JPS58182556A - Anti-a anti-b type hepatitis assay and vaccine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the Invention The present invention relates to the detection and prevention of infection by newly discovered etiological agents for non-A, non-B (, NANB) hepatitis. In particular, the present invention relates to the production of vaccines and the performance of immunoassays for the drug or its antibodies.

NANB hepatitis is a giant cell virus Epstein-B.
It is defined as clinical hepatitis that is not attributable to infection with ARR virus or hepatitis A or B. Transmission from humans to chimpanzees and cascading transmission in chimpanzees provided evidence that NANB hepatitis is due to an infectious agent. NANB hepatitis represents up to 90% of post-transfusion hepatitis. This is because HBsAy positive blood is no longer used for transfusion. The risk of acquiring NANB hepatitis after blood transfusion is estimated to be close to 10% in the United States. It is therefore important to screen the blood of potential donors to detect NANB hepatitis. Additionally, the use of blood donors vaccinated against NANB hepatitis will help reduce the probability of transmission of serum hepatitis.

It has been discovered that NANB hepatitis is associated with a variety of virus-like particles found in serum and tissue extracts. Hollinger et al., “Proc.
.

5econd Symposium on Viral
Hepatitis” fsan Fran-CiS
CO) # p, 699. f1978) is NANB
220-22n and 660-80n associated with hepatitis
have reported previous work by others in the discovery of particles.

Other particles reported to be associated with NANB hepatitis include:
(a) Globules and filaments of diameter 115-25n)
, (b) Hepatitis B 35-diameter similar to DANE particles.
435-40n virion, (C) 27 nm virus-like particles identified in lofts of antihemophilic factor and liver tissue from chimpanzees infected with antihemophilic factor (“Morbidity anaMortali
ty weeklyRepzrt”27(211(19
7B+), and (220-27n diameter particles identified in hepatocytes of dl-infected animals (Shimizu et al.
ce” 205:197-200 (1979)).

Additionally, antigen-antibody systems that do not bind to distinct particles have also been identified as being associated with NANB hepatitis (K
Abiri et al., “LanCet”, A.
August 4゜19791 pp, 221-224
; Vitvitski et al., “Lanc
etll.

December 15.1979. pp, 126
3-1267HDienstag etal, “La
ncet”, June 16.1979+pp,
1265-1267; and 5hirachi et
al,, ”Lancetll, 0ctover
21.1978゜pp, 853-856).

OBJECTS OF THE INVENTION The primary objective of the present invention is to locate and isolate viral particles, the etiologic agent of NANB hepatitis, from their host environment.

One of the additional objectives is to purify the particles without changing their antigenic characteristics.

Another purpose is to produce vaccines from the particles.

Another purpose is to elicit or harvest antibodies against the particles for administration as therapeutic gamma globulin or for use in immunoassays to detect the particles.

Yet another object is to produce insolubilized particle antigens or antibodies thereto, in particular for use in immunoassays or for the purification of particle antigens or antibodies.

Yet another object is to provide labeled, especially enzyme- or radiolabeled, particulate antigens or antibodies.

These and other objects of the invention will be apparent from consideration of this specification as a whole.

SUMMARY OF THE INVENTION The above-mentioned materials are (11(a) substantially free of primate protein;
Togavirus strain ATCC VR-2011, VR-201
2゜One or more antigens of VR-2013 or VR-2014, (b) Togavirus strain ATCC VR that has been inactivated or attenuated so as to be biologically non-infectious.
-2011, VR-2012, VR-201:3 or VR-2014, (c) other primate proteins, and Togavirus strain ATCC.
Primate antibody capable of binding at least one of the antigens of VR-2011, VR-2012, VR-2013 or VR-2014, (dl) Cahirus strain ATCC VR-20
11, VR-2012, VR-2013 or VR-2
A non-primate antibody capable of binding at least one antigen of 014.

and (e) bound to a water-insoluble substance or enzyme.
A composition comprising a composition of any species, (2I) Method for producing the above composition, (3) Togavirus strain ATCC, VR-2011, VR
-2012, VR-2013 or VR-2014 (7
) an immunoassay comprising detecting or measuring at least one antigen; (4) an immunoassay comprising detecting or measuring a NAN B hepatitis particle core antigen or an antibody thereto;
(5) an immunoassay comprising detecting or measuring iPM capable of binding NANB hepatitis particle antigen; (6) a method for recovering NANB hepatitis virus antigen comprising separating NANB hepatitis virus antigen from urine; (7) ) a method for detecting NANB hepatitis comprising assaying 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. Ru.

