CA1237984A

CA1237984A - Supported viral antigen and preparation and use thereof

Info

Publication number: CA1237984A
Application number: CA000529953A
Authority: CA
Inventors: Preston H. Dorsett
Original assignee: Individual
Current assignee: Individual
Priority date: 1984-03-13
Filing date: 1987-02-11
Publication date: 1988-06-14
Also published as: CA1231305A

Abstract

SUPPORTED VIRAL ANTIGEN AND PREPARATION
AND USE THEREOF
ABSTRACT OF THE DISCLOSURE
A solid support is sensitized with soluble rubella virus antigen which is obtained by disruption solubilization of whole (intact) rubella virus. The sensitized support is useful in an assay for rubella virus antibody.

Description

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1 This invention relates to viruses, and more particularly

2 to the purification of virus, production of virus antigens,

3 the use of virus antigens for the production of sensitized solids

4 and the use of virus antigen sensitized solids for testing for virus antibodies. Most particularly, the invention relates to 6 rubella virus, rubella virus antigen and a test for rubella virus .
7 antibody.
8 united States Patent No. 4,195,074 discloses a process for producing soluble rubella virus antigen, and the use thereof in an agglutination test for rubella virus antibody. In 11 accordance with U.S. Patent 4,195,074, the tissue culture from 12 rubella virus infected cells is subjected to immunosorbent 13 separation through a column containing Gig derived from 14 human serum known to contain antibodies reactive with rubella ant c lo followed by elusion of the rubella antigen material from the 16 column and selection of the soluble antigen by gel permeation 17 chromatography. The antigen ma then be el~ploye~ for sensitizing lo erythrocytes, and the sensitized erythrocytes are used to deter-19 mine antibody in human serum samples by direct agglutination In accordance with the aforesaid patent, the so-called 21 rubella antigen is not recovered prom the virus, per so, and, 22 therefore, it is be' loved that such material does not 23 include structural proteins of the virus.
24 In accordance with one aspect of the present invention, there is roved a solid support sensitized with soluble rubella 26 viral antigen which it obtained by disruption and solubili~ation I of whole intact rubella virus.
28 In accordance with another aspect of the invention, soluble 29 rubella virus antigen is obtained from whole rubella virus.
In accordance with still another aspect of the present . I, .
I.'.

Lo invention, there is provided a test or assay for rubella virus antibody and a reagent kit therefore In accordance with a further aspect of the present invent lion, there is provided a process for producing purified virus by the use of an adsorption gel to remove non-viral proteins and nucleic acids.
In accordance with yet a further aspect of the invention, there is provided a method for producing a solid sensitized with a viral antigen.
Thus in one aspect the invention provides a composition comprising solid particles sensitized with soluble rubella virus antigen, said sensitized particles being immunoreactive with early phase rubella antibody, the soluble rubella antigen having been obtained by disruption and solubilization of whole rubella virus.
In another aspect the invention provides a process or producing solid particles sensitized with soluble rubella virus antigen, comprising supporting on the particles soluble rubella virus antigen which is immunoreactive with early phase rubella antibody, the soluble rubella virus antigen having been obtained by disruption and solubilization of whole rubella virus.
In another embodiment the invention provides a kit for determining rubella virus antibody by agglutination, in which the improvement comprises the kit including in a reagent container solid particles sensitized with soluble rubella virus antigen, the sensitized particles being immunoreactive with early phase rubella antibody, the soluble rubella virus antigen having been obtained by disruption and solubilization of whole rubella virus.
In another embodiment the invention provides a direct agglutination assay for rubella virus abodes employing solid particles sensitized with soluble rubella virus antigen, in clue which the improvement comprises employing in the assay solid particles sensitized with soluble rubella virus antigen, the sensitized particles being immunoreactive with early phase rubella antibody, the soluble rubella virus antigen having been obtained by disruption and solubilization of whole rubella virus.
In a further embodiment the invention provides an a say for rubella virus antibody, in which rubella virus antibody immunoreacts with rubella virus antigen, the improvement come prosing Lmmunoreacting in said assay rubella virus antibody with a solid support sensitized with soluble rubella virus anti-gent the sensitized solid support being immunoreactive with early phase rubella antibody, the soluble rubella virus antigen having been obtained by disruption and solubilization of whole rubella virus.
In another embodiment the invention provides a kit for determining rubella virus antibody by agglutination, in which the improvement comprises a kit including in a reagent container solid particles sensitized with soluble rubella virus antigen, the sensitized particles being immunoreactive with early phase rubella antibody.
In a still further embodiment the invention provides a direct agglutination assay for rubella virus antibodies employing solid particles sensitized with soluble rubella virus antigen, in which the improvement comprises employing in the assay solid particles sensitized with soluble rubella virus antigen, the sensitized particles being immunoreactive with early phase rubella antibodies.

