CA1087983A

CA1087983A - Rubella virus immunoassay materials and methods for making and using same

Info

Publication number: CA1087983A
Application number: CA296,720A
Authority: CA
Inventors: John W. Safford, Jr.
Original assignee: Abbott Laboratories
Current assignee: Abbott Laboratories
Priority date: 1977-03-01
Filing date: 1978-02-10
Publication date: 1980-10-21
Also published as: FR2382461A1; GB1592825A; SE7802069L; JPS53107414A; AU513684B2; AU3318178A; JPS6231301B2; DE2808615A1; NL7802216A; CH639207A5

Abstract

ABSTRACT OF THE DISCLOSURE
Purified soluble antigen, specific for rubella virus, is isolated from growth media of rubella-infected cell cultures by affinity and gel permeation chromatography and characterized, inter alia, by its specific activity.
Antigen-sensitized particles are employed as immunoassay reagents in, for example, agglutination assays for detection and quantification of rubella antibodies in body fluids such as serum, spinal fluid and the like.

Description

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B~CKGROU~D OF THE IMV~MTION

The present inVention relates gene~all~ ~o m~teri~ls and methods useful in the detection of antibodies and particu~
larly relates to a novel, soluble, rubella virus antigen.
The antigen of the in~ention is employed to develop specific immunoassay reagents useful for rapid de~ec~ion and quantifi~
cation of rubella antibodies in test fluids. ~aterials and methods of the present invention are useful in establishing the immunological status of a patient, (evg., a woman of ;~
child-bearing age) and are also of value in diagnostic programs.
Procedures commonly employed for determination of anti-rubella antibodies in test fluids are based upon antibody inhibition of baby chick erythrocyte hemagglutination by an insoluble rubella virus particle. Among the essential steps of such procedures is the absorption of test fiuids with kaolin to effect removal of non-specific lipoprotein inhibi-tors and absorption of the sera with baby chicX erythrocytes to remove cross-reacting antibodies present in the fluid --all prior to testing agglutination inhibition. Hemaggluti-nation inhibition (~AI) assays of this type are relatively reliable but are time consuming because of the above-mentioned serum pre-treatment steps. Final test results are ordinarily not available for at least about 5-7 hours after test fluid collection. Other techni~ues for detection of antibody to rubella are summarized, e.g., in Meyer, H.M., et al., Am J.
Clin. PathoL., 57: 803-813 (1972).
Prior attempts have been made to secure a solubLe rubella virus antigen, apart from the insoluble hemagglutinins , .~ :
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~, ~87~83 ùsed in HAI tests. The art descxibe~ identification o~ kwo major ~Isoluble~ ant~gens (~sl~nate~ theta and iot~ bu~
attempts to definitivel~ isolate and characterize these antigens from rubella-infected cell cultures have me~ with limited success and no soluble antigen hereto~ore isolated has been useful in developing an antigen-sensitized particle ef~ective in detection and quantification of antibvdies to rubella.

SUMMP~R~ OF THE IMVENTIO~I -- ' ' '~

According to the present invention a soluble rubella virus antigen is isolated from media supporting growth of tissue culture cells infected with rubella virus.
The antigen has a molecular weight of from about 40,000 to about 60,000 daltons; is insoluble in 50% saturated ammonium sul~ate; and exhi~its ~ mobility in immunoelectrophoresis.
More specifically~ the novel antigen is character-ized by forming a single line precipitate with human se~um reactive ta rubella virus (as shown by hemagglutination inhibition tests). The antigen is ~ur-ther characterized as having a speci~ic rubella antigen activity (S.R.A.A.) o~ from about 500 to about 10,000.
-; The purified antigen is isolated by process steps including: af~inity chromatography; gel permeation chroma-tography; and isolation on the basis of relative reverse passive hemagglutination (RPHA) activity.
Immunological reagents of the invention are pro-vided when the antigen is employed to sensitize immunologi-cally inert particulate materials such as stabilized erythro-cytes, bentonite, collodium, cholesterol crystals, quartz, , . ..