DETAILED DESCRIPTION OF THE INVENTION We have discovered particles resembling Togavirus in the body fluids of patients with NANB hepatitis. The particles range in diameter from about 50 to 60 nm and about 40 nm in diameter.
It has a core of It is made non-infectious to tissue cultures by exposure to ether or heating in aqueous suspension at 250 °C. The particles were unexpectedly found to be more concentrated in urine than in serum. In general, we have discovered that the particles can be cultured in vitro.

Four samples of this particle are in the American Type Culture Collection (ATCC) number VR-201.
1, VR-2012, VR-2013 and VR-20
Uke is accepted as 14.

The edict "particles" hereinafter refers to these A
Refers to TCC Deposit. These four types of samples of particles are
It was discovered using a molecular microscope in the urine of four patients in the acute phase of the NANB hepatitis epidemic in Algeria. The particles were also found in the urine of 5 other NANB hepatitis patients out of a total of 10 patients whose urine was tested for particles. Additionally, 3 of the 5 urine samples were infectious for tissue culture. Paradoxically, no particles could be detected by electron microscopy in the serum of 10 patients with acute NANB hepatitis. Nevertheless, the particle is NAN
It was detected in the serum of patients infected with hepatitis B. Among the eight hemodialysis patients whose serum glutamate/pyruvate transaminase activity (SGPT) increased above normal values, 5G
One person's serum, collected at the peak of PT activity, contained the particles when resolved by electron microscopy. Finally, the particles were detected in a sample of a commercially available blood protein fraction concentrate, which was associated with serum hepatitis infections in recipients of the concentrate.

The particles are a cell lineage capable of being infected by the particles, usually a human or a diploid primate lineage.

Preferably, those of fetal origin are used and can be cultured in vitro. Examples are MRC-5 and Wl-38
It is a systematic. These can be obtained commercially, or similar cell lines can be prepared in the laboratory using conventional techniques.

Any medium commonly available for growing and maintaining susceptible cell lines is satisfactory for sustaining the cells during initial culture and subsequent replication of the virus. M.R.
A preferred medium for use with the C-'5 and Wl-38,-% strains is that of Morgan et al.

1lProc, Soc, Biol, Med,”
73:1 (1950). It is supplemented with antibiotics and a small amount of human or animal serum, usually about 5% to 15% (by volume). If human sera are used, they must be clearly free of antibodies against the particles.

The particles culture host cells to confluence and produce approximately I
I O'slzized I X I O8 particles/ml'wo'
inoculate with a pathogenic sample (preferably urine),
and approximately 30'C while maintaining approximately neutral pH of the medium.
It can be replicated in vitro by incubation for about 2 to 7 days at temperatures ranging from 40°C to 40°C. The air was satisfactory for virus culture, ie no additional atmosphere was required. The medium must be changed on the second day during virus replication. The initial r'culture will produce approximately 1 x 10 to 1 x 10' particles/me. It is believed that these particles were not infectious for subsequent cultivation and were therefore attenuated as a result of replication in the conditions described. As described below, such particles are useful as vaccines.

Particles are recovered from the culture by simply tilting the culture vessel and merging the spent medium, and purified using one or more of the procedures described below.

Purified particles are preferably grown in 50% serum-free growth medium.
and 50% glycerol (by volume).

For various reasons it is desirable to purify particles present in pathogenic materials such as tissues, urine and blood fractions or particles obtained by tissue culture. For example, if a particle is to be processed and used as a vaccine or in an immunoassay, it usually should have as little extra protein contamination as possible. For this reason, the particles or their antigens must be substantially free of primate proteins. The phrase "substantially free" does not mean that the particle antigen is devoid of proteins, such as antibodies, present in its natural environment, either non-covalently or absorbed into it. However, this means that all but trace contaminating concentrations of proteins associated with the particles or particle antigens, such as normal human serum proteins, family albumins, and cellular proteins, have been removed.

A "particle antigen" is defined as a substance that contains at least one epitope site of one of the deposited particles.

Thus, a "-particle antigen" could be found on a particle that is otherwise immunologically distinguishable from the deposited particle. [Particle antigen J may also be present in a fragment or solution without any other antigenic sites present on the deposited particle.

