Note: Descriptions are shown in the official language in which they were submitted.
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The present invention relates to immunoassay reagents, kits and
methods.
More particularly the present invention relates to a novel
reagent for binding and detection of antibodies and to kits and
methods incorporating the same.
Specific antibody binding assay methods, also called immunoassay
methods, are a rapidly emerging analytical technique, most widely used
in diagnosis and research, owing to their high specificity and
sensitivity. The earlier versions of this technique which used
radioisotopic labels have in recent years been increasingly replaced
with enzyme immunoassay(EIA) techniques using enzyme labels, which
techniques are equally sensitive but safer, simpler and cheaper.
.
Among the EIA techniques there are commonly used the so-called
"heterogenous" assay methods employing enzyme-labeled antibodies or
antigens attached to ("immobilized on") sensitized surfaces of solid
carriers, such as test tubes, polystyrene beads or plates provided
with recessed "wells". Such techniques are generally ~e~erred to by
the abbreviation "ELISA" which stands for "enzyme-linked immunosorbent
assay". In accordance with one modification, this technique is
combined with the known use of a so-called "second antibody", namely
an antiserum to the specific "first" antibody. The second antibody
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serves as a non-specific detector for any "first antibody" either in
the Free state or when bound to its specific antigen in an antigen-
antibody complex.
In accordance with one version of the above methodi which reactsalso to the known "coated tube" technique, an antigen is attached to
the inner wall surface of a test tube in which the binding reaction is
then carried out, whereupon the free specific antibody to said antigen
present in the test sample will be attached to the surface of the tube
via the antigen immobilized thereon. Thereafter the liquid reaction
mixture is removed from the test tube, the latter is washed and a
second solution containing an enzyme-labeled second antibody is
introduced into the tube. This labeled second antibody will also be
immobilized by attaching itself to any antibody which is bound to the
surface of the tube via the antigen. The test tube is again emptied,
rinsed and filled with a suitable substrate responsive to the enzyme~
label of the second antibody and the enzymatic activity is measurede
This activity is directly proportional to the antibody concentration
in the test sa~ple.
In recent years it has been found that the ~"second antibody" in
immunoassay techniques can be replaced by a certain protein, namely
the so-called "protein A" which is a major cell wall component of many
strains of Staphylococcus aureus. This protein A is covalently linked
to the cell wall of the staphylococci but can be solubilized and ~is
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capable of binding many classes of immunoglobulin molecules, with high
affinity. It thus also binds antibody-antigen complexes. The
solubilized protein A can, furthermore, be labeled, e.g. with radio-
iodine or with enzymes, and enzyme-labeled protein A preparations are
now becoming an increasingly popular component of immunoassay kits, in
which they serve as the detector.
More recently Lars Bjorck et al. have described in The Journal of
Immunology Vol. 133 No. 2 Aug. 1984 pp. 969-974 their ~indings with
regard to the purification and properties of Streptococcal Protein G,
as a novel IgG-binding receptor.
As described therein Protein G, a bacterial cell wall protein
with affinity for immunoglobulin G(IgG) was isolated from a human
group G streptococcal strain (G148) and found to bind all human IgG
subclasses and also rabbit, mouse and goat IgG.
. . .
In a further article by Bo Akerstrom et al, in The Journal of
Immunology Vol. 135 No. 4 Oct. 1985 pp. 2589-2592 the avidity of
protein G for various monoclonal and polyclonal Ig of the G class was
studied as well as the use of radiolabeled proteln G for binding and
: detection of antibodies~
The present invention constitutes a yet further important
improvement of the abovementioned ELISA, protein A and protein G
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techniques and is based on the inventive concept that
the enzyme-labeled protein A and radiolabeled protein G
can be replaced by intact non-viable stabilized bacter-
ial cells having an active receptor for immunoglobulin
and an active marker enzyme, which have undergone an
appropriate pre-treatment. Thu~, e.g., staphylococcal
cells can effectively and rapidly bind immunoglobulin
(such as any specific "first antibody") by virtue of the
pxotein A in their cell walls and G streptococcal cells
can similarly bind immunoglobulin by virtue of the
protein G in their cell walls. At the same timP the
enzyme-label is provided by enzymes which are naturally
present in the cells (i.e. the endogenous or auto-
chthonous enzymes), the activity of which can be
readily measured, or in especially preferred embodiments
of the present invention, as described hereinafter, the
enzyme label or marker is the result of a gene for said
enzyme having been inserted in a parental cell thereof.
