrifugation
or precipitation. The separated core or envelope antigens
may be labelled as described below or used as immunolgen to
2~ prepare antibodies.
Antibodies to either or both of surface or core antigen
may be assayed. Such antibodies will generally fall into
two dlagnostically interesting classes, immune globulins G
(IgG) and M (igM). It is preferred to assay for IgM as it
is the antibody class containing specificity for particle
antigens which ~irst appears during the course of infection,
when IgG synthesis may not yet have been initiated. IgG assays
may be material for regulatory purposes because this immune
fl;;2~i~l7~73
globulin fraction is detectable for many months after infection,
thus demonstrating that a potential blood donor has at one time
been exposed to the particle antigen even though the donor may
no longer be infectious.
Methods for determining IgG or IgM of specific activity
are conventional; any such method may be employed herein. For
example, the IgG and IgM fractions of a test sample may first
be separated by conventional procedures, most conveniently by
specifically absorbing either fraction to an insoluble surface.
For example, insoluble anti-Ig~ or anti-lgM antibodies of a
first animal will bind the immune globulins of a second animal
against which the first animal is immunized. DEAE or QAE-
cellulose may be used to differentiate IgM from IgG. 80yle
et al., ("J. Immunological Methods" 32 (1):51-8 [1980~) disclose
that immobilized staphylococcal Protein A and concanavalin A
will remove IgG or IgM antibodies, respectively. ~hus, any
of the foregoing specific adsorbents may be employed to
separate IgG from IgM. Then the presence of anti-particle
:
antigen specificity in either globulin fraction can be readily
evaluated by measuring the binding of labelled particle antigen.
Either heterogeneous or homogeneous immunoassay tech-
niques are satisfactory for determining particle antigens. It is
preferred to use heteroganeous assays when determining intact
particles or large antigenic fragments. All heterogeneous
methods by definition include a step of separating antibody-
bound antigen from residual antigen remaining in solution. In
such methods the viral antibody is insolubilized before the
assay, as for example by adsorption onto plastic beads or the
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~2~i~773
inner wa11 of a plastic test tube, or by binding to anti-
antibody already so adsorbed. The antibody may alternatively
be insolubilized after the immune r action with antigen has
taken place, e.g., by precipitation with a second antibody. A
representa~ive heterogeneous assay i5 the "competitive"
method. Here, an unlabelled sample antigen ~if any) and
labelled particle-antigen analogue compete for a limited
number of particle antibody binding sites, the antibody-
bound antigen is separated from the free antigen and the dis-
tribution of labelled antigen determined. This method ispreferred for the assay of antibodies to the part;cle. In
such a case, the antigen is insolubilized, and labelled and
sample antibody compete for limited antigen sites.
The sandwich heterogeneous method i5 another technique
which can be used. This method is preferred for the deter-
mination of particle antigen, particularly when found 7n
intact particles or large fragments. The method comprises
adsorbing the sample antigen onto insoluble antibody to
particle antigen, removing the residual sample, adsorbing
labelled particle antibody onto the antigen, removing excess
labelled antibody and determining the distribution of label,
usually by assaying the amount of label found in the solid
phase. A variant of this method includes directly adsorbing
antigen from the sample onto a nonspecific adsorben~, e.g.,
plastic, followed by labelled particle antibody or, sequentially,
particle antibody and labelled antigen.
Another suitable eechnique is sequential saturation. As
with the sandwich method, a surplus of insoluble antibody to
particle antigen is used to bind the sample antigen, if any.
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19
However, rather than next contacting the insoluble antibody-
antigen complex with labelled antibody, labelled antigen
is added to occupy the remaining antibody sites. Excess
labelled antigen is removed and the label content of
one of the separated phases is determined.
Homogeneous immunoassay methods may also be
employed to advantage. These methods have in common
the elimination of the above-described phase separation;
the complete determination can be conducted entirely
with water soluble reagents. One such method comprises
labellin~ an enzyme with particle antigen to form a con-
jugate, followed by admixing the conjugate, particle
antibody and sample, and determining the change in enzyme
activity brought on by binding of free antibody by the
conjugate. Additional disclosure of this method may
be found in U.S. Patent 3,817,~37.
Another homogeneous assay, which incidently
may also be used in a heterogeneous mode, is disclosed
in U.S. Patent 3,935,074. Here an antigen conjugate
is formed by covalently linking the antigen with a detector
; ligand. Antibodies to the antigen and detector ligand
are then mixed with the conjugate and sample. The usual
practice is to then measure the residual unbound detector
ligand antibody, which will be found in inverse relation
to the amount of particle antigen.
