U.S. patent number RE32,833 [Application Number 07/004,172] was granted by the patent office on 1989-01-17 for screening vaccines and immunization process.
This patent grant is currently assigned to President and Fellows of Harvard College. Invention is credited to Bernard N. Fields, Mark I. Greene.
United States Patent |
RE32,833 |
Greene , et al. |
January 17, 1989 |
Screening vaccines and immunization process
Abstract
Mammals are vaccinated against infectious organisms and
polypeptides are screened for utility as vaccines by a
complementing set of monoclonal antibodies, the first of which
antibodies binds specifically to the site on the organism which
itself binds specifically to a receptor on a host cell of the
mammal, and the second of which binds specifically to the first.
Vaccination is done with the second antibody alone, and screening
is done by determining whether the polypeptide binds to the first
antibody.
Inventors: |
Greene; Mark I. (Penn Valley,
PA), Fields; Bernard N. (West Newton, MA) |
Assignee: |
President and Fellows of Harvard
College (Cambridge, MA)
|
Family
ID: |
26672704 |
Appl.
No.: |
07/004,172 |
Filed: |
December 23, 1986 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
353257 |
Mar 1, 1982 |
04490358 |
Dec 25, 1984 |
|
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Current U.S.
Class: |
424/131.1;
424/147.1; 424/204.1; 424/215.1; 436/548 |
Current CPC
Class: |
C07K
16/10 (20130101); C07K 16/4216 (20130101); G01N
33/686 (20130101); A61K 38/00 (20130101) |
Current International
Class: |
C07K
16/08 (20060101); C07K 16/10 (20060101); C07K
16/42 (20060101); G01N 33/68 (20060101); A61K
38/00 (20060101); A61K 039/395 (); A61K 039/42 ();
G01N 033/54 (); G01N 033/56 () |
Field of
Search: |
;424/85,86,88,89
;435/172.2 ;436/548 |
Other References
Nisonoff et al., Clinical Immunology and Immunopathology, vol. 21,
pp. 397-406, (1981). .
Moshly-Rosen et al., FEBS Letters, vol. 106, pp. 389-392, (1979).
.
Letvin et al., The J. of Immunology, vol. 127, pp. 2334-2339,
(1981). .
Nepom et al., J. Exp. Med., vol. 155, pp. 155-167, (1982)..
|
Primary Examiner: Hazel; Blondel
Claims
What is claimed is:
1. The method of immunizing a host mammal against an infectious
organism having a site which binds specifically to a receptor site
on a host cell which method comprises
providing a first monoclonal antibody which binds specifically to
said organism site,
providing a second monoclonal antibody which binds specifically to
the first, and
inoculating said mammal with an effective amount of said second
antibody to .[.bind specifically to said receptor site and block
binding of said organism thereto..]. .Iadd.immunize said mammal.
.Iaddend.
2. The method as claimed in claim 1 in which the infectious
organism is a virus.
3. The method of screening polypeptides for potential vaccine
activity against an infectious organism having a site which binds
specifically to a receptor site on a host cell, which method
comprises
providing a first monoclonal antibody binding specifically to said
organism site,
and determining whether said polypeptide binds to said
antibody.
4. The method as claimed in claim 3 in which said organism is a
virus.
5. The method as claimed in claims 3 or 4 in which the binding is
determined by measuring competitive binding of said polypeptide and
of a labelled specimen of said organism to said antibody.
6. The method of claim 4 in which the binding is determined by
measuring competitive binding to said antibody of said polypeptide
and of radiolabelled viral hemagglutinin containing a site which
binds specifically to a receptor site on a host cell.
7. The method of screening polypeptides for potential vaccine
activity against an infectious organism having a site which binds
specifically to a receptor site on a host cell, which method
comprises
providing a first monoclonal antibody binding specifically to said
organism site,
providing a second antibody binding specifically to the first,
and determining whether said polypeptide interferes with the
binding of said second antibody to said first antibody.
