CA1231305A

CA1231305A - Supported viral antigen and preparation and use thereof

Info

Publication number: CA1231305A
Application number: CA000449487A
Authority: CA
Inventors: Preston H. Dorsett
Original assignee: Individual
Current assignee: Individual
Priority date: 1984-03-13
Filing date: 1984-03-13
Publication date: 1988-01-12
Also published as: CA1237984A

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 and solubilization of whole (intact) rubella virus. The sensitized support is useful in an assay for rubella virus antibody.

Description

Lowe This invention relates to viruses, and more particularly to the purification of virus, production of virus antigens, the use of virus antigens for the production of sensitized solids, and the use of virus antigen sensitized solids for testing for virus antibodies. Most particularly, the invention relates to rubella virus, rubella virus antigen and a test for rubella virus antibody.
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 accordance with U.S. Patent 4,195,074, the tissue culture from rubella virus infected cells is subjected to immunosorbent separation through a column containing Gig derived from human , serum known to contain antibodies reactive with rubella antigen followed by elusion of the rubella antigen material from the column and selection of the soluble antigen by gel permeation chromatography. The antigen may then be employed for sense-tiring erythrocytes, and the sensitized erythrocytes are used to determine antibody in human serum samples by direct aggluti-nation.
In accordance with the aforesaid patent, the so-called rubella antigen is not recovered from the virus, per so, and, therefore, it is believed that such material does not include structural proteins of the virus.
In accordance with one aspect of the present invention, there is provided a solid support sensitized with soluble rubella viral antigen which is obtained by disruption and solubilization of whole (intact) rubella virus.
In accordance with another aspect of the invention, soluble rubella virus antigen is obtained from whole rubella virus.

In accordance with still another aspect of the present " I, :~23~L3t~

invention, there is provided a test or assay or 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 for 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 antibodies employing solid particles sensitized with soluble rubella virus antigen, in 3~3~S

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 ox whole rubella virus.
In a further embodiment the invention provides an assay for rubella virus antibody, in which rubella virus antibody immunoreacts with rubella virus antigen, the improvement come prosing immunoreacting 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 beer. obtained by disruption and solubilization of whole rubella virus.
In a 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.

pa-I

More particularly, the rubella virus antigen is isolated 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 antigenic characteristics thereof. The detergent is employed in an amount that is sufficient to disrupt and syllables the whole virus without destroying its anti~enic characteristics.
The surfactant or detergent which is used for disrupting 10 the whole rubella virus may be any one of a wide variety of surfactants or detergents which disrupt and syllable the virus, without destroying the antigenic characteristics, including. -cationicJ anionic and non-ionic surfactants. Such surfactants are well known in the art, and as representative examples, there may be mentioned alkali metal salts of sulfates, soaps, sulfated or sulfonated oils, various amine, qua ternary salts, ~ondensa-lion products with ethylene oxide, etc. Such detergents and surfactants and the use thereof for disrupting whole virus are known it the art. Preferred detergents for such use are alkali 20 (lithium or sodium) dodecyl sulfate; sulfobetain, deoxylcholate and laurolylsarcosine (Sarcosyl~. --2b-r s 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 6 a particle, the sensitized particle remains mono-dispersed. In 7 general, when using the rubella virus antigen for the sensitize-8 lion of a particle, the soluble antigen does 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 em~lo-~ed 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).
In general, the surfactant to virus weight ratio is an amount 16 of from 0.201 to about 5:1, preferably from about 0.5:1 to tot.
17 The selection of an optimum amount is deemed to be within 18 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 no-t exceeding about 30C, with a temperature 22 of from 20C to 25C 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 about 9Ø
26 The treatment of the purified virus with the surfactant 27 is for a period of time sufficient to disrupt the virus and 28 effect solubilization thereof. In general, such disruption 1.2;~13~5 1 and solubilization can be accomplished in time periods in the ore r 2 of from 5 to 120 minutes, however, in some cases longer or 3 snorter times may be applicable.
4 The selection of an optimum treatment time is deemed to be I within the scope of those skilled in the art from the teachings 6¦ herein.
71 Applicant has found that by using a surfactant to disrupt 81 and syllables the whole rubella virus, as hereinabove described, I it is possible to provide soluble rubella virus antigen which lo retains its antigenicity;
11¦ - A procedure for disruption and solubilization of 12¦ whole virus, as hereinabove described has keen previously pray-13¦ tired in the art; for example, Voyeur et at. "Structural Proteins 14¦ and Subunits of Rubella Virus", Journal of Virology, P. 10-16 15¦ (Jan. 1972). In addition, it it known that such a procedure 16¦ is capable of recovering the structural proteins of the whole 17¦ rubella voyeurs with there being three principal structural 18¦ proteins, namely a structural protein with a molecular weight in lug¦ the order of from 60,000 to 65,000 Dalton, a structural protein 20¦ with a molecular weight in the order of from 40,000 to 50,000 21¦ Dalton, and a structural protein having a molecular weight in 22 the order of from 32,000 to 38,000 Dalton. Applicant has also 23 found evidence of a structural protein having a molecular 24 weight of from 100,000 to 120,000 Dalton.
Applicant has found that the structural proteins recovered 26 by such a procedure retain antigenic characteristics, and in 27 addition, such structural proteins can be used in an assay for 28 rubella antibody. Furthermore :, applicant has found that such 29 structural proteins are capable of detecting early phase 30 rubella-antibody, i.e., the rubella antibody present in serum 31 or plasma within ten days of onset of rubella rash. The 32 term "rubella virus antigen" as used herein encompasses 33 one or more of such structural proteins recovered such _ . . - . _ - _ - . -- . - - - , . . .
.

