Note: Descriptions are shown in the official language in which they were submitted.
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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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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 -
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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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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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~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
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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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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
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at room temperature. The pH is adjusted to
'!'lj approximately 7.3 with sodium hydroxide.
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(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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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
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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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(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
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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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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-
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vide an assay based on co~pler,~ent-mediated lysis.
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