Thus, the present invention in one aspect, provide~
a method of immunoassay for binding and detecting anti-
bodies which u~es both an active receptor for an
immunoglobulin, and an endogenous active marker enzyme
as a label, both being present in non~viable stabilized
bacterial cells and both having been heritable and
inherently formed therein befora stabilization.
In a first preferred embodiment of the present
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invention said reagent compris~s non-viable stabilized
staphylococcus cells.
In another preferred embodiment o~ the present
invention said reagent comprise~ non-viable stabilized
5treptococcus cells.
The reagent used in accordance with th~ present
invention, namely a suspension of appropriately treated
bacterial cells, has considerable advantages as a
"detector suspension" over all known detectors of
antibodies or their complexes, in that it obviates the
work~ time and costs involved in:
(a) isolating the binding protein or preparing
anti-immunoglobulins;
(b) preparing the enzyme to be used as label:
(c) linking the enzyme-label to the binding
protein - a notoriously wasteful ~tep, and/or
(d) radiolabeling of proteins.
The present invention also provides a test kit for
enzyme immunoassay according to the above-described
method comprising one or more containers holding an
aqueous suspension of non-viable stabilized bacterial
c~lls having an active receptor for i~munoglobulin and
an endogenous, heritable active marker enzyme formed and
produced by bacterial cells, or a dry s~abilized
preparation o~ such cells, suitable for in-situ
reconstitution.
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The test kit for carrying out immunoassay may
further comprise, in a packaged combination, one or more
solid carriers having ~ixed on at least a portion of the
surface thereon a capture antibody or a hapten or
antigen the specific antibody to which is to be assayed,
one or more containers holding standardized solutions of
a speci~ic antibody to the hapten or antigen which i5 to
be assayed and/or an adsorbant reagent carrier
incorporating therein a reagent specific to the
endogenous active marker enzyme.
In accordance with one embodiment of the
immunoassay method, there is provided a heterogenous
specific binding assay m~thod for determining an
unbound antibody in a liquid medium, comprising the
steps of:
i) incubating a sample of the liquid medium in
contact with a solid carrier having fixed on
its surface an antigen or hapten specific to
: the antibody;
ii) separating the solid carrier from the liquid
medium sample and rinsing it with aqueous
solution to remove all traces o~ the sample;
iii~ incubating the solid carrier in contact with
an aqueous suspension of non-viable stabilized
bacterial cells having an endogenou~ active
marker enzyme, the enzyme having been
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heritabl~ and inherently formed in the cells
before stabilization;
iv) separating the solid carrier from the
suspension and washin~ it with aqueous
solution; and
v) incubating the solid carrier in contact with a
suitable substrate and assaying for enz~matic
activity of the endogenous active marker
enzyme on cells retained on the solid carrier
surface.
The specific binding assay method according to the
invention can be applied to the determination o~ a
hapten or antigen in a liquid medium by adding to a
sample of said liquid medium an antibody specific to the
hapten or antigen which is to be determined and
incubating the reaction mixture in contact with a solid
carrier having ~ixed on its sur~ace the same hapten or
antigen which is to be assayed wherein in step (i) a
: specific antibody to said hapten or antigen is added to
the test sample, and as the result of binding of said
hapten or antigen in said sample, the amount o~ antibody
available for binding ~o the hapten or antigen fixed on
the solid sur~ace is proportionately reduced. The
above-described steps ii) to v) above are then carried
out and the enzymatic activity which is determined in
step v~ is inversely proportional to the concentration
of the hapten or antigen in the sample~ ~his
8a
concentration can be calculated by methods well known
in immunoassay techniques, e.g. by comparison with a
series reference samples having known standard
concentrations of the hapten or antigen.