1;~313~

1¦ procedure.
21 The hereinabove described technique for disruption and soul- _ 31 bilization of whole rubella virus to provide soluble rubella 41 virus antigen is also applicable to providing virus antigen from 51 other viruses; e.g., those hereinafter disclosed with reference 61 to a purification of virus. Such viral antigens may then be 71 supported on a solid support, as hereinafter describe to prove 81 a solid sensitized with the viral antigen for use in an assay.
I In accordance with an aspect of the present invention, 10¦ applicant has found that disruption and solubilization of whole 11¦ rubella virus produces a soluble product which is 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 for rubella antibody; and 15 ¦ in particular on a solid support, it is possible to detect rubella 16 ¦ antibody even during the early phase.
17 ¦ As hereinafter described, 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 20 ¦ supporter on a latex particle tin particular a polystyrene) 21 ¦ without the problem of self agglutination, 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 non-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 produce the rubella virus includes 31 Veto cells, Baby Hamster Kidney, Procaine Stabile Kidney, Serum :~Z3~3~S

Institute Rabbit Cornea and the like. In general, tissue gut-lures 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 art; e.g. as disclosed by Burr 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 hydroxyl appetite gel in an aqueous solution of con-trolled ionic strength and phi More particularly, after filtration and concentration, the virus is contacted with hydroxyl appetite gel in an aqueous soul-lion having an ionic strength which is great enough to minimize or prevent adsorption of the virus by the gel, and which is low enough to allow the non-virus proteins to be adsorbed by the gel The ionic strength is maintained by the use of phosphate ions, with the phosphate ions being present at a polarity of from 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 is at least 0.08 M.
In addition, the adsorption is conducted at a pi in the order of from 6 to 9, most generally in the order of from 7 to 8. The pi of the solution is maintained by the use of a suitable buffer. The adsorption may be conducted in the 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 r,', 3L~,.3~ it's l 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 accomplished by centrifugation through a barrier layer or 6 cushion as known in the art. 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 lo barrier, and the virus being centrifuged through the if barrier, as a separate layer. The fluid containing the low 12 molecular weight proteins and the barrier layer are then 13 removed leaving a virus protein essentially free of non-virus lo proteins, nucleic acids, lipids, and the like. In general, lo the purified virus contains less than 1%, most generally less lo than 0.1% of non-virus lipids, nucleic acids and proteins.
17 The above procedure may be employed for purifying a 18 wide variety of viruses, including, but not limited to: rubella, lo virus; rubella virus, hereto viruses herpes simplex variously Roster, cytomegalovirus, Epstein-Barr [infectious mononucleosis 21 parainfluenza viruses; influenza virus; dying virus, etc.
22 Such purified virus may then be treated with a surfactant :3 to disrupt the virus and effect solubilization thereof to 24 thereby provide a virus antigen, as hereinabove desk cried.
I 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 treatment with surfactant to thereby produce 31 the soluble rubella virus.
I

... , .. . . .. ... . .

