Process for producing a soluble rubella antigen

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.

BACKGROUND OF THE INVENTION 
The present invention relates generally to materials and methods useful in 
the detection of antibodies and particularly relates to a novel, soluble, 
rubella virus antigen. The antigen of the invention is employed to develop 
specific immunoassay reagents useful for rapid detection and 
quantification of rubella antibodies in test fluids. Materials and methods 
of the present invention are useful in establishing the immunological 
status of a patient, (e.g., 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 fluids 
with kaolin to effect removal of non-specific lipoprotein inhibitors and 
absorption of the sera with baby chick erythrocytes to remove 
cross-reacting antibodies present in the fluid all prior to testing 
agglutination inhibition. Hemagglutination inhibition (HAI) 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 techniques for detection of antibody to rubella are 
summarized, e.g., in Meyer, H. M., et al., Am. J. Chin. Pathol., 57: 
803-813 (1972). 
Prior attempts have been made to secure a soluble rubella virus antigen, 
apart from the insoluble hemagglutinins used in HAI tests. The art 
describes identification of two major "soluble" antigens (designated theta 
and iota) but attempts to definitively isolate and characterize these 
antigens from rubella-infected cell cultures have met with limited success 
and no soluble antigen heretofore isolated has been useful in developing 
an antigen-sensitized particle effective in detection and quantification 
of antibodies to rubella. 
SUMMARY OF THE INVENTION 
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 
sulfate; and exhibits .beta. mobility in immunoelectrophoresis. 
More specifically, the novel antigen is characterized by forming a single 
line precipitate with human serum reactive to rubella virus (as shown by 
hemagglutination inhibition tests). The antigen is further characterized 
as having a specific rubella antigen activity (S.R.A.A.) of from about 500 
to about 10,000. 
The purified antigen is isolated by process steps including: affinity 
chromatography; gel permeation chromatography; and isolation on the basis 
of relative reverse passive hemagglutination (RPHA) activity. 
Immunological reagents of the invention are provided when the antigen is 
employed to sensitize immunologically inert particulate materials such as 
stabilized erythrocytes, bentonite, collodium, cholesterol crystals, 
quartz, synthetic resins, various kinds of synthetic latex, and liposomes 
prepared from phospholipids and sterols. Sensitized 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 and quantified by standard dilution techniques. 
This passive agglutination method does not ordinarily require removal from 
test fluid of non-specific inhibitors or antierythrocyte antibodies as do 
the HAI methods of the prior art. 
Sensitized particles of the invention may also be employed in 
radioimmunoassay (RIA) and enzyme immunoassay (EIA) techniques. Further, 
the soluble antigen of the invention is expected to be useful in practice 
of well known immunoprecipitation assay technique. 
Advantages attending the use of the antigen and reagents and practice of 
immunological assay methods of the invention will be apparent upon 
consideration of the following detailed description. 
DETAILED DESCRIPTION 
The soluble antigen of the invention is isolated from the culture medium of 
rubella virus infected cells. Cell lines suitable for tissue culture 
growth to obtain the antigen may include Baby Hamster Kidney (BHK-21), 
Porcine Stabile Kidney (PS), Serum Institute Rabbit Cornea (SIRC) and 
others well known in the art. In 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 of insoluble rubella hemagglutinins for 
HAI tests are well suited for use according to this invention. 
Isolation of the antigen proceeds by two-step chromatographic separation of 
growth medium components. As previously noted, the culture medium, 
preferably first concentrated by forced dialysis, is initially subjected 
to affinity chromatographic separation by passage through a column 
consisting of a solid phase to which IgG, derived from human serum known 
to contain antibodies reactive with rubella virus, has been conjugated or 
covalently bonded. Preferred solid phase materials for the column include 
agarose 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. Where particularly high pH buffers are 
employed, neutralization 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 accomplished 
by gel permeation chromatography involving, e.g., a Sephadex G-150 column. 
The effluent from the column is monitored on a UV spectrophotometer at 280 
nm and reverse passive hemagglutination by standard techniques is employed 
to identify fractions containing the purified antigen. The agglutination 
employs, e.g., erythroctyes sensitized with human IgG from the same source 
as employed for preparing the affinity column. 
The antigen so obtained is insoluble in 50 percent ammonium sulfate; 
displays a sedimentation coefficient of approximately 3.4S; has an 
estimated molecular weight of about 40,000 to 60,000 as determined by 
Sephadex G-150 chromatography; and exhibits .beta. mobility in 
immunoelectrophoresis. 
The antigen is more particularly characterized by forming a single line 
precipitate 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.A. values are developed according to the 
following criteria. Any given crude tissue culture medium from growth of 
rubella-infected cells will display absorbancy at 280 nm. A typical crude 
medium from infected BHK-21 cells displays an absorbancy of about 1.1 when 
compared to water. The crude medium will also have a relatively fixed 
titer as determined by RPHA. Once again, a typical crude medium from 
infected BHK-21 cells will display a titer of 1:32. The "total A.sub.280 
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 
reciprocal of the RPHA titer by the total A.sub.280 units, the S.R.A.A. is 
determined. S.R.A.A., therefore, equals the reciprocal of the RPHA titer 
divided by the total A.sub.280 units.