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~79~33 synthetic xesin3, various kinds o~ ~nthetlc latex, and liposomes pxeparecl ~rom phospholipids ~nd stexols. .Sensi~ized i particles are employed in direct agglutination assays wherein rubella antibodies present in a given test fluid sample are rapidly detected by observation of particle agglutination phenomena ancl ~uantified by standard dilution techniques.
This passive agglutination method does not ordinarily require removal from test fluid of non-specific inhibitors or anti-erythrocyte antibodies as do the HAI methods of the prior art.
Sensitized particles of the invention may also be -~emp].oyed in radioimmunoassay (RIA) and enzyme immunoassay (EIA) techniaues. Further, the soluble antigen of ~he invention is expected to be useful in practice of well known immunoprecipi-`i tation assay technique.
Advantages attending the use of the antigen and re-agents and practice of i~munological assay methods of the in-vention will be apparent upon consideration of the following detailed description.
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DETAILED DESCRIPTION

The soluble antigen of the invention is isolated ~rom the culture medium of rubella virus infected cells.
Cell lines suitable for tissue culture growth to obtain the antigen may include Baby Ramster gidney (BHI~-21), Porcine Stabile Kidney (PS), Serum Institute Rabbit Cornea (SIRC) and others well known in the art. I~ general, tissue cultures employed [according to the methods of Stewart, et al., N E.

.
Jour. Med. 276, No. 10 pp. 554-7 (1967)] for production o~
insoluble rubella hemagglutinins for HAI tests axe well suited for use according to this invention.

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~01379B3 Is~lation of the antigen ~xoceeds b~ t~o-step chromatographic sepaxation o~ growth medium components. ~s ~.;
previously noted, the culkuxe medium, preferably irst con-centrated by forced dialysis, is initially subjected to affinity chromatographic separ~tion by passage through a column consisting of a solid phase to which IgG, derived from human serum known to con-tain antibodies reactive with rubella virus, has been conjugated or covalently bonded.
Preferred solid phase materials for the column include aga~ose beads. -After washing through unbound material, the antigen bound to the IgG is eluted with a suitable buffer. A glycine-sodium hydroxide buffer with a pH in excess of 8 is preferred.
~ere particularly high pH bu~ers are employed, neutrali-zation of eluted materials may be advisable.
Separation of the antigen from higher molecular weight material which may bind to the affinity column and be eluted with the buffer is àccomplished by gel permeation chromatography involving, e.g., a Sephadex G-150 column.
The effluent from the column is monitored on a UV spectro-photometer at 280 nm and reverse passive hemagglutination by ` standard techniques is employed to identify fractions con-taining the purified antigen. The agglutination employs, e.g., erythxocytes sensitized with human IgG from the same source as employed for preparing the affinity column.
The antigen so obtained is Lnsoluble in 50 percent ammonium sulfate; displays a sedimentation coefficient of approximately 3.4S; has an estimated molecular weight of ~ ' :
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i37983 ~bout 40,000 to 60,000 as det:e~min~d b~ Seph~dex ~ 150 chrom~-tography; alld exhibits ~ mobili~y ln immunoelec~rophoresis.
The antigen is more parti~ularl~ characterized b~
formin~ a single line precipi~ate with human serum reactive to rubella virus in hemagglutination inhibition tests. The antigen is also precisely characterized by having a specific rubella antigen activity (S.R.A.A.) of from about 500 to about 10,000. As employed herein, S.R.~.A. values are developed according to the following criteria. Any given crude tissue culture medium from growth of rubella-infected cells will dis-play absorbancy at 280 nm. A typical crude medium from in fected BHK-21 cells displays an absorbancy o~ about 1.1 when compared to water. The crude medium will also have a rela-tively fixed titer as determined b~ RPHA. Once again, a typical crude medium from infected BHK-21 cells will display a titer of 1:32. The "total A280 units" of material found in ,. .
the crude culture medium is defined as the volume (in ml) ;~
multiplied by the absorbance at 280 nm. By dividing the re-ciprocal of the RPHA titer by the total A280 units, the S.R~A.A.
is dekermined. S.R.A~A., therefore, equals the reciprocal of the RPHA titer divided by the total A28o units.
The following illustrative examples relate to:
(1) preparation of a "concentrated" cell culture medium con-taining the antlgen of the invention; (2) preparation of human I~G for use in affinity chromatography and reverse passive hemagglutination; ~3~ preparation of the affinity gel; ~4) preparation of the gel permeation chromatography column; (5) purification of the antigen from the "concentrated"

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)87983 medium, (6) preparation o~ ~ubella antlgen-~en~itized er~thro-cy~es. ~