8. The method as claimed in claim 7 in which said organism is a
virus. .Iadd.
9. The method as claimed in claim 1 in which said second antibody
binds specifically to said receptor site and blocks binding of said
organism thereto. .Iaddend. .Iadd.10. The method as claimed in
claim 9 in which the infectious organism is a virus. .Iaddend.
.Iadd.11. The method of immunizing a host mammal against an
infectious organism having a site which binds specifically to a
receptor site on a host cell which method comprises
providing a first monoclonal antibody which binds specifically to
said organism site,
providing a second monoclonal antibody which binds specifically to
the first, and
inoculating said mammal with an effective amount of said second
antibody to serve as a surrogate antigen for the infectious
organism in providing
immunization of the host mammal. .Iaddend. .Iadd.12. The method of
immunizing a host mammal against an infectious organism having a
site which binds specifically to a receptor site on a host cell
which method comprises
providing a first monoclonal antibody which binds specifically to
said organism site,
providing a second monoclonal antibody which binds specifically to
the first, and
inoculating said mammal with an effective amount of said second
antibody to block the binding of the infectious organism to said
receptor site. .Iaddend.
Description
The invention described herein was made in the course of work under
a grant from the Department of Health and Human Services.
This invention relates to immunization of mammals against
infectious organisms having sites binding specifically to host
cells of the mammals by inoculating the mammals with a second
successive monoclonal antibody against such sites and also relates
to a method of screening polypeptides for potential vaccine
activity against such infectious organisms by determining whether
they bind to the first monoclonal antibody to the organism at the
site which binds specifically to a receptor site on a host
cell.
Infectious organisms such as bacteria, funguses, parasites, viruses
and the like function by binding specifically to mammalian host
cells, each organism having a site which binds specifically to a
complementary portion, called a receptor site, of the host cells.
It has now been found that by providing a monoclonal antibody
specific to the binding site of the organism, then providing a
second monoclonal antibody specific for the combining site of the
first, the second antibody can be used as a surrogate for the
infectious organism in providing immunization of a host mammal by
inoculation. In this case, in which a host mammal is inoculated
with the second monoclonal antibody, a decrease in infectivity of
the mammal by the organism is brought about without any contact of
the mammal with the infectious organism itself. This procedure is
of particular importance in the case of a dangerous infectious
organism such as the rabies virus. In the screening polypeptides
for potential vaccine activity, whether the polypeptide be a
synthetic polypeptide or an infectious organism of attenuated
virulence, only those polypeptides which bind specifically to the
first monoclonal antibody are capable of functioning as vaccines
against the original infectious organism.
While the methods of the present invention can be employed with any
infectious organisms such as bacteria, fungi, parasites, viruses or
the like, they are particularly applicable to viruses, including
reoviruses and rotaviruses as well as those viruses against which
it has been particularly difficult in the past to provide effective
nonvirulent vaccines, such as respiratory syncitial virus, and
dangerous viruses such as rabies.
The present invention provides a method of immunizing a host mammal
against an infectious organism having a site which binds
specifically to a receptor site on a host cell, which method
comprises providing a first monoclonal antibody which binds
specifically to said organism site, providing a second monoclonal
antibody which binds specifically to the first, and inoculating the
mammal with an effective amount of said second antibody to bind
specifically to said receptor site and block binding of said
organism thereto. The present invention also provides a method of
screening polypeptides for potential vaccine activity against an
infectious organism having a site which binds specifically to a
receptor site on a host cell, which method comprises providing a
first monoclonal antibody binding specifically to said organism
site, and determining whether said polypeptide binds specifically
to said first antibody. The extent of such polypeptide binding can
be measured by conventional immunoassay procedures. Extent of
binding can be determined directly by labelling either the unknown
polypeptide or the first monoclonal antibody of the pair and
immobilizing the other of the pair of adsorbing on a solid surface
of synthetic plastic. Extent of binding can also be determined
indirectly by immobilizing one member of the pair, for example the
first monoclonal antibody, and allowing the other, for example the
unknown, to compete with a known quantity of labelled second
antibody (whether monoclonal or not) or of labelled infectious
organism in binding to the immobilized first monoclonal antibody.