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In yet a further aspect, the invention provides an improved process for purifying whole virus to separate the virus from non-viral proteins, whereby the whole virus is contacted with hydroxyl appetite in an aqueous solution of controlled strength and phi The ionic strength is maintained by the use of phosphate ions being present in a polarity of from 0.05M to 1.5M and at a pi of from 6 to 9 to provide for adsorption of non-viral proteins without significant adsorption of viral protein.
More particularly, the rubella virus antigen is isolate from intact rubella virus by treating purified whole rubella virus with a surfactant or detergent which disrupts the virus to provide the soluble rubella virus antigen, without destroying the antigen characteristics thereof. The detergent us employed in an amount that it sufficient to disrupt and ~olubilize the whole virus without destroying its antigenic characteri~ti~s.
The suxfactant or detergent which is used for disrupting the whole rubella virus may be any one of a wide variety of 20 surface tents or detergents which disrupt and syllables the virus, without destroying the antigenic characteristics, including avionic anionic and anionic surfactants~ Such sur~actants are well known in the art, and as representative examples, there may by mentioned alkali metal salts of sulfates soaps, sulfated or sulfonated oils, various amine, guaternary salts, condense-lion product with ethylene oxide, etch Such detergents and surfactants and the use thereof I disrupting whole virus are known in the art. Preferred detergents for such use are alkali (lithium or sodium) dodecyl sulfate, sulfobetain, dPoxylcholate and lauxolylsarc~sine (Sarcosyl~.

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1 In the case where the rubella virus antigen is to be 2 supported on a solid support for use in an agglutination assay 3 technique, the detergent or surfactant is one which is capably 4 of disrupting and solubilizing the virus to provide soluble virus antigen having a molecular weight such that when supported on a particle, the sensitized particle remains mono-dispersedO In _ 7 general, when using the rubella virus antigen for the sensitize-8 lion of a particle, the soluble antigen dyes not have a molecular weight in excess of 125,000 , and most generally not in excess of Lucy determined by acrylamide gel electrophoresis.
11 As hereinabove indicated, the surfactant is employed in 12 an amount which is sufficient to disrupt and syllables the virus 13 and which does not destroy the antigenic characteristics thereof 14 too much detergent may destroy the antigenic characteristics).
lo In general, the surfactant to virus weight ratio is an amount 16 of from 0.2:1 to about 5:1, preferably from about 0.5:1 to 1:1.
17 The selection of an optimum amount is deemed to be within lo the scope of those skilled in the art from the teachings herein.
19 The treatment of the purified virus is effected at a temperature which does not denature the virus proteins, with such 21 temperature generally not exceeding about Cathy a temperature 22 of from 20C to 25~C being most convenient. Similarly, the 23 pi is selected so as to maintain stability with the pi being 24 generally at 8.5, with the optimum pi generally being in the order of from 8.0 to abut 9~0.
26 The treatment of the purified virus with the surfactant 27 is or a period of time sufficient to disrupt the virus and 28 effect solubilization thereof. In general, such disruption I ~37~
and solubillzation can be accomplished in time periods in the order of from 5 to 120 minutes, however, in some cases longer or shorter times may be applicable.
The selection ox an optimum treatment time is deemed to be within the scope of those skilled in the art from the teachings herein.
Applicant has found that by using a surfactant to disrupt and syllables the whole rubella virus, as hereinabove described, it is possible to provide soluble rubella virus antigen which retains its antigenicity.
A procedure for disruption and solubilization of whole virus, as hereinabove described, has been previously practiced in the art; for example, Voyeur et at. "Structural Proteins and Subunits of Rubella Virus", JQurn~l of Vernal, P. 10-16 (Jan.
15 1972). In addition, it it known that such a procedure is capable of recovering the structural proteins of the whole rubella virus, wealth there being three principal structural proteins, namely a structural protein with a molecular weight in the order of from 60,000 to 65,000 Dalton, a structural protein with a molecular 20 weight in the order of from 40,000 to 50,000 Dalton, and a structural protein having a molecular weight in the order of from 32,000 to 38,000 Dalton. Applicant has also found evidence of a structural protein having a molecular weight of from 100,000 to 120,000 Dalton.
Applicant has found that the structural proteins recovered by such a procedure retain antigenic characteristics, and in addition, such structural proteins can be used in an assay for rubella antibody. Furthermore, applicant has found that such structural proteins are capable of detecting early phase rubella antibody, i.e., the rubella antibody present in serum or plasma within ten days of onset of rubella rash. The term "rubella virus antigen" as used herein encompasses one or more of such structural proteins recovered by such 1 ¦ procedure.
2 ¦ The hereinabove described technique fur disruption and soul-3 ¦ bilization of whole rubella virus to provide soluble rubella 4 ¦ virus antigen is also applicable to providing virus antigen from