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EX~MPLE I
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Pre~a _t on of Concen~rated ~edium Contain_ng An~i~en BHK-21 cells were monolayered in 20 liter roller bottles and innoculated with Gilchrist strain rubella virus.
Ater three to four days of incubation, the medlum was har-vested and subjected to zonal centrifugation and effluent is saved. This effluent is concentrated 100-fold at 2-8 C. in an Amicon DC-2 hollow fiber dialyzer concentrator. The concentrated material is clarified by centrifugation at 9000 rpm for 30 minutes followed by ultracentrifugation at 29000 ~ rpm for 6 to 18 hours. The resulting concentrated material j~ may be stored at -20 C.
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EXAMPLE II
Preparation of I~G
Human recalcified plasma wi-th a rubella titer of approximately 1:640 is precipitated with ammonium sulfate, dialyzed and purified according to the following procedural sequence.
(1) E~ual 150 ml volumes of saturated ammonium sulfate and human recalcifiea plasma which is rubella positive are admixed at a rate of 6 to 10 ~"~ ml/minute while stirring with a magnetic stirrer !1 .
at room temperature. The pH is adjusted to '!'lj approximately 7.3 with sodium hydroxide.
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~ 37983 `
, , (2) The mixture is stirred or approxima~el~
one hour and the precipitat~ form is collected by centrifugation at 9000 rpm for 30 minutes at 2-8 C.
(3) Centrifuged precipitate is added to dial~sis tubing and dialyzed against two liters of O.OlM K2HP04/O.OlM ~H2P04 bufer, pII of 8.0, with five changes of two liters each of the same buffer over 48 hours.
' (4) The dialyzed material is recovered and is further purified through use of a Whatman DE52 Diethylaminoethyl Cellulose ~licrogranular (pre-swo~len) Anion Exchanger and the IgG pool collected , is subjected to further concentration, clarified by centrifugation and stored.
~6) The final yield of IgG recovered ranges from 150 mg to 160 mg per 70 ml of whole serum.

EX~PLE III
Preparation_of Affinity Gel Sepharose 4b (Pharmacia) or any suitable agarose solid phase is activated with cyanogen bromide, 97% ~Aldrich) in acetonitrile and subsequently coupled with human IgG (as prepared in Example II). The coupling of IgG to the solid phase is accomplished by practice of the method of Cuatrecasas, 8iol. Chem., 245: 3059-65 (1970) as modified in March, S.C., et al., Analyt. Biochem , 60: 149-52 (1974).

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EX~MPL~ XV
Preparation of Gel Permeation Column The gel permeation column for use in purifying antigen eluted from the af~inity column is prepared by the following procedure.
(1) Eighteen gm of Sephadex G-150 (Pharmacia~
is added to 1 liter 0.05M Tris-HCl in 0.15M NaCl~0.02ONaN3 p~
8.0 buf~er, mixed, allowed to swell at room tempe~ature ~or 3 days and d~gassed.
(2) The swollen gel slurry is added to a column to whlch the buffer has been added and partially drained, with hydrostatic pressure at 3-5 cm during the packing.
(3) The column is operated with use of a perls-taltic pump, equilibrated with two bed volumes o~ the buffer at a flow rate of a~8 mlJcm2/hour, and tested for homoseneity with 6-8 ml of 0.2% Blue Dextran 2000, collecting 12 ml frac- ;

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~ tions.
, EXAMPLE V
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Purification of Anti~en from Concentratea Medium Purification of soluble antigen from the concen-trate o~ Example I proceeds by (A) afinity chromatography and ~B) gel permeation chromatography as follows:
! ` A~ A~finity Chromatography , tl) The affinity column of Example III is warmed to room temperature and the buffer is drained to ` the top o~ the bed.
~2) The column is loaded with 3 bed volumes of ultracentrifuged "concentrate" of Example I at - about 1 ml/minute flow rate.

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~oB7s83 (3) The colunn is washed w.ith 5~bed ~olumes o~
0.05M Tris-HCl in 0. 15M NaCl~0.02% NaN3 pH 8.0 ~-buffer at 2-3 ml/minute.

(4) Following the wash, the column is eluted wi~h ~-: 6 bed volumes of 0.1M glycine-NaOH in 0.15M NaCl pH 12 buffer at about l ml/minute. The elu~ed - material is collected.

(5) With min}mal delay, the eluted material is neutralized to pEI 8.0 b~ adding lN HCl, dropwise, with constant stirring.
; (6) The neutralized material is concentrated S-fold of the original load volume in a single hollow ~ fiber concentrator and clarified by centrifugation.
B. Gel Permeation Chromatography The material eluted from the af~inity colun~n is chromatographed on a Sephadex G-150 column as prepared in Example IV. The fractions eluting in a volume èxpected to contain material with a molecular weight of 40,000 to 60,000 - daltons are collected and RPHA titers are determined. Frac-tions with a titer equaL to or in excess of 1:6400 are pooled. S.R.A.A. values may be determined based on the ;~ A280 value and RPHA titer of the pooled fractions. Typically, the antigen is concentrated to a 3 to 8 ml volume from an original 80 to 100 liter volume of crude growth medium and has a S.R.A.A. value of from 500 to 10,000.