In an alternative example the second antibody (whether monoclonal
or not) is immobilized and the unknown polypeptide is allowed to
compete with a known quantity of labelled first monoclonal antibody
in binding to the second immobilized antibody.
In the drawings,
FIG. 1 is a schematic showing symbolically the preparation of the
two monoclonal antibodies and their use for screening polypeptides,
and
FIG. 2 is a graphical summary of radioimmunoassays showing
measurement of the extent of binding of the first monoclonal
antibody to viral hemagglutinin and to other polypeptides.
As represented in FIG. 1, the infectious organism, the binding site
of a virus, depicted as the viral hemagglutinin, binds specifically
to a host cell surface receptor, while the first monoclonal
antibody (MC.sub.1) also binds specifically to the same site of the
viral hemagglutinin to neutralize it. The second monoclonal
antibody (MC.sub.2) binds specifically to the same binding site of
MC.sub.1 as does the neutralization domain of the viral
hemagglutinin. Since any polypeptide to be used as a vaccine must
bind specifically to the host cell surface receptor, and since
polypeptides which are incapable of bonding to MC.sub.1 are
incapable of specific binding to the cell surface receptor,
determining extent of binding of polypeptides to MC.sub.1 is useful
for screening polypeptides as potential vaccines.
The monoclonal antibodies employed in the present invention are
made by conventional procedures. An animal such as a mouse is first
injected with the infectious organism, its spleen cells are removed
and fused with myeloma cells to form hybridoma cells, the latter
are cloned in a serum-containing medium, and the monoclonal
antibodies produced are separated from the medium. The monoclonal
antibodies are then screened by neutralization assay to select
those antibodies which bind to the site on the infected organism
which binds specifically to the receptor site on the cells of the
host, as measured by determining the decrease in infectivity of the
organism after treatment with the antibody.
The neutralizing monoclonal antibodies are then injected into other
animals such as mice, the spleen cells are removed and fused with
myeloma cells to produce second hybridoma cells which are then
cloned in a serum-containing medium to produce second monoclonal
antibodies. The second monoclonal antibodies thus produced bind
specifically to the same receptor sites on the host cells as do the
original infectious organisms.
The second monoclonal antibodies thus produced can be injected into
mammals in an amount effective to bind specifically to the receptor
sites on the host cells and block binding of the infectious
organism thereto, thus decreasing the infectivity of the organism
without any contact between the organism and the host.
The first monoclonal antibody can also be used to screen unknown or
test polypeptides (either synthetic polypeptides or live infectious
organisms of attenuated virulence) for potential utility as
vaccines, only those polypeptides which bind specifically to the
first monoclonal antibody being capable of acting as vaccines.
Any conventional immunoassay procedure can be used to determine the
extent to which the unknown polypeptides bind specifically to the
first monoclonal antibody. Among suitable procedures are the
following in which radioactive labelling is used:
1. Immobilize the first monoclonal antibody by adsorbing it on the
surface of a synthetic plastic test tube or well, and incubate in
contact with it an aqueous medium containing both the unknown
polypeptide and a quantity of the labelled infectious organism,
washing, and subjecting the tube to counting. A low count means
that the polypeptide may be an effective vaccine.
2. Immobilizing the first monoclonal antibody, then incubating it
with both the unknown polypeptide and a labelled quantity of a
second antibody (whether monoclonal or not) specific to the first
monoclonal antibody, washing, and counting. A low count means that
the polypeptide may be an effective vaccine.
3. Immobilizing the first monoclonal antibody, then incubating it
with a quantity of labelled unknown polypeptide, washing, and
counting. A low count means that the unknown is ineffective as a
vaccine.