"Particle antibody J is defined as an antibody capable of binding said particle antigen. It should be noted that said term does not refer to the aqueous solubility of the antigen or antibody, but to their origin or specificity. be.

The particles may also be injected into chimpanzees that do not have antibody titers against the particles from pathogenic material (usually or preferably IO to 10°9 particles) and daily serum and aliquots for the first expression of the particles. It can be reproduced in vivo by testing. Plasma separation methods can then be used to collect Taisha's infected plasma. Alternatively, the animal can be sacrificed, the liver dissected and homogenized as a source of particles.

Particle antigens, whether soluble or in the form of intact particles, are initially separated from water-insoluble contaminants that have larger dimensions or a different density than the intact particles. Such contaminants include, for example, animal cell debris from tissue culture, cellular microorganisms such as bacteria in urine, or chylomicrons in serum. This rough separation is generally carried out by low speed centrifugation or filtration, the parameters of which obviously vary depending on the nature and extent of the contaminants and can be determined by routine experimentation. Both techniques can be supplemented by precipitation through a sucrose density gradient. However, oral filtration is preferred for particle suspensions of persons j and i. Typically, filters with an average pore size of 08 microns are useful for retaining large contaminants and passing particles.

After removing large contaminants, the particles can be separated from unwanted bathing materials. If it is desired to recover only the intact particles or their water-insoluble fragments, such as separated core or outer membrane fragments, it is convenient to simply remove all water-bathable components from the sample. This preferably involves the replacement of contaminant solutes with one or more protein stabilizers such as hydroxy compounds, such as sugars, glycerol, or carbohydrates, sulfhydryl compounds such as cysteine, and proteins such as serum albumin. Buffers may also be included. The above objectives are achieved by ultrafiltering the sample through a membrane having an intermediate molecular weight cut-off of about 50 million particles and about 1 million of the commonly encountered tanx contaminants.

The ultrafiltrate is then washed with a buffered solution of the stabilizer, the ultrafilter is washed with the solution, and the washed ultrafiltrate is collected.

It is more convenient to precipitate the particles or high molecular weight fragments thereof using one or more of a variety of well-known flocculants or protein precipitating agents, particularly salting-out agents or flocculating polymers. . Examples include polyethylene glycol and ammonium sulfate. The precipitant can be easily removed from the precipitate by dialysis, and the particles can be resuspended for further purification or use. The concentration of polyethylene glycol that should be used will depend on whether the particles are intact, the electrolyte composition 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 weight i6000 can be added to the serum at a concentration of about 10 to 15% by weight to precipitate the particles at about 1'C to 5C. Precipitation from urine and serum will not require any amount of polyethylene glycol.

The ammonium sulfate concentration to be used depends on the factors mentioned above for polyethylene glyfur, except that the molecular weight of the precipitant remains unchanged. Ammonium sulfate at a saturation of about 12 to 18% is generally sufficient to precipitate the particles.

Several other methods are also well suited to obtaining particles of high purity, although they are usually useful only on small scales. Such highly purified particles are useful in immunoassays, particularly as immunogens or labeled reagents. Suitable methods include ultracentrifugation, eg sedimentation through sucrose gradients or isopycnic centrifugation through cesium chloride, zone electrophoresis, and chromatography on ion exchange resins, gels or insolubilized specific binding materials.

Chromatography is the most useful method because it can be easily scaled up to commercial production of particles. Kel chromatography systems that use cross-linked dextran beads are preferred because of their relatively low cost; they allow proteins and low molecular weight substances to diffuse through the void volume of Kel, thereby eliminating the column of these contaminants. A suitable column of gel can be selected that will inhibit the passage of particles through the particle, but will allow the particles to pass through without significant obstruction; the gel selected is thus a matter of routine experimentation.

Any of the methods described above can be combined as desired. For example, gel chromatography of contaminated serum on Biogel ASM followed by isopycnic centrifugation in cesium chloride is very effective in removing human serum proteins.

If a large proportion of the particle antigens present in the sample are particle fragments or water-soluble protein antigens, the most convenient technique for their separation is affinity chromatography. Antibodies capable of binding particle antigens are covalently attached or adsorbed to an insoluble support using conventional techniques. The insoluble antibody is then placed into the column and the sample is passed through. Immunologically bound antigen is washed with a buffer and then released by changing the ionic strength or pH of the wash buffer. Generally acidic pH is effective in releasing bound antigen. This technique has been highly effective in separating closely related L f:j proteins from particulate antigens.