Other suitable assay systems are disclosed in
U.S. Patents 4,006,360; 4,134,792 and 3,996,345; Yorde
et al, and "Clinical Chemistry" 22(8): 1372-1377 (1976).
Additional methods will be apparent to those skilled
in the art.
The labels to be used in some of the above methods
may be dictated by the technology underlying the assay,
as will be recognized by the skilled artisan. In most
; cases, however, the method is not dependent upon use
of a particular label. Thus, any known label will be
satisfactory, e.g., radioisotopes, enzymes, coenzymes,
phages, stable free radicals, and fluorescent and luminescent
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~ 7 73
substituents. Preferred labels are radioisotopes, particu-
larly 125I, or enzymes.
The particle antigens or antibodies may be labelled
with radioiodine in any conventional manner. Suitable
methods use chloramine-T or lactoperoxidase, e.g., as
disclosed by Dermody et al., "Clinical Chemistry" 25
(6): 989~995 (1979) or Parsons et al., "Analytical Bio-
chemistry" 95:568-574 (1979), externally radio-iodinated
small molecules such as (125I) iodohydroxyphenyl propionate-
N-hydroxysuccinimate ester (Bolton et al., "Biochem.
Journal" 133: 529-533 [1973]), (125I) diiodofluorescein
isothiocyanate (Gabel et al., "Analytical Biochemistry"
86:396-406 ~1973]), tertiary-butyloxycarbonyl-L-(125I)
iodotyrosine N-hydroxysuccinimide ester (Assoian et al.,
"Analytical Biochemistry 103:70-76 [1980]), or ICI (Montelaro
et al., "Analytical Biochemistry" 99:92-96 [1973])~ While
the chloramine-T method is preferred for ~adiolabelling
antibody or the particle core, the technique disclosed
by Montelaro et al is preferred for labelling the particle
envelope.
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The invention will be more fully understood by reference
to the following contemplated examples.
Example 1
Urine procured rrom a hospitalized patent diagnosed to be
in the acute phase of NANB hepatitis is preliminarily centri-
fuged at 8500 9 for 10 min. 1 ml of the supernatant is layered
in a 4 ml linear gradient of sucrose (10-?0%), then ultracentri-
fuged for 3 hours at 240,000 9 using an SW 50 rotor in a
Beckman~L5.65B centrifuge. The pellet is resuspended in 50
to 100 ul of 0.01 ~ tris saline buffer at pH 7.2. The specimen
is applied to carbon grids, negatively stained with 1~
uranyl acetate and examined in a Jeol lOO~electromicroscope. A
serum sample from an acute phase haemodialysate pat'ent is
; treated in the same manner. In both samples togavirus-like
particles are observed having an outer diameter of 54-57 nm
and a nucleocapsid of about 40 nm.
Example 2
A urine sample in which the togavirus-like particles
described in Example 1 had been visualized is centrifwged at
8500 9 for 10 min. An eqwal volume of 28~ po!yethylene glycol
(average molecular weight 6000) solution is added to the
supernatant. The resulting precipitate is centrifuged at low
speed until the suspension is clarified. The pellet is dis-
solved in tris buf~er, sufficient ammon;um sulfate added to
.~
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,
f-~
~L2~ 7
22
produce a concentration of 15~ of saturation, the resulting
precipitate centrifuged until the suspension is clarified,
the pellet dissolved in tris buffer and lyophilized or frozen
for storage at -70 C.
Example 3
A urine sample containing the particles is purified by
filtering the tris suspended pellet described in Example l
through a Romicon filter membrane having an average pore
diameter of o.8 microns, followed by ultrafiltration with a
Milipore~PTHK membrane having a retention capability for
molecules and particles of molecular weight greater than lO0,000.
The ultrafiltration is conducted at a slow rate so as to minimize
damage to the particles by fluid shear at the membrane. After
three v~lumes of tris buffer are passed through the retentate,
thereby washing the particles free of lower molecular weight
proteins, the retentate Is ultrafiltered to one-tenth of the
applied sample volume, the membrane briefly back-flushed with
tris buffer, the retentate recovered and lyophilized or frozen
at -70 C.
Example 4
: The particle may be radioiodinated by the following method.
The particles are suspended in phosphate buffered saline at
pH 7.4. A stock solution of ICl is prepared by diluti~g 0.56 9 Kl,
`:
- .-- ~ . .