The detector cell suspension of the present
invention can be similarly used in "sandwich" type
immunoassays, namely in assays where a "capture
antibody" is immobilized on a solid surface so as to
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capture antigen present in the test sample, and a "tracer" antibody to
that antigen is then applied to form a "sandwich". The binding o~ the
"tracer" antibody is conventionally revealed by a label incorporated
in that antibody molecule.
In the present method the detector suspension can be used to
eliminate the need for labeling the second antibody. The only
requirement is that the detector cells must not bind to the capture
antibody. This requirement can be met by using antibodies which do not
bind~Protein A. (e.g. IgY from chicken serum or from yolk) or modified
antibody in which the Fc segment is missing (e.g. Fab or F(ab')2).
Selective binding to the second antibody can often be observed
with conventional antibody preparatlons, indicating that the capture
antibody is fixed in a configuration which makes the Protein A binding
moiety (Fc segment) of the immunoglobulin unavailable for binding of
the detector cell.
A test kit adapted for carrying out the assay method according to
the invention for determining a hapten or antigen in a liquid medium
may additionally comprise one or more containers holdiny standardized~
solutions of a specific antibody to said hapten or antigen which is to
be assayed~ The test kits according to the invention optionally
further comprise packaged auxiliary reagents or solutions for carrylng
out the assay method, such as buffer solutions, standardized referer)ce
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solutions of the analyte to be determined~ substrate solutions and
detector reagents for measuring the activity of the endogeneous enzyme
serving as label, etc. Such test kits which comprise a lyophilized
stabilized preparation of Staphylococcus aureus cells may also
additionally comprise packaged aqueous medium ~or forming the required
aqueous suspension of said cells in situ.
In preferred embodiments of the present invention there is also
provided a test kit additionally comprising an absorbent reagent
carrier incorporating thereon a reagent specific to said marker
enzyme.
As stated above9 the staphylococcal cells of the present
invention are appropriately treated before use in order to ensure that
the cell suspension is safe ti.e. non-pathogenic), standardized and
stable whilst preserving its binding capacity and the activity of the
marker enzyme which is selected to serve as the label~ It has been
found that these requirements can be met, e.g. by fixing the
staphylococcal cells by treatment with 0.5% formaldehyde solution at
room temperature for 2 hours followed by heating for about 5-15 mins.
at about 50-70C~ The treated cells can be preserved, e.g., as a 10%
suspension with a preservative such as sodium azide at ~C or as a
freeze-dried pellet of staphylococcal cells stored in sealed ampoules~
Alternatively, a safe and stable reagent can be obtained by freeze-
drying a suspension of staphylococcal cells and storing the product in
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3L~9~
sealed ampoulesO The freezedried cells can be
reconstituted in situ for use by suspending them in a
suitable liguid medium.
It is to be noted that while stabilized
staphylococcal cells are commercially available the
standard treatment for stabilization thereof normally
inactivates any endogenous enzymes found thereon and
thus non-viable stabilized staphylococcal cells having
an active receptor for immunoglobulin and an active
marker enzyme heretofore have not been available and
also have not been suqgested for use as immunoassay
reagents.
In one embodiment of the present invention said
marker enzyme is endogenous catalase and tha catalase
activity of the staphylococcal cells is assayed.
In especially preferred embodiments o~ the present
invention there are used a~ reagents non-viable
stabilized staphylococcal cells having an active
receptor for immunoglobulin and an act~ve marker enzyma,
the gene for said enzyme having been inserted into a
parental cell thereof by genetic engineering procedures
known per se.