4. Immobilizing the second antibody (whether monoclonal or not),
incubating with both the unknown polypeptide and a labelled
quantity of first monoclonal antibody, washing, and counting. A low
count means that the polypeptide may be effective as a vaccine.
5. Immobilizing the infectious organism, incubating with both the
unknown polypeptide and labelled first monoclonal antibody,
washing, and counting. A low count means that the unknown may be
effective.
6. Immobilizing the unknown polypeptide, incubating with both the
infectious organism and labelled first monoclonal antibody,
washing, and counting. A low count in this case means that the
unknown is ineffective as a vaccine.
In each of the foregoing assay procedures a standard or control
specimen known to be effective or known to be ineffective as a
vaccine may be used for purpose of comparison.
Two of the foregoing assay procedures are particularly preferred,
the second and the fourth, because each can be carried out without
the need for coming into contact with an infectious organism and
without the need for any special treatment of the unknown
polypeptide such as labelling or immobilizing it. The fifth
procedure can be used when the infectious organism can be obtained
in a pure form; it is desirable only if the organism is not
dangerous for laboratory use.
Enzyme or fluorescent labels can be used instead of radioactive
labels, as is well known. In addition, when infectious organism is
used in the assay, as for example in the first procedure described
above, there may be used only a fraction of the organism containing
a binding site which binds specifically to a receptor site on the
host cells (such as I.sup.125 labelled viral hemagglutinin).
This screening procedure, as pointed out above, can also be
effectively carried out using in place of the second monoclonal
antibody any ordinary antibody such as rabbit antibody grown by
injecting rabbits with the first monoclonal antibody and harvesting
in the usual manner. However, best results are obtained using
monoclonal antibody as the second antibody.
The screening procedure, by eliminating many potential vaccines
without the need for testing by inoculation of host mammals,
greatly facilitates development of vaccines.
The following examples will serve to illustrate more fully the
nature of the present invention without acting as a limitation upon
its scope.
EXAMPLE
Immunization of mice
Adult mice were immunized with mammalian reovirus type 3 (Dearing),
an infectious organism which results in a highly fatal encephalitis
in suckling mice. BALB/c mice were obtained from the National
Institutes of Health. The mice did not have neutralizing antibody
against reovirus at the time of the initial immunization. The mice
were immunized by intraperitoneal injection of 5.times.10.sup.7 PFU
of gradient purified type 3 reovirus (Dearing) in gelatin-saline
(0.8% NaCl, 0.003% CaCl.sub.2, 0.07% MgCl.sub.2, 6H.sub.2 O, 0.12%
H.sub.3 BO.sub.3, 0.005% Na.sub.2 B.sub.4 O.sub.7, 10H.sub.2 O,
0.3% gelatin). Injection occurred initially four months and again
one month prior to the myeloma cells fusions. Three days prior to
the fusion, the animals were inoculated intravenously with
3.times.10.sup.7 PFU of virus in gelatin-saline. The spleens of
three immunized animals were aseptically removed and the spleen
cells were dispersed by repeated flushing with Dulbecco's modified
minimal essential medium (DMEM) at 4.degree..
Myeloma Cells
The mouse myeloma cell line BALB/c P-3-NS1/1-Ag4-1 (NS1) (Kohler
and Milstein, 1976) was maintained in suspension culture in DMEM
supplemented with 7.5% heat inactivated (56.degree., 30 min.) fetal
calf serum (FCS) and 7.5% heat inactivated agamma newborn calf
serum (DMEM)-15%).