Substances that can be removed from urine during particle purification include urea, bilirubin, uric acid, albumin, mucin, and calcium, iron, sodium, potassium, magnesium, chlorine,
Contains one or more electrolytes including phosphoric acid, ammonia, and sulfuric acid.

All of the above substances can be separated from the serum or plasma that retains the particles. In addition, one or more of the following lipids or proteins can also be removed from serum during particle purification. Namely, cholesterol, phosphatides, fatty acids, albumin, α1-globulin, n2-globulin, β-globulin 1, fibrinogen, gamma globulin and theta, and l-goglobulin, the glutinous group.

Particles obtained from pathogens such as serum or urine can be conjugated with antibodies. Particle antigens without attached antibodies are desirable for use as reagents in immunoassays. They can be obtained by growing the particles in tissue culture and then passing the used culture through a column of insolubilized Staphylococcal protein A prepared in a known manner. The protein A specifically absorbs most of the remaining immune complexes of particle antigen and antibody.

Purified antibodies against particle antigens are also useful in immunoassays. The impure antiserum source may be the serum of a patient recovering from a NANB hepatitis infection or an animal immunized against the particle antigen. The immunized animal may be a non-primate, generally a guinea pig, rabbit, goat or horse, which is non-infectious to hepatitis and so can be injected with particles that would otherwise be infectious and fatal. The antibody-containing gamma globulin fraction can be purified by conventional protein fractionation methods previously employed to separate gamma globulin from plasma, such as alcohol, polyethylene glyfur or ammonium sulfate precipitation.

The desired degree of antibody purification depends on the source and its intended use. Gamma globulin for therapeutic or palliative administration in the treatment or prevention of NANB hepatitis must be of human origin and must be as highly purified as commercially practicable. Usually this involves removing at least other desirable fractions of human plasma, such as anti-hemophilic factors or prothrombin complex, from the donor plasma. For immunoassays, on the other hand, it is usually sufficient to use unfractionated serum from non-primates as an insoluble or labeled reagent, as described below.

The purified particle antigen is further processed for use as a vaccine. As starting material for vaccine production it is preferred to use particle antigens produced by tissue culture of infectious viruses. The antigen is preferably recovered as intact particles by purifying the intact particles as described above. However, it is possible to produce suitable vaccines from particles purified from the serum or urine of acutely ill human patients, or from fragments of particles from any source that contain water-soluble antigenic proteins. Proteins such as those naturally occurring in the viral outer membrane are preferred.

These proteins can be purified by affinity chromatography as described above. It is desirable, but not necessary, that the particle antigen be purified substantially free of any attached gamma globulin when human proteins are used as starting material, particularly the circulating fluid of patients with acute NANB hepatitis. However, it is important that the antigen be free of proteins of non-human origin that may be introduced by the nutrient medium, cell line or tissue in which the virus is cultured.

Although it is conceivable that non-infectious particle antigens free of infectious particles can be isolated without further processing, particle antigen-containing suspensions should be prepared in a manner that attenuates or tastes inactivates any potentially infectious virions. Preferably, it is treated. This can be accomplished by adding the inactivating agent in a manner that does not significantly affect the immunogenic or antigenic properties of the formulation.

Formalin is effective for this purpose. A satisfactory procedure involves adding formalin to the antigen-containing solution or suspension and incubating under conditions that destroy the infectivity of the composition, usually at 25°C to 40°C for about 4 to 5 hours.
with incubation for days. The formalin is then removed or neutralized, but a bacteriostatically effective amount of formalin, eg, IO, may remain active. Virus particle counts can be made approximately by addition of a physiologically acceptable carrier such as saline, or by removing excess diluent by ultrafiltration or lyophilization and subsequent reconstitution with an aqueous solution of the carrier. 1×10 or 1×
Adjusted to 10 particles.

Vaccine injection is carried out in a conventional manner, e.g.
can be administered subcutaneously for 14 weeks at two-week intervals.

Any of the immunoassay methods previously described in this field can be used to measure particle antigens or antibodies. The following description is generally expressed with respect to the measurement of antigens. However, antibodies can also be measured using the same method.

The analytical methods described herein for quantifying particle antigens or antibodies are not to be construed as requiring measurement of all particle antigen or epitope sites. Similarly, it is not necessary to quantify all of the antibodies capable of binding the population of epitope sites provided by the particle.