Thus for use in the present invention there wa~
prepared a genetically engineered strain o~ the Cowan
staphylococcus which differs from the original strain in
having a gene for the formation of B-lactamase.
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12
The general advantage of using this approach o~
inserting a new gene is in not having to rely on enzyme
markers which happen to be present in the original
strain. Thus there can be added to the existing
repertoire o~ the endogenous enzymes a new heritable
marker which is more suitable for immunoassay.
The specific advantage~ of the gene for ~-lactamase
are in that it fulfills all of the following
requirements:
1. The enzyme is largely cell~ound.
2. The enzyme is fully accessible to the
substrate.
3. The enzyme is very stable and its activity can
be well preserved in the processing of the
cells.
4. The enzyme has a very high turnover rate~
5. The substrate i~ stable, inexpensive and its
use involves no health hazards or safety
precautions.
6. The assay of the B-lactamase reaction is
simple, rapid and very sensitive.
7. The assay require~ no instrumentation and a
permanent record of the results can be
retained without any additional manipulations.
~s indicated hereinbefore several types of
Streptococcus pyoqenes are known to produce cell wall
proteins which can serve as receptors for the Fc portion
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o~ immunoglobulins. The receptor specificity of such
proteins may vary with the type of the producing
strain. Of
12~37:~
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special interest are streptococci of types C and G which produce
powerFul receptors of broad specificity. Such bacterial cells can be
used as reagents in a manner analogous to that described for the Cowan
I cells. The following differences must be noted, however:
1. The broader speciFicity of streptococcal Fc receptors allows
the extension of the present methodology to the detection of
immunoglobulins which do not adequately bind Protein A.
2. The streptococcal cells have no catalase, but have a rich
variety of other cell bound enzymes including hemolysins,
which can serve as endogenous indicators of binding to the
immunoglobulin molecule.
3. In analogy to the genetic modification of the Cowan strain, as
described above, insertion of suitable genes will provide
additional enzyme labels which may be more advantageous in
immunoassay diagnostic kits.
From the aforementioned, it will be obvious that streptococcal
cells possessing Fc receptors can by themselves serve as a source of
reagent for detecting a variety of immunoglobulins. When preferable,
such cells can be used in combination with the Cowan reagent described
before. The combined use of both types of cells may take one oF
several forms:
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1. The reagent may consist of a mixture of cells
of both typesO Each type may carry its own
distinctive enzyme label or a single assay may
be used if a similar enzyme activity is chosen
as a label for both cell types.
2. The mixed cells are linked together (e.g. with
glutaraldehyde) to form a combined reagent,
which now carries a single enzyme label (e.g.
B-lactamase) but two kinds of Fc receptors.
lo 3. A combined reagent is formed through
interaction with immunoglobulins present in
the assay or deliberately added for that
purpose.
While the invention will now be described in
connection with certain preferred embodiments in the
following examples so that aspects thereo~ may be more
fully understood and appreciated, it i5 not intended to
limit the invention to these particular embodiments. On
the contrary, it is intended to cover all alternatives,
modifications and equi~alents as may be included within
the scope of the invention as de~ined by the appended
claims. Thus, the ~ollowing examples which include
preferred embodiments will serve to illustrate tha
practice of this invention, it being understood that
the particulars shown are by way of example and for
purposes of illustrative discussion o~ preferred
embodiments of the present invention only and are
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presented in the cause of providing what is believed to
be the most useful and readily understood description o~
formulation procedures as well as of the principles and
conceptual aspects of the invention.