Construction of Hybridomas
The spleen cells were washed with DMEM and mixed with MS1 myeloma
cells at a ratio of 4:1. The cells were pelleted by centrifugation
and fused with 50% polyethylene glycol (PEG 1500) (Nowinski et al.,
1979, Lostrom et al., 1979). After fusion, the cells were pelleted
and resuspended in DMEM containing 20% fetal calf serum (DMEM-20%)
and were dispersed into 96 well microtiter plates (Linbro). On day
one, a half volume of DMEM-20% containing 10.sup.-4 M hypoxanthine,
4.times.10.sup.-7 M aminopterin, 1.6.times.10.sup.-5 M thymidine
and 3.times.10.sup.-6 M glycine (HAT media) was added. On day 2, 3,
6, 9, 12 and 15 half the media was removed and replaced with HAT
media. After day 15, media was replaced with DMEM-20% containing
10.sup.-4 M hypoxanthine and 1.6.times.10.sup.-5 M thymidine
(Nowinski et al., 1979). Colonies became visible between the second
and third week after initiation of the culture. Culture
supernatants were screened for anti-viral antibodies by a
radioimmunoassay procedure. The reovirus was added to polyvinyl
chloride plates (Cooke Engineering Co.) at 10.sup.9 pfu/well in 25
.mu.l PBS and allowed to air dry. The plates were then incubated
with 50 .mu.l of methanol per well until dry; and then with 5%
bovine serum albumin (BSA) for 2-3 hours. Culture fluids (25 .mu.l)
from the hybridoma cells were added to each well and kept at
37.degree. for one hour. The wells were then washed 4 times with
PBS-1% BSA (Lostrom et al., 1979). Rabbit antimouse IgG antisera
(Cappel) that had been iodinated with chloramine T (Greenwood et
al., 1963) was added (10.sup.5 cpm/well) and the plates were
incubated overnight at 4.degree.. The specific activity of the
iodinated antiserum was 1.8.times.10.sup.6 cpm/mg of protein. Wells
were washed five times with PBS, cut out, and counted in a gamma
counter.
Hybridoma Cell Cloning
Hybrid cell lines producing monoclonal antibodies, as determined by
the foregong radioimmunoassay, were cloned by dilution and growth
in 0.3% agar (Cotton et al., 1973). Colonies were allowed to grow
until readily visible by eye. They were then transferred with a
pasteur pipette to one well of a 96-well plate containing 0.1 ml of
IMEMZO (improved minimal essential media supplemented with zinc,
insulin, and Hepes buffer) media (International Biological
Laboratories, Inc.) containing 15% FCS. Cells were grown up to mass
culture and subsequently maintained in IMEMZO containing 5% heat
inactivated fetal calf serum and 5% agamma newborn calf serum.
Isolation of First Monoclonal IgG Antibody
Cloned hybridoma cell cultures, grown up to a 400 ml volume by
daily feedings with fresh media were maintained at this volume for
48 hours and the cells were removed by centrifugation at 400 g for
20 min. The supernatants were removed and phenylmethylsulfonyl
fluoride (PMSF) was added to a concentration of 100 .mu.g/ml. The
supernatant was applied to a 3 ml bed volume column of protein
A-Sepharose CL-4B (Sigma) at a flow rate of approximately 25 ml/hr
(Lee et al., 1981). The column was washed with PBS until no protein
was detectable in the eluent and the IgG was eluted with 0.1M
glycine-HCl (pH 2.5). The fractions containing IgG were pooled (4
ml) and dialyzed against PBS (4 liters). Aliquots were stored at
-20.degree.. All antibodies were used at a concentration of 1-2
mg/ml.
Neutralization Assays
Virus preparation were diluted to 2.times.10.sup.3 PFU/ml in
gelatin-saline. Purified and isolated hybridoma IgGs prepared as
described were diluted serially in 5-fold steps in gelatin-saline
and 0.2 ml of the IgG dilutions was mixed with 0.2 ml of the virus.
The neutralization was carried out at 34.degree. for 1 hr (Weiner
and Fields, 1977). Duplicate 0.1 ml samples were then plated in 6
well cluster dishes (Costar) and allowed to adsorb for 1 hr. The
cells were then overlaid with agar and the plaque assays were
performed as reported previously (Fields and Joklik, 1969). The
neutralization titer is expressed as the higest dilution of
antibody which neutralized 80% of the input virus (Weiner and
Fields, 1977).