5 ¦ other stresses; ego, those hereinafter disclosed with reference

6 ¦ It a purification of virus. Such viral antigens may therm be .

7 ¦ supp~xted on a solid support, as hereinafter Ascribe, to prove

8 ¦ a solid sensitized with the viral antigen for use in an assay.
¦ In accordance with an aspect of the present invention, 10 ¦ applicant has found that disruption and solubilization of whole 11 ¦ rubella virlls produces a soluble product which it antigenic 12 and which is capable of reacting with rubella antibody, including 13 the early phase antibody. thus, by using a product prepared by 14 such a procedure in an assay or rubella antibody; and in particular on a solid support, it is possible to detect xubellc 16 antibody even during the early phase.
17 As hereinafter describe, the recovered product is of 18 particular value for a direct agglutination assay, and applicant 19 has found that such soluble rubella virus antigen may be supportecq on a latex particle in particular a polystyrene) 21 without the problem of self a~glukination, i.e., the sensitized 22 particles remain mono-dispersed.
23 The purified whole virus which is treater with surfacants 24 is a virus which is produced in a tissue culture by procedures known in the art, and which is subsequently purified to remove 26 non-virus lipids, nucleic acids, and nQn~viral proteins.
27 The tissue culture growth of rubella virus wherein 28 rubella virus infected cells are raised in a suitable culture 29 medium is well known in the art the cells that are suitable 30 for tissue culture growth to prc~tluce the rubella virus includes 31 Very cells, Bay Lowry ton Kidney Prison Stabile Kidney, Serum I
Institute Rabbit Cornea and the like. In general, tissue cultures conventionally used for producing rubella virus are also suitable for the purposes of the present invention.
The virus may then be purified by procedures known in the S art; e.g. as disclosed by awry et at., swooper. In accordance with a preferred embodiment, the virus is purified in accordance with a procedure of the present invention.
More particularly, the procedure for purifying virus in accordance with the invention, involves, treating concentrated virus with hydrox~l appetite gel in an aqueous solution of controlled ionic strength and phi More particularly, after filtration and concentration, the virus is contacted with hydroxyl appetite gel in an aqueous solution having an ionic strength which is great enough to minimize or prevent adsorption of the virus by the gel, and which it low enough to allow the non-virus proteins to be adsorbed by the gel. The ionic strength is maintained by the use of phosphate ion, with the phosphate ions being present at a polarity of prom 0.05M to 1.5M to provide for effective adsorption of non-virus proteins and nucleic acids, without significant adsorption of the virus. The phosphate polarity in most cases it at least 0.08 M.
In addition, the adsorption is conducted at a pi in the order of from 6 to Y, most generally in the order of from 7 to 3.
The pi of the solution is maintained by the use of a suitable buffer. The adsorption may be conducted in thy presence of ETA
at a concentration from Old to .OOOlM. ETA as well as other chelating agents increases adsorption of non-viral proteins and nucleic acids, and aids in minimizing the adsorption of viral proteins.
By proceeding in accordance with the purification of the invention, the high molecular weight proteins and nucleic acids are adsorbed by the gel to thereby separate the virus protein from the non-viral I