EXAMPLE 1
Pre~aration of stabilized suspension of Staphylococcus
aureus cell~
Staphylococcus aureus (strain Cowan I, ATCC), was
grown in TSB (Trypticase Soy Broth, Difco), in 500 ml
Erlenmeyer flasks, on a reciprocal shaker, for 18 hours
at 37C. The cells were then spun down (10 min at 10,000
RPM), washed twice with phosphate buffered saline (PBS),
pH 7.3 and suspended in 10 volumes of PBS (pH 7.3)
containing 0.5% formaldehyde. After 2 hours at room
temperature the cells were spun down and washed as
before, and then suspended in 10 volumes o~ PBS (pH
7.3). The suspension wa~ incubated for lQ minutes at
60C under gentle shaking, spun as before and
resuspended in 10 volume~ of P~S (pH 7.3). Samples of
that suspension, streaked on AB3(Difco) plates, failed
to show growth after 24 hours at 37C. After 12 months
storage at 4~C the suspension showed no 3ign5 of change
in binding and catalytic properties. Before use the
suspension was diluted 1:10 in PBS.
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EXAMPLE 2
Preparation of freeze-dried stab;lized Staph,ylococcus au~ c~a~
Staphylococcus aureus (strain Cowan I, ATCC) was cultivated and
harvested as described in ~xmaple 1 above. The cells were washed with
PBS, resuspended in 10 volumes of fresh TSB~ inc~bated for 10 minutes
at 60C and dispensed in 0.5 ml portions into lyophili~ation-ampoules.
After freeze-drying the sealed ampoules were stored at room
temperature t22-25C). For use, the contents of one ampoule were
suspended in 5.0 ml PBS containing 0.01% merthiolate.
EXAMPLE 3
Detection of Rabbit anti-BSA
Microtitr~e plates (U type, Nunc) wre activated by filling the
wells with 100 1 of 0.2% glutaraldehyde solution in bor~te buffer, pH
9.0, and incubation for 3 hours at 56C. The plates were then washed
10 times with twice-distilled water and coated with antigen by filling
the wells with 100 1 of bovine serum albumin (BSA) (50 g/ml),
incubating for 2 hours at 37C and storing at 4C for 18 hours. After
3 washes with PBS the unoccupied surfaces were blocked by filling the
wells with 100 1 of ovalbumin (1.0 mg/ml) and incubating for 2 hours
at 37C, followed by 3 washes with PBS.
Control wells were prepared as above with the BSA being omitted.
~' .
Antibody to BSA (Anti-BSA) was prepared in rabbits~ Normal rabbit
serum (NRS) served as a non-specific control. Serial dilutions were
17
made in PBS, and 100 ~1 of each were added to the
antigen coated wells. After 30 minutes incubation at
37C the wells were washed thrice with PBS. For
detection o~ the antibody retained by the antigen, an
ali~uot (100 ~1) of a staphylococcal cell suspension
- (prepared in accordance with Examples 1 and 2 above) was
added to each well. After 20 minutes incubation at 37-C
the wells were washed once with 1% Tween* 20 and twice
with PBS. The retention of the cells by the bound
antibody was detected by acid phosphatase or catalase
activity, in accordance with the method described in
Israeli Patent Specification No. 36496.
~SS~E
The test system dsscribed above was used to compare
the sensitivity o~ the detectors, i.e., the
staphylococcal cell suspensions prepared in accordance
with Examples 1 and 2 above with the sensitivity of a
radioiodinated protein A preparation, conventionally
used in sensitive solid-phase radioimmunoassays. The
results are summarized in Table I.
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~E~
Detector
prepared Anti-BSA ~nti-BSA Anti-BSA NRS
according to1:1000 1:3QOO 1:10000 1:1000
Example 1* Pos. Pos. Pos. Neg.
Example 2* Pos. Pos. Pos. Neg.
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10 Radioiodinated
Protein A Pos. Pos. NegO Neg.
(*) Based on catalase activity displayed 5 minutes
after th~ addition of the substrate, namely 50 1 of a 6%
hydrogen peroxide solution.