This is because mutant strains and variants of viruses are often found in nature. Certain antibodies originally obtained by immunizing animals against particles in this manner may not bind, or bind very weakly, to the relevant NANB hepatitis strain. Fortunately, it is exactly as described in ＼
The particles are large so that a large number of epitope sites on or in the particles can bind the same number of different specific antibodies. Thus, changes in one or more epitope sites due to mutation or disruption of particle structure will not adversely affect immunoassays using antibodies raised against the entire population of epitope sites. This is because a certain proportion of the antibody population continues to be capable of binding an equivalent proportion of the antigen. Similarly, even antibodies raised against a single antigen common to the particle and sample antigen can be effectively used to assay the seed particle or any fragment thereof containing that antigen.

This means that (a) the antibodies used to detect the antigen are actually cross-reactive to the particles deposited by the applicant, or to a newly isolated strain from nature, or to a cross-reactive strain of said strain or a new whether or not raised by immunization against the 7 fragment of the isolate and
whether the antigen used to detect the antibody is derived from deposited particles, from new isolates of cross-reactive strains, or from fragments of deposited particles or new isolates; That applies. If the antibody raised against the antigen of the deposited particle is able to bind the antigen in question with at least about 40% of the ability that the antibody would have been able to bind the antigen against which it was originally raised, It is assumed that reactivity exists.

Particle antigens are divided into two groups: surface and core antigens. Surface antigens are antigens that are represented by intact particles. These antigens are generally beer proteins that are exported to the environment through a lipid-containing outer membrane. These are of greatest interest to us.

This is because intact particles have a high potential for infection and are generally found early in the disease.

Incomplete particles consisting only of a 40 nm diameter core can also be found or produced. Stresses that disrupt the outer membrane or incomplete synthesis of particles can expose the core antigen. These core antigens can be measured in the same manner as surface antigens.

Core antigens can be produced from intact particles by contacting the particles with a detergent, such as sodium dodecyl sulfonate, which ruptures the outer membrane by acting on its lipid components. The core can be easily separated from the ruptured remaining outer membrane by conventional operations such as ultracentrifugation or precipitation. Isolated core or outer membrane antigens can be labeled as described below or used for antibody production.

Antibodies to either or both surface or core antigens may be assayed. Such antibodies generally fall into two diagnostically interesting classes: immunoglobulin G (IP
G> and M (IgM). It is preferred to assay 17M since IvM is the antibody class that contains specificity for particle antigens that first appear during the course of infection, when IPG synthesis has not yet been initiated. IPG assays can serve as material for regulatory purposes.

After all, this immunoglobulin fraction can be detected for months after infection, so although the donor may no longer be infectious, the potential donor may have previously been exposed to particle antigens. This is because it shows that.

Methods for measuring specific activity of 19G or IPM are conventional and any such method can be employed here. For example, the IP, G or IPM fractions of a test sample can be first separated in conventional manner, conveniently by specific adsorption of either fraction onto an insoluble surface.

For example, insoluble anti-IPG or anti-1. PM
The antibody will bind the immunoglobulin of the second animal against which the first animal has been immunized. DEAE or QAE-cellulose can be used to separate IPM from IPG. Boyle'et al.
.

("J, Engineering Mmmunological Methods
``32 +i+:51-s (1980)+ is immobilized staphylococcal protein A and concanavalin A
describe the removal of IPG or IPM antibodies, respectively. Thus any of the above specific adsorbents can be used to separate IyG from IyM. The presence of anti-particle antigen specificity in either globulin fraction can then be readily assessed by measuring binding of labeled particle antigen.

Both heterogeneous or homogeneous immunoassay techniques are satisfactory for measuring particle antigens. It is preferred to use a heterogeneous assay when measuring intact particles or large antigenic fragments. All heterogeneous methods by definition include the step of separating antibody-bound antigen from residual antigen remaining in solution. In such methods, the viral antibodies are made insoluble prior to the assay, eg, by adsorption to plastic beads or the inner walls of plastic test tubes, or by binding to the antibodies so adsorbed. The antibody is instead insolubilized after an immune reaction with the antigen has taken place, for example by precipitation with a second antibody. A typical heterogeneous assay is the “competitive method”
It is. In this method, unlabeled sample antigen (if any) and labeled particle antigen analogs compete for a limited number of particle antigen binding sites, and antibody-bound antigen is separated from free antigen and labeled. The distribution rate of the antigen is measured. This method is preferred for assaying antibodies against particles. In such cases, the antigen is insolubilized and labeled and the sample antibody competes for limited antigen sites.