The desired first monoclonal antibody, designated as G5, exhibited
no detectable neutralization of reoviruses of serotype 1 (Lang) or
2 (Jones) at a 1:10 dilution of antibody, but exhibited effective
neutralization titer of 12,500 against the reovirus serotype 3
(Dearing) which had been used to immunize the mice, as well as
neutralization of additional type 3 isolates from natural sources
(Hrdy et al., 1979). The antibody subclass was determined by
ouchterlony analysis to be IgG.sub.2a. It was determined to be
specific to the viral .sigma.-1 protein, a viral outer capsid
protein of serotype 3 by immunoprecipitation of .sup.35
S-methionine labelled infected cell lysates, and thus was specific
to the site which has been well known to be involved in binding the
virus to host cell receptors.
Isolation of Second Monoclonal IgG Antibody
The development and screening of the second monoclonal antibody
involved the immunization of BALB/c mice with purified G5
monoclonal antibodies in complete Freund's adjuvant. 100 .mu.g of
G.sub.5 monoclonal antibody was used to prime and then boost three
weeks later the same mouse. Five days later the spleen was removed
and splenocytes subjected to fusion using the same procedure as
described above for construction of hybridomas except that another
generally available mouse myeloma cell line (653) was used in place
of NS1. Culture conditions were the same. Positive wells were
determined by their ability to secrete antibody which would
selectively bind the G5 monoclonal antibody fixed to a polyvinyl
chloride plate as described above. Controls used included 653
supernatant and irrelevant monoclonals of the same isotype and
allotype. Positive clones were those which were found to
selectively bind G5; They were then shown to bind to the
hemagglutinin binding site of reovirus on a variety of somatic cell
lines including the R1.1 (H-2.sup.k) thymoma line, the R1.E
(H-2.sup.k thymoma line), human T cells but not YAC
(H-2.sup..alpha.) lymphoma cells. All of these lines bind or do not
bind the hemagglutinin (HA) in the same manner as the second
monoclonal antibody. In this manner a G5 binding monoclonal
antibody (anti-G5 antibody) was selected which could selectively
bind to sites on cells which were themselves structured to bind
specifically the HA of reovirus.
Competition of Second Monoclonal Antibody With Virus in
Infectivity
The anti-G5 antibody was then evaluated to examine its ability to
compete for virus in an infectivity assay. BW 5147 AKR lymphoma
cells were plated in linbro wells at a density of 2.times.10.sup.6
cells/well. The BW 5147 cell expresses the receptor for the
neutralization domain of the HA. Consequently the BW 5147 cell will
bind to reovirus and become infected with reovirus. The BW 5147
cell also binds the anti-G5 monoclonal antibody. Incubating
1.times.10.sup.7 plaque forming units (pfu) of reovirus with the BW
5147 cells led to large numbers >10.sup.7 pfu infections virus
recoverable from 100% of these cells several days later. Incubation
of the virus, the BW 5147 cells and the monoclonal anti-G5 antibody
(100 82 l of ascites) resulted in 0% of infected cells. Thus
protection is absolute for local infections in tissue culture.
Use of Second Monoclonal Antibody As Immunization Agents
Hybridoma cells bearing the Anti-G5 antibody prepared as described
above were used to prime naive BALB/c mice. The spleens of these
mice were evaluated for cyto-toxic activity against reovirus
infected targets after in vitro restimulation for 5 days at
37.degree. C. with reovirus infected stimulator cells. The cells
were cultured in 2 ml of RPMI 1640 medium (M.A. Bio-products,
Walkersville, Md.) supplemented with 5% fetal calf serum, 100
.mu./ml penicillin, 100 .mu.g/ml streptomycin and 5.times.10.sup.-5
M 2-mercaptoethanol in 16 mm wells (Linbro Scientific, Hamden,
Conn.) in a 5% CO.sub.2 humidified air atmosphere. Cell density was
10.sup.6 responders/10.sup.5 stimulators. In vitro stimulation of
spleen cells with infected stimulators but without in vivo priming
does not produce cytotoxic activity (.ltorsim.5%). Anti-G5 primed
mice developed significant (.gtorsim.30%) cytotoxicity at an
effector target ratio of 100:1, when assayed on reovirus infected
target cells. The targets were reovirus infected L cells and a
standard Cr.sup.51 release assay was used. Thus the anti-G5
antibody is completely protective for local infection and can
readily prime for reovirus specific CTL.