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EXAMPLE 4
A gene for constitutive penicillinase Pormation
(pen I-) was inserted into the Cowan I strain of
Staphylococcus aureus. The gene originated in a plasmid
(PI258 pen I443) hosted by strain (RN 453) of
Staphylococcus au~eus (Novick. R.P., and Brodsky, R.,
1972)~ Studies on plasmid replication: I. Replication
of unestablished plasmids in S ~ureu~. J.Mol.Biol 68:
285-302), and was transduced with phage o 11. The
transduction was as des~ribed by Novick ~Novick, R.P.,
1967). Properties of a cryptic high frequency
transducing phage in StaPhylococcus aureus; Virology
33:155-166. A stabilized suspension of thes~ cells
having B-lactamase as their marker enzyme was then
prepared by the procedure of Example 1.
EXAMPLE S
Detection of an~ibodies to ~rucella i~ bovine sera
A polystyrene Petri dish (standard size, Miniplate)
was activated at 8 preselected, equidistant spots by
20- placing 50 ~1 of a 25% solution o~ glutaraldehyde
(Merck) at each spot. Aftsr 150 sec at 24C the dish
was rinsed with water and 10 ~1 of the antigen
suspension wa added to each spot. The antigen
suspension consisted of heat-killed (90 min at 72C
cells o~ B~ucella abortus, suspended in PS (0~5% phenol
and 0.85% NaCl in distilled water) at optical density
corresponding to 200 Klett Units. The spots were
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extensively rinsed and the Petri dish overlayed with 5
ml of a blocking solution consisting of BSA (20 mg/ml)
in PBS. After 2 hrs at 37~ and 16 hre at 4 the
blocking solution was poured o~f and the Petri dish was
rinsed twice with PBS, then twice with O.05% ~ween* 20
in PBS and again with PB~. The dish was then air-dried
and 20 ~1 samples of bovine sera, diluted as indicated
in Table 2, were added to the marked spots. After 60 min
at 37, the dish was rinsed and dried as before, and
each spot received 20 ~1 o~ a detector suspe~sion. The
detector suspension consisted of the genetically
modified staphylococcal cells of example 4 suspended in
PBS at OD corresponding to 200 XU. After rinsing and
air drying the B-lactamase activity of the det~ctor
cells retained on the spots was tested by placing on
each spot a developer ætrip. The dQveloper strip was a
section (12x15 mm) of Whatman No. 3 filter paper
impregnated with a reagent solution containing iodine
(12 mM), potassium iodide (63 mM), soluble starch (1.0%
w/v) and penicillin (50 mM) in P~S. The activity o~ the
marker enzyme of the d~tector cells, namely B-lactamase,
was assayed by determining the time required for the
appearance o~ a white circle in the center of the
blue-hlack detector strip. The white circle indicated
that iodine has been taken up by penicilloic acid and
thus removed from the complex which gave the dark color
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20a
to the detector strip. The penicilloic acid is the
product of hydrolysis of a ~-lactam (here penicillin)
catalysed by B-lactama~e. Hence the rate o~ *he
decolorization of the circular area above the marked
spot is inversely proportional to the amount of B-
lactamase retained on that spot.
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TABLE 2
Serum Agglutination Complement Present
No. Titre* Fixation Test
Titre* Titre*~
2 1:10 1:5 1:200
6 1:20 1:5 1 200
32 Neg Neg l:1000
53 1:80 1:5 l:1000
77 Neg Neg Neg
26 1:40 1:50 1:2000
111 1:1250 - 1:16000
Normal Neg Neg Neg
Bovine Serum
.
* As determined at the Kimron Veterinary Institute, Beth Dagan,
- Israel
** The highest dilution (in PBS) which showed decolorization in ~ 10
mi n.
- It will be evident to those skilled in the art that the invention
: is not limited to the details of the foregoing illustrative examples
and that the present invention may be embodied in other specific forms
without departing from the essential attributes thereof, and it is
therefore desired that the present embodiments and examples be
considered in all respects as illustrative and not restrictive,
reference being made to the appended claims, rather than to the fore-
going description, and all changes which come within the meaning and
range of equivalency of the claims are therefore intended to be
embraced ~herein. ~ :
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