The sandwich heterogeneous method is another technique that can be used. This method is preferred for the measurement of particle antigens, particularly those found in intact particles or large fragments. This method adsorbs the sample antigen onto an insoluble antibody to the particle antigen, removes the remaining sample, adsorbs the labeled particle antibody to the antigen, removes the excess labeled antibody, and then The distribution of the label can be determined by quantifying the amount of the label. Variations on this method include adsorbing the antigen from the sample directly onto a non-specific adsorbent, such as plastic, adsorbing the labeled antibody, or sequentially adsorbing the particulate antibody and the labeled antigen.

Another suitable technique is sequential saturation. As in the Sand-Deutsch method, excess insoluble antibody against the particle antigen is used to bind the sample antigen, if any. However, instead of then contacting the insoluble antibody/antigen complex with labeled antibody, labeled antigen is added to occupy the remaining antibody sites.

Excess labeled antigen is removed and the label content of one of the separated phases is determined.

Homogeneous immunoassay methods can also be used advantageously.

These methods have in common that they do not involve the aforementioned phase separation and can perform complete measurements with water-soluble reagents. Such methods involve labeling an enzyme with a particle antigen to form a complex, mixing the complex, particle antigen, and sample, and measuring the change in enzyme activity resulting from binding of free antibody by the complex. It's okay. Further description of this method can be found in US Pat. No. 3,817,837.

Other homogeneous assays that can sometimes be used in a heterogeneous mode are described in US Pat. No. 3,935,074. In this method, antigen complexes are generated by covalently linking the antigen with a detector ligand.

Antibodies against the antigen and detector ligands are then mixed with the complex and sample. The usual procedure is then to measure the remaining unbound detector ligand antibody found which is inversely proportional to the amount of particle antigen.

Other suitable assay systems are disclosed in U.S. Pat. No. 4,006.
360, No. 4,134,792 and No. 3,996.3
45+ Yordeetal,, “C1inicC1
1nicalChe” 22 (81: 1372-1
377 (19761 and U.S. Patent Application A372,0
It is described in 08.

Other methods will be apparent to those skilled in the art.

The labels to be used in some of the above methods will depend on the technology involved in the assay, as will be appreciated by those skilled in the art. However, in many cases the method does not rely on the use of a particular label. Thus, any known label is satisfactory, such as radioisotopes, enzymes, coenzymes, phages, stable free radicals, and fluorescent and luminescent substituents. Preferred labels are radioisotopes, especially
I and the enzyme.

Particle antigens or antibodies can be labeled with radioiodine in any conventional manner. A suitable method is, for example, Dermo
dy et al, ``C11nical Che
mistry” 25 +61=989-995 (1
979), Parsons et al., “A.
95
:56B-574 (1979) Chloramine T or lactoperoxidase, as described in G, Z.
Externally radioactively iodinated small molecule example L! '(12
51) Iodo-hydroxyphenyl-, propionate-N-hydroxysuccinimate ester A/ (Bol
ton et al., ``Biochem, Jou.
133: 529-533 (1973)
1. (1) Short fluorescein inocyanate (Gabel et al., “Analyti
calBiochemistry” 86:396-4
0'6 (1973)), tert-butyloxycarbonyl-L-(1251)yo)'Tyrosine-N
-Hydroxysuccinimide ester (Assoian
et al,, “Analytical Bioch
emistry” 103 Near 0-76 +1980)
L or ICl (Montelaro et al.
,.

“Analytical Biochemistry”
¥92-96 Use f1979+1. Although the chloramine T method is preferred for radiolabeling of antibodies or particle cores, Montearo et al.

The technique disclosed by M. et al. is preferred for labeling outer particle membranes.

The invention may be more fully understood by reference to the following examples.

Example I Urine obtained from an inpatient diagnosed with the acute phase of NANB hepatitis is centrifuged at 8500 G for 10 molecules. 1 me in a linear gradient simisaccharide (10-20%) ame and then ultracentrifuged for 3 hours at 240,000 G using a 5W50 rotor in a Beckman L5.65B centrifuge. Pellet 0. OIM) Squirrel saline buffer pH 7,
Resuspend in 50-100 μl of 2. The specimen was applied to a carbon grid and stained negative with 1% uranium acetate.
Examine with a Jeol electron microscope. Specimen samples from acute hemodialysis patients are processed in the same manner. Togavirus-like particles with an outer diameter of 54-57 nm and nucleocybids of about 40 nm are observed in both samples.