~(Z3~13~S

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 support-Ed 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 where-in 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 immunochemical reaction. Moreover, the support should be one which is stable; i.e., not adversely affected by the pro-pared antigen. The antigen may be supported on the support by an adsorption technique, or by covalent coupling, of 1 either by activation of the support, or by the use of a suitable 2 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., amounted 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 ~x~iment, the Peculate support is 14 either a pulsator, amounted Polystyrene, cæboxylat~d Folysytrene or a no vinyl chloride, although, it is to be understood that toe scope of the 16 invention is jot limited to such swooper.
17 As hereinabove inducted 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 employs of suitable coupling agents there may be mentioned:
21 dialdehydes; for example glutaraldehyde, succinaldehyde, melon-22 alluded, eta; unstirred alluded, e.g., acrolein, methacrolein, 23 crotonaldehyde, etc.; carbodiimides; diisocyanates; dimethyladi-24 primate; cyan uric chloride etc. The selection ova suitable 25 coupling agent should be apparent to those skilled in the art 26 from the teachings herein.
27 Similarly, the antigen may he supported by activation 28 of a suitable support; for example, cyanogen, bromide activated 29 agaxose.
In accordance with a preferred embodiment, as hereinabove I! 3 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 8 preferably prepared for use in an assay in which rubella virus antibody is determined by an agglutination technique. The 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 of 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 to be used in the assay. As examples of suitable proteins there 26 may be mentioned: bovine serum albumin, ovalbumin, and the like.
27 The selection of a suitable protein to saturate the spaces 28 between 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.

1 It is to be understood that such coating with protein 2 is not required for producing sensitized particles for use in 3 an agglutination assay.
4 After the rubella virus antigen has been supported on a solid support, as generally practiced in the art fur the p unction of sensitized particles for use it an agglutination assay, the 7 sensitized particles are treated with a liquid containing p~lyc~e+~ylene son-B Titan nolaurate (Winnie 20~ at a weight ratio to the polystyrene of 0.1:1 to it :
9 The sensitized particles are preferably a synthetic polymer and in particular a polystyrene [substituted (carboxylated or 11 laminated) or unsubstituted] or polyvinyl chloride latex. Applicant 12 has found that sensitization of such particles with soluble rubella 13 virus antigen prepared, as hereinabove described, produces a 14 sensitized particles which remains mono-dispersed (no self agglutination), whereby such sensitized latex particles may be 16 effectively employed in a direct agglutination assay for rubella 17 antibody. Such sensitized particles are capable of detecting 18 early phase rubella antibody. In addition, such sensitized particles are capable of providing a direct agglutination assay having a high sensitivity for rubella antibody.
21 The rubella virus antigen sensitized particle prepared in 22 accordance with the invention are suitable for use in a kit and 23 assay for rubella virus antibody by a direct agglutination 24 procedure. Such kit may include, in addition to the sensitized rubella virus particles, as hereinabove described, in a suitable 26 container thrower, a reactive serum control (contains rubella 27 antibody) and a non-reactive serum control (no rubella antibody) 28 in suitable containers therefore In accordance with a preferred 29 embodiment, in addition to the reagents, there is provided a test card on which the assay is effected. The test card has a 31 flat testing surface which include suitably marked areas (for 32 example, a test circle) for placing one us more samples to be 33 assayed, as well as suitably marked areas for each of the serum * trade mark. -11-,!~ ,~, ,1.