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'' . "~ .' ' ~0137983 X~MPL~ Vl Rubella Anti~eri-Sensitized Erythrocy~es Human er~throc~tes are stabilized according to the procedures disclosed in U~S. Patents 3,714,3~5, 3,715,427 and/or 3,925,541; made up in 2.0 ml, 10~ suspensions, and centrifuged for 2 to 3 minutes at 500-1000 rpm. The buffer is decanted and the cells are resuspended in 2.0 ml of 0.01 acetate-pH 4.0 buffer. 0.2 ml of aqueous chromic chloride ... . .
solution ~10 mg CrC1~6H2O/ml) is added to the erythrocyte -suspension. 0.05 to 0.50 ml of antigen ~rom Example 5 is added to the erythrocytes, the suspension is incubated at 30-32 C. for 2 hours with mixing at 30 minute intervals.
Sensitized er~throcytes are pelleted by centrifugation and the supernatant is discarded. The erythrocytes are washed twice by re-suspending in O.lM phosphate buf~er and centri-fuging as before. The pellet is re-suspended in O.lM phos-phate buffer in quantlties providing a 0.125~ (vJv) sus-pension of sensitized erythrocytes.

EXAMPLE VII
Sensitized erythrocytes~ essentially according to Example VI, were employed to determine the antibody titers of random human blood donor serum samples and results were compared to titers obtainèd by HAI techniques. 1336 serum samples were tested and the correlation coef~icient (r) was determined as 0.99 by linear regxession analysis. Using the sensitized erythrocytes there is no need to pre-absorb the serum samples to remove antibody cross-reacting with heterologous erythrocytes. Additionally it is unnecessary to pre-treat the serum samples to remove non-specific lipo-protein inhibitors.

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t7983 Numer~u~ modl~icakions and V~ria~ion~ o~ ~he above-described in~ention will occur to those skilled in the art.
Fox exa~ple, the antigen may be employed to sensitize immunologically inext particles o-f varying types well l;nown in the art as use~ul in antigen-antibody detection schemes.
In this regard, sensitized particles may be used in the detection o antibody by agglutination techniques, by radioimmunoassay techniques, by fluorescent techniques, and by enzyme immunoassay techniques. Additionally, particles such as erythrocytes and liposomes may be sensitized to pro-.
vide an assay based on co~pler,~ent-mediated lysis.
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Claims

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

1. A process for producing a soluble rubella antigen, said process comprising, in sequence:
(a) subjecting growth media of a tissue culture of rubella virus inrected cells to chromatographic separation through a column consisting of a solid phase to which IgG, derived from human serum known to contain antibodies reactive with rubella antigens, has been covalently coupled;
(b) eluting said column to separate materials bound to the IgG therein;
(c) subjecting the separated eluted material to gel permeation chromatography to achieve material separation on the basis of molecular size;
(d) isolating rubella antigen from said sized, separated eluted material on the basis of relative reverse passive hemagglutination activity.

2. The process of claim 1 wherein, in step (a), the solid phase consists of agarose beads.

3. The process of claim 1 wherein, in step (b), the eluting material is a buffer solution having a pH greater than about 8, and further including the step of neutralizing said eluted material prior to step (c).

4. Purified rubella virus antigen characterized by forming a single line precipitate with human Serum reac-tive to rubella virus in hemagglutination inhibition tests and further characterized as having a specific rubella anti-gen activity of from about 500 to about 10,000.

5. The antigen of claim 4 further characterized by having a molecular weight of from about 40,000 to about 60,000, by being insoluble in 50 percent saturated ammonium sulfate and by exhibiting .beta. mobility in immunoelectrophoresis.

6. A reagent having immunochemical determinations comprising discreet particles, surface portions of which are activated with a rubella antigen characterized by forming a single line precipitate with human serum reactive to rubella virus in hemagglutination inhibition tests and further char-acterized as having a specific rubella antigen activity of from about 500 to about 10,000.

7. A reagent according to claim 6 wherein said particles are erythrocytes.

8. A method for detecting the presence of rubella virus antibodies in a fluid to be tested, said method comprising (a) contacting said fluid with a solution con-taining discreet particles, surface portions of which are activated with a rubella antigen char-acterized by having a specific rubella antigen activity of from about 500 to about 10,000, to form a reaction mixture; and (b) monitoring said reaction mixture for evidence of an antigen-antibody reaction.