Example 2 A urine sample in which the togavirus-like particles described in Example 1 were visible is centrifuged at 8500 G for 10 minutes. Add an equal volume of 28% polyethylene glyfur (average molecular weight 6000) solution to the supernatant. Centrifuge the resulting precipitate at low speed until the supernatant becomes clear.

Dissolve the pellet in Tris buffer, add enough ammonium sulfate to give a concentration of 15% saturation, centrifuge the resulting precipitate until the suspension becomes clear, and dissolve the pellet in Tris buffer. and lyophilize or freeze for storage at -70°C.

Example 3 Pellets of urine containing particles suspended in Tris as described in Example 1 were placed in a Rani c with an average pore size of 08 microns.
on filter membrane, and purified by ultrafiltration through a millibore PTHK membrane, which has the ability to not pass molecules and particles with a molecular weight of 100°,000 or more. Ultrafiltration is performed at slow speeds to minimize particle damage due to liquid shear forces in the membrane. After passing 3 volumes of Tris buffer through the retentate, thereby washing the particles free of low molecular weight proteins, the retentate is ultrafiltrated into 1/10 of the sample volume used and the membrane is washed with Tris buffer. Backflush with liquid, collect retentate, and lyophilize or freeze at -70°C.

Example 4 Particles can be radioiodinated in the following manner.

K suspending the particles in phosphate buffered saline pH 7.4
A stock solution of Ice is made by diluting I O,56P, NaC129,2G' and concentrated hydrochloric acid 21 Ome to 25' Ome with distilled water. Immediately before use, the solution is extracted several times for 2 minutes with chloroform 3me until the chloroform no longer has a pink color. Residual chloroform is removed by bubbling the ICl stock solution with water-saturated air for 5 minutes. Finally, IC1! Mix well 1 mf of the stock solution with 110 ml of 2M NaC. Mix 0.1 me of the disrupted particle suspension with an equal volume of IM glycine buffer pH 8.5 and add O.l.
Mix with 1 mci of Na125I in M glycine buffer. Iodination is initiated by adding 0.1 me of final IC/formulation and allowed to proceed for 5-10 seconds. The product was transferred to a column of Sephadex G-25 beads (0.9X
It is rapidly separated from unreacted 125I by gel filtration through 15 cm) and eluted with phosphate buffered saline.

Example 5 Suspension of purified particles in phosphate buffered saline (IXIO
'/d) to formaldehyde at a final concentration of 1:40
00 and the mixture was incubated at 37°C for 1 day, then at 4°C.
Incubate the hearts at 0°C for 5 days with continuous agitation. The reaction mixture is partially neutralized with formaldehydeurium. This vaccine was stored at 4°C.

Inactivated particles are administered subcutaneously to chimpanzees in a protocol of 3 injections at 1-month intervals and a booster 1 year later.

Example 6 Medium of Morgan et al (Proc. S
oc. Exp. Biol.

Med. "73: l (1950)) with 10% horse serum, dihydrostreptomycin 50%)";'/
Me! 6 Hengylpenicillin 100 units/'me
Correct by adding. The medium is sterilized by passing through a 0.22 micron filter. An aliquot of this medium is placed in a plastic Falcon flask G and inoculated with Wl-38 human diploid cells. The inoculated medium is incubated at 37°C until cell confluence is achieved. A urine sample containing particles at a concentration of approximately 1 x 107 particles/me is added to the medium at a volume ratio of 5. Nozomi N(
The culture is incubated at 37° C. while maintaining the pH at approximately 7.4 with 70 H or HCl.

After about 7 days of incubation, a cytotoxic effect on the tissue culture was observed, in particular the transformation of fibroblasts into virtually spherical birefringent cells, and finally cell lysis.

Product particles are passed through by decanting the supernatant from the cell culture flask, centrifuging at low speed to remove cell debris, precipitating the virus by adding polyethylene glycol 6000 to a concentration of approximately 15%, and centrifuging. Harvested from culture. The beer pellets were resuspended in a composition consisting of half volume of the medium without horse serum and half volume of glycerol. The particles were stable in this composition when stored at -20°C.