-` l 1 controls. The test card and reagents may be included in a single 2 Kit package.
3 ¦ In the agglutination assay, undiluted serum or dilute 4 serum (e.g. 1:10) is contacted with the sensitized particles S followed by mixing with the presence of the antibody 6 against rubella virus being evidenced ho visible agglutination. Jo 7 ¦ Such rubella virus antigen sensitized particles may also 8 Abe employed in a quantitative assay for rubella virus antibody.
¦ In a quantitative assay, the sample to be assayed is 10 serially diluted, as appropriate, and to each serial dilution 11¦ there is added the particles sensitized with the soluble rubella 12¦ antigen. The quantity of antibody in the sample is determined fro 13¦ the highest dilution giving any agglutination of the sensitized 14 particles.
15¦ The quantitative or qualitative assay or rubella 16 antibody may be effected on a card surface wherein the surface 17¦ includes suitably marked areas for placing the sample and 18 control to which the sensitized particles are added.
19 ¦ The invention will be further described with respect to the 2~1 following examples; however, the scope of the invention is not 21 It be limited thereby:
22¦ EXAMPLE I
23 ¦ Production and Purification of Rubella Virus.
I ¦ Confluent roller cultures (680 cm2) of Veto cells (a 25 continuous culture line of cells derived from African Green monkey 26 kiwi) were i~xx~ated with apc~i~ately 0.01 PFU of n~x~la viny -27 per cell and maintained in a standard culture medium (Medium 28 ¦199) containing .025 M hopes buffer, pi 7.4, and 2% Volvo of 29 the filtrate obtained by forcing fetal bovine serum through a 30 membrane designed to retain molecules of 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 and 4 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 Jo 7 20 minutes, the pi was adjusted to 7.6 at 22C and 1/10 volume 8 of hydroxylapatite suspension was added, and the slurry was incubated at 4C with mixing, overnight. The hydroxylapatite was removed by centriguation at 5,000 x g for 15 minutes, after which 11 30 ml of the concentrate was layered over 9 ml of 69% ~wt/wt) 12 glycerol in a Beckman SUE tube. The virus was sediment Ed at 13 82,000 x g for 16 hours at 4C, and the resultant pellet was 14 resuspended in 0.01 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~ (w/v) SO and was 22 incubated for 30 minutes at zoom temperature.

24 Preparation of Sensitized Latex.
Commercial suspensions of polystyrene latex (0.9 micron 26 diameter particles) were washed four times with 25 volumes each of the coating buffer and were resuspended in the coating buffer 28 to provide 3% solids ( vowel.). The latex suspension was aided 29 directly to the solubili~ed virus at a ratio of 2 volumes of the - - . - .

1 3% latex to 1 volume ox solubili~ed virus and the suspension was 2 mixed by jumbling for 16 yours at room temperature. The sense-3 tired latex was washed twice with I volumes of 1% bovine serum 4 albumin in phosphate buffered saline ASPS and resuspended at 005% in 1% BSA-PBS contained 0.05~ polyoxyethylene sorbitan 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.
10 Serial fulled dilutions of serum were prepared in 1% BLABS
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 lo suspension was mixed and rotated 100 rum for 5 minutes The 14 presence of antibody against rubella virus was evidenced by 15 visible agglutination.

_ .
17 Purified virus prepared in accordance with Example 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 Z0 the virus.

21 The pi ox 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 6.5) for 1 hour a 24 4C.

To the solution was added 10 my of a carbodiimide 26 coupling agent and the mixture was mixed overnight at 4C.

27 After centrifugation, the solids were resuspend in 28 phosphate buffered saline PUS followed by centriguation and 29 resuspension in PUS containing 1% USA and 0.05~ Tweet 20.

The procedure covalently gourd the soluble rubella virus 31 antigen to the latex.

* trade mark.

, ;11.~3.~ I

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 in their respective circles.
With a micropipettor,there is 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 wet state under 13 a high intensity incandescent lamp.
14 The reactive control should show definite agglutination lo and the nonreactive control should show no agglutination.
16 Any serum samples showing any agglutination should be no-17 ported as reactive.
18 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 2 2 duskier ire ..
, . ., . . . . - .

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A composition, comprising:
solid particles sensitized with soluble rubella virus antigen, said sensitized particles being immunoreactive with early phase rubella antibody, said soluble rubella antigen having been obtained by disruption and solubilization of whole rubella virus.

2. The composition of claim 1 wherein the soluble rubella virus antigen has a molecular weight of no greater than 125,000 daltons as determined by acrylamide gel electro-phoresis.

3. The composition of claim 1 wherein the solid particles are a polystyrene latex.

4. The composition of claim 1 wherein the solid particles are a synthetic polymer.

5. The composition of claim 4 wherein the synthetic polymer is selected from the group consisting of polyvinyl chloride, polystyrene, aminated polystyrene and carboxylated polystyrene.

6. The composition of claim 4 wherein the soluble rubella virus antigen is covalently coupled to the solid particles.

7. The composition of claim 4 wherein the soluble rubella virus antigen is adsorbed on the solid particles.

8. The composition of claim 1 wherein the soluble rubella virus antigen is obtained by disruption and solubili-zation of whole rubella virus with a detergent.

9. The composition of claim 8 wherein the detergent is an alkali dodecyl sulfate.

10. A process for 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, said soluble rubella virus antigen having been obtained by disruption and solubilization of whole rubella virus.