Screening of Polypeptides
In providing a screening for determining potential vaccine activity
of polypeptides, it was first demonstrated that the first
monoclonal antibody (G5 ) could be immobilized while retaining its
ability to bind specifically to infectious organism, i.e., to the
hemaglutinin portion (HA) of reovirus type 3, the hemagglutinin
containing the binding site which is capable of binding
specifically to the receptor on the host cell. There was used
purified HA isolated from reovirus type 3 infected cell lysates.
The HA was affinity-purified by passage through Sepharose 4B beads
to which G5 was coupled. The HA was selectively eluted from the
beads by acid, pH 2.8 glycine HCl. A radioimmunoassay was developed
in which the binding of G5 antibody to radiolabelled HA was
measured. Purified hemagglutinin protein (HA) from reovirus type 3
was radiolabelled with I.sup.125 by the chloramine-T method to a
specific activity of 3000 cpm/ng. As a control antibody, there was
used a monoclonal antibody 4S produced from an anti-Sendai virus B
cell hybridoma which secretes an anti-Sendai antibody of the same
isotype as anti-G5 (IgM). Monoclonal antibody G5 or control
monoclonal antibody 4S were partially purified from culture
supernatants from hybridoma cell lines and adsorbed onto the bottom
of polyvinyl microtitre plates (Cooke, Dynatech) as previously
described. After incubation with phosphate buffered saline (PBS)
containing 10 mg/ml of bovine serum albumin (BSA) to saturate
unbound sites on the plastic adsorbent, 10 ng of radiolabelled HA
in 20 .mu.l PBS were then added to each well for 60 minutes
incubation at room temperature. Following extensive washing and
drying, well bottoms were removed and counted in a Beckman 4000
gamma counter; determinations represent triplicate
means.+-.standard error. The results are shown in FIG. 2A of the
drawing.
The extent of binding of several different polypeptides to G5 was
measured by immobilizing the G5 , then incubating it with both a
polypeptide and with labelled HA. The G5 was immobilized by
adsorbing 250 ng of G5 onto the bottom of each well of a microtitre
plate and incubating with BSA as described above. Test antisera,
either normal sera, antibodies to antibodies to reovirus type 1
(anti-anti-R1), or antibodies to mouse antibodies to reovirus type
3 (anti-G5 ), were then incubated in the wells for 60 minutes at
room temperature. After extensive washing, 10 ng of radiolabelled
HA in 20 .mu.l PBS was added and the assay performed as before,
with the results shown in FIG. 2B. As is seen, only the potential
vaccine anti-G5 bound to the G5 , as shown by the decreased count,
whereas the other polypeptides, normal sera and the anti-anti-R1
did not. Stated another way, the decrease in the count for anti-G5
antibody measures the extent to which the binding of G5 to anti-G5
is disrupted by competition of the labelled HA with the anti-G5 .
Conversely, the HA can be immobilized by adsorption, incubated with
the test serum and with labelled G5 antibody; again, a decrease in
count indicates that the test serum is a potential vaccine.
By substituting other polypeptides, such as attenuated virus or
synthetic polypeptides, for the normal sera or anti-anti-R1 in the
foregoing assay, their potential effectiveness as vaccines against
reovirus type 3 can be measured.
* * * * *