Example 7 Rabbits are injected with purified particle antigen in Freund's adjuvant every two weeks at 14 weeks.

Serum is collected from the animals and globulins are precipitated with 15% ammonium sulfate. The precipitate is dissolved and dialyzed overnight in 200 volumes of 0.04M phosphate buffer pH 7.2. The solution is passed through a DEAE-cellulose column (l x 20σ) and the 19'G fraction is eluted with the same buffer. 5o)iy
A subsample was prepared by Greenmod et al.

"Biochan, J-' 8th: l'14-12
3 (1963) with 1251. Labeled 117G is separated from free radioactive iodine by passage through a Cef Adex G-50 column. Labeled ■ri G
(approximately 3) in a diluent of 50% calf serum 9 antibody-negative human serum and 30.1% NaN.
”Ci/me. The formulation is stored at 4°C.

Example 8 1me of guinea pig serum obtained from an animal immunized against the particles is applied to the inner wall of a polypropylene test tube according to US Pat. No. 3,646,346. acute NANB
A 0.1 me urine specimen from a hepatitis patient and a control specimen known to be free of particles are placed in coated test tubes and incubated for 12 hours. Unbound elements in the sample are rinsed from each tube with water, the radiolabeled antibody of Example 7 is added to each tube, incubated for 12 hours, unbound labeled antibody is rinsed from the tube with water, and the Count the radioactivity bound to.

Particle antigen could be detected satisfactorily by measuring the extent of bound radioactivity in assay tubes compared to control tubes.

Claims (1)

Claims: (1) A method characterized by assaying a sample suspected of containing non-A, non-B hepatitis virus antigen for non-A, non-B hepatitis virus surface or core antigen. (2) A method characterized in that a sample suspected of containing immunoglobulins for non-A, non-B hepatitis is assayed for immunoglobulins capable of binding non-A, non-B hepatitis virus surface or core antigens. (3) The method according to claim 2, wherein the immunoglobulin is 19M. (4) The method according to claim 2, wherein the immunoglobulin is 19G. (5) An immunoassay for determining non-A, non-B hepatitis infection in a patient, characterized in that a sample from the patient is assayed for IPM capable of binding non-A, non-B hepatitis antigen. (6) A method for collecting non-A, non-B hepatitis antigen, which comprises separating the non-A, non-B hepatitis virus antigen from urine. (7) A method for detecting non-A, non-B hepatitis, which comprises assaying for non-A, non-B hepatitis particle antigen in urine. (8) A method for propagating non-A, non-B hepatitis virus, which comprises culturing the non-A, non-B hepatitis virus in an animal cell culture. (9) The culture is a primate diploid cell line W138 or M
9. The method of claim 8 which is RC-5. QQ Togavirus strains that are substantially free of primate proteins ATCC VR-2011, VR-2012, VR20
13 or VR-2014, an acid product containing one or more antigens. 11. The composition of claim 10, wherein said composition is a core of one or more of said strains. (12) Togavirus strains ATCC VR-2011, VR-2012, VR-20 substantially free of core antigen
13 or VR-2014. (13) Togavirus strain AT that does not substantially contain outer membrane antigens
CCVR-2011, VR-2012, VR-2013
Or a composition comprising one or more cores of VR-2014. (14) The composition according to claim 12 or 13, which does not substantially contain any primate protein. (I5) Togavirus strain ATCC VR-2011° VR-2012, VR-2013 or V, R-201 inactivated or attenuated so as to be biologically non-infectious
4(7) A composition comprising at least one antigen. (16) The composition according to claim 15, which contains a biologically acceptable carrier. (1η) The composition of claim 15 comprising a bacteriostatic agent.
゜At least one of VR-2013 or VR-2014
A composition comprising a primate antibody capable of binding a species antigen. (+9]) Gabilus ATCC VR-2011,V
R-2012°VR-2013 or VR-201'4
A composition comprising a non-primate antibody capable of binding at least one of one or more antigens. (b) Radiolabeled claims 10 and 1
The composition of item 2, item 13, item 15, item 18 or item 19. ■1) Claims 10 and 12 made insoluble
The composition of paragraph 13, paragraph 14, paragraph 1d or paragraph 19.・Toga virus ATCC VR'-2 in 2 samples
011°VR-2012, VR-2013 or VR-
A method for assaying a sample suspected of containing non-A, non-B hepatitis virus antigens, the method comprising detecting or measuring at least one antigen of 2014.