11. The process of claim 10 wherein the soluble rubella virus antigen has a molecular weight of no greater than 125,000 daltons, as measured by acrylamide gel electro-phoresis.

12. The process of claim 10 wherein the solid particles are a polystyrene latex.

13. The process of claim 10 wherein the particles are a synthetic polymer.

14. The process of claim 13 wherein the synthetic polymer is selected from the group consisting of polyvinyl chloride, polystyrene, aminated polystyrene and carboxylated polystyrene.

15. The process of claim 13 wherein the soluble rubella virus antigen is covalently coupled to the particles.

16. The process of claim 10 wherein the soluble rubella virus antigen is obtained by disruption and solubili-zation of whole rubella virus with a detergent.

17. The process of claim 16 wherein the detergent is an alkali dodecyl sulfate.

18. In a kit for determining rubella virus antibody by agglutination, the improvement comprising:
said kit including in a reagent container solid particles sensitized with soluble rubella virus antigen, said sensi-tized particles being immunoreactive with early phase rubella antibody, said soluble rubella virus antigen having been obtained by disruption and solubilization of whole rubella virus.

19. The kit of claim 18 wherein said kit further includes a test card having a flat surface for receiving assay samples.

20. The kit of claim 19 wherein the solid particles are a polystyrene latex.

21. The kit of claim 19 and further comprising in separate reagent containers a reactive serum control of rubella antibody and a non-reactive serum control free of rubella antibody.

22. In a direct agglutination assay for rubella virus antibodies employing solid particles sensitized with soluble rubella virus antigen, the improvement comprising:
employing in the assay solid particles sensitized with soluble rubella virus antigen, said sensitized particles being immunoreactive with early phase rubella antibody, said soluble rubella virus antigen having been obtained by disruption and solubilization of whole rubella virus.

23. The assay of claim 22 wherein the soluble virus rubella antigen has been obtained by disruption and solubili-zation of whole rubella virus.

24. The assay of claim 22 wherein the particles are a polystyrene latex.

25. The assay of claim 22 wherein the particles are a synthetic polymer.

26. The assay of claim 25 wherein the synthetic polymer is selected from the group consisting of polyvinyl chloride, polystyrene, aminated polystyrene and carboxylated polystyrene.

27. The assay of claim 25 wherein the soluble rubella virus antigen is adsorbed on the solid particles.

28. The assay of claim 25 wherein the soluble rubella virus antigen is covalently coupled to the solid particles.

29. The assay of claim 22 wherein the soluble rubella virus antigen has been obtained by disruption and solubili-zation of whole rubella virus with a detergent.

30. The assay of claim 29 wherein the detergent is an alkali dodecyl sulfate.

31. In an assay for rubella virus antibody, wherein rubella virus antibody immunoreacts with rubella virus antigen, the improvement comprising:
immunoreacting in said assay rubella virus antibody with a solid support sensitized with soluble rubella virus anti-gen, said sensitized solid support being immunoreactive with early phase rubella antibody, said soluble rubella virus antigen having been obtained by disruption and solubilization of whole rubella virus.

32. A composition, comprising:
solid particles sensitized with soluble rubella virus antigen, said sensitized particles being immunoreactive with early phase rubella antibody.

33. The composition of claim 32 wherein the solid particles are a polystyrene latex.

34. The composition of claim 32 wherein the solid particles are a synthetic polymer.

35. The composition of claim 34 wherein the synthetic polymer is selected from the group consisting of polyvinyl chloride, polystyrene, aminated polystyrene and carboxylated polystyrene.

36. The composition of claim 32 wherein the soluble rubella virus antigen is adsorbed on the solid particles.

37. In a kit for determining rubella virus antibody by agglutination, the improvement comprising:
said kit including in a reagent container solid particles sensitized with soluble rubella virus antigen, said sensitized particles being immunoreactive with early phase rubella anti-body.

38. The kit of claim 37 and further comprising in separate reagent containers a reactive serum control of rubella antibody and a non-reactive serum control free of rubella antibody.

39. The kit of claim 38 wherein the solid particles are a polystyrene latex.

40. In a direct agglutination assay for rubella virus antibodies employing solid particles sensitized with soluble rubella virus antigen, the improvement comprising:
employing in the assay solid particles sensitized with soluble rubella virus antigen, said sensitized particles being immunoreactive with early phase rubella antibodies.