1 proteins having similar molecular weights.
2 ¦ After such adsorption the lower molecular weight proteins 3 still remaining in the fluid may be separated by conventional 4 procedures. Thus, for example, further separation may be 5 accomplished by centrifugation through a barrier layer or 6 cushion as known in the auto In particular, the virus protein 7 is centrifuged through a suitable barrier layer such as sucrose, 8 glycerol, sesame chloride, sesame sulfate and the like, with the lower molecular weight proteins remaining above the 10 barrier, and the virus being centrifuged through the 11 barrier, as a separate layer. The fluid containing the low I molecular weight proteins and the barrier layer are then 13 removed leaving a virus protein essentially free of non-virus 14 proteins, nucleic acids, lipids, and the like. In general, the purified virus contains less than I most generally less 16 than 001% of non-virus lipids, nucleic acids and proteins.
17 The above procedure may be employed for purifying a lo wide variety of viruses, including, but not limited to: rubella;
19 virus; rubella virus, hereto viruses herpes simplex variously Roster, cytomegalovirus, Epstein-Barr [infectious mononucleosis : 21 parainfluenza viruses; influenza virus; dying virus, etc.
22 Sigh purified virus may then be treated with a surfactant :3 to disrupt the virus and effect solubilization thereof Jo I thereby provide a virus a~tige~, as hereinabove dyes cried.
26 It is to be understood that although the hereinabove 27 described procedure for purifying the rubella virus is preferred, 28 other procedures for separating non-virus proteins, lipids and 29 nucleic acids can also be employed for purifying the rubella virus for subsequent trea~nent with surfactant to thereby produce I the soluble rubella virus.
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The viral antigen which is prepared by disruption and solubilization of whole virus may be supported on a solid support for use in an assay. The following description is particularly directed to rubella virus antigens; however, the teachings are also applicable to other viral antigens.
The rubella virus antigen prepared by disrupting and solubilizing whole rubella virus may then be supported on a solid support for use in an assay for rubella virus antibody. Such supported rubella virus antigen is capable of reacting with early phase rubella virus antibody. In accordance with the preferred embodiment, the rubella virus antigen is supported on a particulate support for use in an agglutination assay; however, it is to be understood that the rubella virus antigen may be supported on a non-particulate support (or for that matter on a particulate support) for use in an assay for rubella virus antibody by procedures other than the agglutination technique.
Thus, for example, the supported rubella virus antigen may be supported on a solid support for use in an assay for rubella virus antibody by a radio immunoassay, fluorescent or enzyme assay technique. Similarly, the rubella virus antigen of the present invention may be employed for the assay of rubella virus antibody in unsupported form by use of such techniques. Thus, the scope of the invention is not limited to the preferred embodiment wherein the rubella virus antigen is supported on a particulate support for use in an agglutination assay for rubella virus antibody.
The antigen may be supported on any one of a wide variety of solid supports which are capable of supporting the antigen, and which can be used in the assay procedure without interfering with the immunoahemical reaction. Moreover, the support should be one which it stable; i.e., not adversely affecter by the prepared antigen. The antigen may be supported on the support by an adsorption technique, or by covalent coupling, I

1 either by activation of the support, or by the use of suitable coupling agent, or by use of reactive groups on the support.
3 Such procedures are generally known in the art.
4 The support may be any one of a wide variety of supports, and as representative examples of suitable supports there may 6 be mentioned: synthetic polymer supports, such as polystyrene, 7 polypropylene substituted polystyrene (e.g., laminated or 8 carboxylated polystyrene), polyacrylamides, polyamides, polyp vinyl chloride, etc.; glass beads, agrees; etc. The supports .
10 may include reactive groups; e.g., carboxyl.groups, amino groups 11 etc. to permit direct linking of the virus antigen to the 12 support 13 In accordance with preferred exigent the pi ate support is 14 either a polystyrene am noted Folks one, Charlotte poly~trene or a no inylc~oride, at ugh, it is to be sty thaw toe scope of the 16 inven~on is notlimi to such Cypriot.
17 As hereinabove indicated the antigen may be supported lo on the support by the use of an adsorption technique, or by co-19 valet coupling with a coupling agent. As representative 20 examples of suitable coupling agents there may be mentioned:
I dialdehydes; for example glutaraldehyde, succinaldehyde, melon-22 alluded, etch unstirred alluded, e.g., acrolein, ~ethacrolein, 23 crotonaldehyde, etc.; carbides; diisocyanates; dimethyladi--24 primate; Sonora chloride etc. The selection ofa.suitable 25 coupling agent should be apparent to those skilled in the art . 26 from the teachings herein 27 Similarly, the antigen may be supported by activation 28 of a suitable support; for example, oyanogen, bromide activated 29 agrees.
30 ¦ If. age ante with a preferred embodiment, as hereinabove . go !