7~73
0.33 rng ~aI03, ~9.2 9 NaCl and 210 ml of concentrated HCl
to 250 ml with distilled water. Immed;ately before use, this
stock solution is extracted several times for 2 min. with 3 ml
oF chloroform until the chloroform is no longer colored pink.
~esidual chloroform is removed from the ICl stock by bubbling
water saturated air for 5 min. Finally, 1 ml of ICl stock is
thoroughly mixed with 10 ml of 2M NaCl. 0.1 ml of disrupted
- particle suspension is diluted with an equal volume of 1 M
glycine buffer ~pH 8.5) and mixed with lmCi of Nal25I in 0.1 m.
glycine buffer. The iodination is started immediately by
adding 0.1 ml of the final ICl preparation and allowed to
proceed for 5-10 seconds. The product is rapidly separated
from unreacted 125I by gel filtration through a column (0.9 x
15 cm) of Sephadex G-25 beads and eluted with phosphate
buffered saline.
Example 5
; I To a purified suspension of particles (1 x 109/ml) in
phosphate buffered saline is added formaldehyde to a final
concentration of 1:4000 and the mixture incubated with continuing
20 agitation for 1 day at 37 C and then 5 days at 40 C. The
reaction mixture is partially neutralized with sodium bisulfite
to leave a final concentration of lOu/ml formaldehyde. This
vaccine was stored at 4 C.
The inactivated particles are subcutaneously administered
to chimpanzees with a protocol of three injections at one month
interval and a booster one year later.
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773
24
Example 6
The culture medium of Morgan et al., ("Proc. Soc. Exp.
Biol. Med.", 73: 1 [1950]) is modified by adding 10% foal
serum, 50 ug of dihydrostreptomycin/ml and 100 units of
benzylpenicillin/ml. The medium is sterilized by filtration
through a 0.22 micron filter. An aliquot of the medium is
placed into a plastic Falcon flask, followed by an inoculum
of Wl38 human diploid cells. The inoculated medium is in-
cubated at 37 C until cellular confluence is achieved. A
urine sample containing particles at a concentration of about
1 x 10' particles/ml is added to the medium in a volumetric
proportion of 1:75. The culture is incubated at 37 C while
maintaining the p~ at about 7.4 with dilute NaOH or HCl as
appropriate. After about 7 days of incubation, cytotoxic
effects on the tissue culture were noted, particularly conversion
of the fibroblast cell line into substantially spherical,
birefringent cells and, ultimately, cell lysis.
The product particles are harvested from the passage
culture by decanting the supernatant from the cell culture
flask, centrifuging at low speed to remove cell debris, pre-
cipitating the virus by adding polyethylene glycol 6000 to a
concentration of about 15~ and centrifuging. The virus pellet
was resuspended in a composition consisting of one half part
by volume of the culture medium without foal serum and one half
part glycerol. The particles w~re stable in this composition
when stored at -20 C.
~`\ ~
r73
Example 7
_
Purified particle antigen in Freunds adjuvant is subcu-
taneously injected into rabbits on a biweekly basis for 14 weeks.
The serum is harvested from the animals and the globulins pre-
cipitated with 15~ ammonium sulfate. The precipitate isdissolved and dialyzed overnight in 200 volumes of 0.04 M
phosphate buffer (pH 7.2). The solution is passed through a
DEAE-cellulose column (1 x 20 cm) and the IgG fraction eluted
with the same buffer. 50 ug alignots are radiolabeled with 51
following Greenwood et al., "Biochem. J." 89:114-123 (1963).
The labelled IgG is separated from free radioiodine by passage
through a column of Sephadex G-50. The labelled IgG is diluted
to about 3 uCi/ml in a diluent of 50~ calf serum, 5~ human
serum negative for antibody particle antigen and 0.1~ NaN3.
C 15 This preparation is stored at 4 C.
Example 8
1 ml of guinea pig serum obtained from animals immunized
against the particles is coated onto the inner surface of a
polypropylene test tube in accordance with U. S. Patent 3,646,346.
20 0.1 ml of a urine specimen from an acute NANB hepatitis patient
and 0.1 ml of a control specimen known to be free of the parti-
; cles are then placed into coated tubes and incubated for 12 hours.
The unbound elements of the sample are washed from each tube
! with water, radiolabeled antibody of Example 7 added to each tube
C1,.~ ','t' "~ ~
773
and incubated for 12 hours, unbound labelled antibody washed
from the tube with water and the radioactivity bound to the
tube counted. The particle antigens could be satisfactorily
detected by determining the extent of bound radioactivity in the
assay tube compared to the control ~ube.
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