The following illustrative examples relate to: (1) preparation of a 
"concentrated" cell culture medium containing the antigen of the 
invention; (2) preparation of human IgG 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" medium; (6) 
preparation of rubella antigen-sensitized erythrocytes. 
EXAMPLE I 
Preparation of Concentrated Medium Containing Antigen 
BHK-21 cells were monolayered in 20 liter roller bottles and innoculated 
with Gilchrist strain rubella virus. After three to four days of 
incubation, the medium was harvested and subjected to zonal centrifugation 
and effluent is saved. This effluent is concentrated 100-fold at 
2.degree.-8.degree. 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 may be stored at 
-20.degree. C. 
EXAMPLE II 
Preparation of IgG 
Human recalcified plasma with a rubella titer of approximately 1:640 is 
precipitated with ammonium sulfate, dialyzed and purified according to the 
following procedural sequence. 
(1) Equal 150 ml volumes of saturated ammonium sulfate and human 
recalcified plasma which is rubella positive are admixed at a rate of 6 to 
10 ml/mixture while stirring with a magnetic stirrer at room temperature. 
The pH is adjusted to approximately 7.3 with sodium hydroxide. 
(2) The mixture is stirred for approximately one hour and the precipitate 
form is collected by centrifugation at 9000 rpm for 30 minutes at 
2.degree.-8.degree. C. 
(3) Centrifuged precipitate is added to dialysis tubing and dialyzed 
against two liters of 0.01 M K.sub.2 HPO.sub.4 /0.01 M KH.sub.2 PO.sub.4 
buffer, pH 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 Microgranular (preswollen) 
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. 
EXAMPLE 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, 
J. Biol. Chem., 245: 3059-65 (1970) as modified in March, S.C., et al., 
Analyt. Biochem., 60: 149-52 (1974). 
EXAMPLE IV 
Preparation of Gel Permeation Column 
The gel permeation column for use in purifying antigen eluted from the 
affinity column is prepared by the following procedure. 
(1) Eighteen gm of Sephadex G-150 (Pharmacia) is added to 1 liter of 0.05 M 
Tris-HCl in 0.15 M NaCl/0.02%NaN.sub.3 pH 8.0 buffer, mixed, allowed to 
swell at room temperature for 3 days and degassed. 
(2) The swollen gel slurry is added to a column to which 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 peristaltic pump, equilibrated 
with two bed volumes of the buffer at a flow rate of 4.8 ml/cm.sup.2 
/hour, and tested for homogeneity with 6-8 ml of 0.2% Blue Dextran 2000, 
collecting 12 ml fractions. 
EXAMPLE V 
Purification of Antigen from Concentrated Medium 
Purification of soluble antigen from the concentrate of Example I proceeds 
by (A) affinity chromatography and (B) gel permeation chromatography as 
follows: 
A. Affinity Chromatography 
(1) The affinity column of Example III is warmed to room temperature and 
the buffer is drained to the top of the bed. 
(2) The column is loaded with 3 bed volumes of ultracentrifuged 
"concentrate" of Example I at about 1 ml/minute flow rate. 
(3) The column is washed with 5 bed volumes of 0.05 M Tris-HCl in 0.15 M 
NaCl-0.02% NaN.sub.3 pH 8.0 buffer at 2-3 ml/minute. 
(4) Following the wash, the column is eluted with 6 bed volumes of 0.1 M 
glycine-NaOH in 0.15 M NaCl pH 12 buffer at about 1 ml/minute. The eluted 
material is collected. 
(5) With minimal delay, the eluted material is neutralized to pH 8.0 by 
adding 1 N HCl, dropwise, with constant stirring. 
(6) The neutralized material is concentrated 5-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 affinity column is chromatographed on a 
Sephadex G-150 column as prepared in Example IV. The fractions eluting in 
a volume expected to contain material with a molecular weight of 40,000 to 
60,000 daltons are collected and RPHA titers are determined. Fractions 
with a titer equal to or in excess of 1:6400 are pooled. S.R.A.A. values 
may be determined based on the A.sub.280 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. 
EXAMPLE VI 
Rubella Antigen-Sensitized Erythrocytes 
Human erythrocytes are stabilized according to the procedures disclosed in 
U.S. Pat. Nos. 3,714,345, 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 M 
acetate-pH 4.0 buffer. 0.2 ml of aqueous chromic chloride solution (10 mg 
CrCl.6H.sub.2 O/ml) is added to the erythrocyte suspension. 0.05 to 0.50 
ml of antigen from Example 5 is added to the erythrocytes, the suspension 
is incubated at 30.degree.-32.degree. C. for 2 hours with mixing at 30 
minute intervals. Sensitized erythrocytes are pelleted by centrifugation 
and the supernatant is discarded. The erythrocytes are washed twice by 
re-suspending in 0.1 M phosphate buffer and centrifuging as before. The 
pellet is re-suspended in 0.1 M phosphate buffer in quantities providing a 
0.125% (v/v) suspension 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 obtained by HAI techniques. 1336 serum 
samples were tested and the correlation coefficient (r) was determined as 
0.99 by linear regression 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 
lipoprotein inhibitors. 
Numerous modifications and variations of the abovedescribed invention will 
occur to those skilled in the art. For example, the antigen may be 
employed to sensitize immunologically inert particles of varying types 
well known in the art as useful in antigen-antibody detection schemes. In 
this regard, sensitized particles may be used in the detection of 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 provide an assay based on complement-mediated lysis.