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1 noted the soluble rubella virus antigen is supported on a 2 particulate support which is either polystyrene (substituted or 3 unsubstituted) or polyvinyl chloride: most preferably polystyrene.
4 In some cases, the soluble antigen may be supported by an adsorption technique, in other cases, it may be necessary to 6 employ covalent coupling .
7 The virus antigen sensitized particulate support is preferably prepared for use in an assay in which rubella virus antibody is determined by an agglutination technique. The 10 particulate support is provided with an effective amount of the 11 antigen for the assay, while preventing excessive amounts 12 which may result in bridging of the antibody to a single particle.
13 In general the weight ratio of soluble rubella antigen to support 14 is from 1:100 to 1-5000. The selection of an optimum amount is deemed to be within the scope ox those skilled in the art from 16 the teachings herein.
17 In accordance with one technique, after the antigen is .
18 adsorbed on the particles, the support, including the adsorbed 19 antigen, is further coated with protein which does not adversely affect the subsequent immunochemical reaction in 21 order to provide a protein coating on the portion of . the support 22 which does not including the antigen. As should be apparent, 23 the protein coating should not immunologically 24 react with either the rubella virus antigen or with sofa I to be used in the assay. As examples of suitable proteins therm 25 may be mentioned: bovine serum albumin/ ovalbumi~, and the like.
27 The selection of a suitable protein to saturate the spaces 28 ennui the rubella virus antigen on the support is deemed to 29 be within the scope of those skilled in the art from the teachings herein.

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It is to be understood that such coating with protein is not required for producing sensitized particles or use in an agglutination assay.
After the rubella virus antigen has been supported on a solid support, as generally practiced in the art for the production of sensitized particles for use in an agglutination assay, the sensitized particles are treated with a liquid containing polyoxyethylene sorbitan monolaurate (Tweet * 20) at a weight ratio to the polystyrene of 0.1:1 to loll.
The sensitized particles are preferably a synthetic polymer and in particular a polystyrene [substituted (carboxylatea or laminated) or unsubstituted] or polyvinyl chloride latex.
applicant has found that sensitization of such particles with soluble rubella virus antigen prepared, as hereinabove described, produces a sensitized particles which remains mono-dispersed (no sell agglutination), whereby such sensitized latex particles may be effectively employed in a direct agglutination assay for rubella antibody. Such sensitized particles are capable of detecting early phase rubella antibody. In addition, such sensitized particles are capable of providing a direct agglutination assay having a high 8 sensitivity for rubella antibody.
The rubella virus antigen sensitized particle prepared in accordance with the invention are suitable for use in a kit I and assay for rubella virus antibody by a affect agglutination procedure. Such kit may include, in addition to the sensitized rubella virus particles, as hereinabove described, in a suitable container therefore a reactive serum control (contains rubella antibody) and a non-reactive serum control (no rubella antibody) in suitable containers therefore In accordance with a preferred embodiment, in addition to the reagents, there is provided a test card on which the assay is effected. The test card has a flat testing surface which include suitably marked areas (or example, * Trademark I
a test circle) for placing one or more samples to be assayed, as well as suitably marked areas for each of the serum controls.
The test card and reagents may be included in a single kit package.
In the agglutination assay, undiluted serum or diluted serum (e.g. 1:10) is contacted with the sensitized particles followed by mixing, with the presence of the antibody against rubella virus being evidenced by visible agglutination.
Such rubella virus antigen sensitized particles may also be employed in a quantitative assay for rubella virus antibody.
In a quantitative assay, the sample to be assayed is serially diluted, as appropriate, and to each serial dilution there is added the particles sensitized with the soluble rubella antigen. queue quantity of antibody in the sample is determined from the highest dilution giving any agglutination of the sensitized particle.
The quantitative or qualitative assay for rubella antibody maybe effected on a card ~urfaae wherein the surface includes suitably marked areas for placing the sample and control to which the sensitized particle are added.
The invention will be further described with respect to the following examples; however, the scope of the invention is not to be limited thereby:
EXAMPLE I
Production and Purification of Rubella Virus.
Confluent roller cultures (680 cm2) of Veto cells (a continuous culture line of cells derived from African Green monkey kidney) were inoculated with approximately 0.01 PFU of rubella virus per cell and maintained in a standard culture 30 medium (tedium 199~ containing .025 M hopes buffer, pi 7.4, and 2% Volvo of the filtrate obtained by forcing fetal bovine serum through a membrane designed to retain molecules ox 100,000 molecular I

1 weight and greater (Am icon XM-100 membrane). The medium was 2 changed daily, and the culture fluids having a hem agglutination 3 titer greater than 16 were made to contain 0.01 M Uris base anal 0.01 M ETA. After incubation at 4C for 1 hour, they were concentrated.. in an Am icon hollow fiber dialyzer-concentrator to 6 1/10 the original volume. After clarification at 5,000 x g for .
7 20 minutes, the pi was adjusted to 7.6 at 22C and 1/10 ~oiume of hydroxylapatite suspension was added, a the slurry was

9 incubated at 4C with mixing) overnight. the hydroxylapatite was remove by centriguation at 5,000 x g for 15 minutes, after which 11 30 ml of the concentrate was layered over 9 ml ox 69% (wt/wt) 12 glycerol in a Beckman SUE tune. The virus was cemented at 13 82,000 x g for 16 hours at 4C, and the resultant pellet was 14 resuspended in 0.01 M carbonate buffer, pi 9.5 (coating buffer).
the purified virus was assayed for hemagglutinin content and 16 stored at -70C~

18 Solubilization of Purified Virus.
19 The purified virus in 0.01 M carbonate buffer, pi 9.5, was solubilized by treatment with sodium dodecyl sulfate (SDS).-21 The purified virus was made to contain 0.05% (wove SO and was 22 incubated for 30 minutes at room temperature.

I Preparation of Sensitized attics Commercial suspensions of polystyrene latex (0.9 micron 26 diameter particles were washed four times with 25 volumes each 27 ox the coating buffer and were resuspended it the coating buffer .
28 to provide 3% solids ( l/vol.9. The latex suspension was aided 29 directly to the syllables virus at a ratio of 2 volumes of the 1 3% latex to 1 volume of solubilized virus and the suspension was 2 mixed by tumbling for 16 yours at room temperature. The sense-3 tired latex was washed twice with 20 volumes of 1% bovine serum 4 albumin in phosphate buffered saline SAPS and resuspended at 0.5% in 1% BSA-PBS contained 0.05% polyoxyethylene orbital 6 monolaurate surface active agent (Tweet 20~ and 0. 02% gentamiacin.

8 Latex Agglutination Test for Rubella Virus Antibodies.
9 Glass plates with 1.4 cm fused circles were employed.
Serial 2-fold dilutions of serum were prepared in I BSA-PBS-11 Tweet 20 and 25 us of each dilution was placed in separate wells.
12 After adding 25 us of sensitized latex, the serum and latex 13 suspension was mixed and rotated 100 rum fur 5 minutes. The 14 presence of antibody against rubella virus was evidenced by lo visible agglutination.
I EXAMPLE
..... _ 17 Purified virus prepared in accordance with ~x~nple I was 18 treated with a 1% aqueous solution of sarcosyl for 30 minutes 19 at room temperature in coating buffer to disrupt and syllables 20 the virus.
21 The pi of the solubilized virus was adjusted to 6.5 with 22 hydrochloric acid and mixed with two volumes of 3% carboxylated 23 polystyrene latex (in phosphate buffer, pi I for 1 hour at 24 4C.
To the solution was added 10 my of carbc~diimide 26 coupling agent and the mixture was mixed overnight at 4C.
27 Peter centrifugati~n, the scolds were resuspend in 28 phosphate buffered saline PUS followed my centriguation and I resuspension in PBS containing 1% USA and 0.05~ Tweet 20.
The procedure covalently wound the soluble rubella virus 31 antigen Jo the latex.

* trade mark.

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2 In accordance with a preferred procedure, there is provided 3 a test card for rubella antibody. The test card includes a marked 4 circle for a reactive control, a marked circle for non-reactive 5 control, as well as one or more test sample circles.
6 25 us of undiluted serum sample is placed in an appear-7 privately marked sample circle, and 25 us of the reactive and non-8 reactive controls are placed n their respective circles.
With a micropipettor,there it added sensitized latex of

10 Example III (approximately 15 us), followed by rotation on

11 a rotator (about 8 minutes), and gentle hand rotation.

12 The card is read microscopically in the jet state under

13 a high intensity incandescent lamp.

14 The reactive control should show definite agglutination

15 and the non-reactive control should show no agglutination.

16 Any serum samples showing any agglutination should be no-

17 ported as reactive.
lo Numerous modifications and variations of the present 19 invention are possible in light of the above teachings and, 20 therefore, within the scope of the appended claims, the 21 invention may be practiced otherwise than as particularly 22 described.

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Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. In a process for purifying whole virus to separate the virus from non-viral proteins, the improvement comprising:
contacting the whole virus with hydroxyl apatite in the presence of phosphate ion and at a pH of from about 6 to 9, said phosphate ion being present in a molarity of from 0.05 M to 1.5 M to provide for adsorption of non-viral proteins without significant adsorption of viral protein.

2. The process of claim 1 wherein the virus is rubella virus.

3. The process of claim 2 wherein the pH is from 7 to 8.