Note: Descriptions are shown in the official language in which they were submitted.
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This invention relates to a system for diagnosing
microbial diseases in humans, animals and plants, and
in particular for measuring the amount of antibodies
produced as a result thereof~
The most common method used for the diagnos is of
certain types of viral disease is the 'haemagglutination-
inhibition (H.I.) test'. This test has the disadvantage
that it depends on the fact that certain viruses will only
agglutinate,certain types of erythrocytes, e.g. influen~a,
mumps and parainfluenza viruses,agglutinate chicken, human and
guinea-pig erythrocytes adenoviruses rat and monkey
erythrocytes, and reoviruses and many enteroviruses only human
erythrocytes. The test is performed by mixing virus with
the appropriate erythrocytes in the presence of patientls
serum, i.e. the fluid which is expressed when blood coagulates
to form a clo~. If antibodies ,to the virus are present
in the serum, the virus will be unable to agglutinate the ',~
cells. The haemagglutination inhibition t~st has the further
disadvantage that it is tedious and not accura~e as
doubling dilution series need to be used with the consequent
loss of accuracy, w~ich makes it impossible to detect
small differences in antibody titre. Furthermore the !sera
must be pretreated to remove non-specific inhibitors that
prevent the virus binding to the erythrocyte and therefore
result in a false positive reading of the test. , ''
Recently a new test for detecting influenza virus
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antibodies, called the 'single radial-diffusion test~
has been proposed by SChild et al. (Symp. Series
Immunobiol. Standard, Vol. 20, pp, 39-46) in which the
appropriate influenza virus (67 to 100 ~g purified virus
particles/ml gel) is incorporated into agarose gel.
When the gel has set circular wells are cut out and the
patient's or animal's serum to be tested is introduced into
the wells. The presence in the test serum of viral
antibodies is detected by the appearance of zones of opale-
scence surrounding the wells. The opalescence i9 caused b~
the diffraction of light owing to a halo of antibody
molecules surrounding each virus particle.
The single radial diffusion test has better resolu-
tion than the H.I. method and is quicker to perform.
However, it does have the disadvantage that the influenza
virus preparation used for the purpose has to be purified
and highly concentrated in the gel, which makes the test -
expensive. Additionally large amounts of virus are required
for the test.
It has now been found that by incorporating
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~ into a gel erythrocytes which have microbe particles
;~ adhering to their surfaces, allowing a sample of body fluid~
` which may contain specific antibodies to the microbe to
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' diffuse into the gel, introducing complement into the
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system, which is fixed, i.e. co~bines, where specific
antibodies have interacted with the microbe particles, a
visible circular zone of lysis will result, the size of which
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is dependent on the amount of antibody presentO Lysis is
the rupture ofanerythrocyte with the resultant liberation
of haemoglobin.
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sroadly the invention provides a system for detecting
the presence of specific microbiol antibodies in body fluids
such as blood, serum, plasma, nasal washings, cerebrospinal
fluid, saliva or milk, which comprises a gel containing in-tact
- erythrocytes having appropriate microbe particles adhering
to the surface thereof. In particular, the system as herein-
before defined also comprises a complement.
In accordance with one aspect of the invention there
is provided aspecific viral antibody detecting diagnostic
system suitable for measuring the amount of antibodies in
body fluids comprising a thin layer of gel containing intact
erythrocytes having appropriate viruses adhering permanently
to the surface thereof.
In anotheraspect of the invention there is provided
a method of preparing a specific viral antibody detecting a
diaynostic system suitable for measuring the amount of
antibodies in body fluids which method comprises mixing intact
erythrocytes with appropriate viruses in order that they become
permanently attached to the erythrocytes, incorporating the
resultant suspension into a gel, and allowing the gel to solidify.
The gel that is used is preferably made from 0.5 to 2.0%
agarose but agar, gelatin or any suitable matrix which allows
diffusion and does not have anti-complement activity can alter-
natively be used. The gel concentration should preferably be
strong enough so that it is easy to handle, but not so concentrated
that diffusion cannot take place.
The erythrocytes may be obtained by venipuncture of a
vertebrate animal and collected in a medium that prevents clotting.
Several types of erythrocytes may suitably be used for the pur-
poses of the present invention, for instance bovine erythrocytescan be used with certain virus. Sheep erythrocytes, are however,
usually used, however they have the disadvantage of having the
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Forssman erythrocytes antigen on their surface, and therefore
the fluid to be tested may be treated prior to testing with
sheep erythrocytes at 37C for 30 minutes in order to absorb
antibodies to the Forssman antigen.
Other types may be preferred in association with
particular microbes, for example when measles virus is
being used Patas Monkey erythrocytes are usually very convenient
and preferred although certain erythrocytes can be too fragile
;~ or larger than necessary causing less defined zones of lysis~
: 10 On storage erythrocytes tend
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to become more fragile with age and more susceptible to
spontaneous lysis, therefore in order to prolong the 'life'
of the erythrocytes chemical substances may be incorporated
into the gel in order to stabilize the cells. Alternatively
the erythrocytes may be pretreated with a chemical substance
such as gluteraldehyde, in order to stabilise them.
Another method of 'prolonging the life' of the
erythrocytes is to incorporate dimethylsulphoxide (DMSO) into the
gel at the pouring stage and then freeze the gel in the vapour
above liquid nitrogen. By incorporating about 10% DMSO the
integrity of the gel is maintained.
If the microbe particles to be used in the system are
viruses then these may be prepared by conventional techniques
from animals, egg or tissue culturé, see for example 'Textbook
of Virology' by A.J~ Rhodes and C.E. Van Rooyan, 5th Edition
published by Williams and Wilkins,Baltimore, U.S.A. 1968.
With certain types of viruses the erythrocytes require
pretreatment with a chemical substance that promotes the binding
of the virus particles to the erythrocytes surface, for example,
influenza virus will agglùt'inateerythrocytes but this is
followed by elution at 37C, i.e. the virus particles will soon
detach themselves from the erythrocytes spontaneously, owing to
the action of the viral enzyme neuraminidase on the erythrocytes
receptors~ In order to ensure that the influenza virus remains
permanently attached to the erythrocytes, they may be pretreated
with potassium periodate solution, the concentration of which will
be dependent on the species of erythrocytes used and will vary
from 1.25 to S.5 x 10 M. This results in the formation of
aldehyde or similarly modified group9 on the erythrocytes -
surface and prevents the action of neuriminidase. With othertypes of viru~, e.g. rubelIa, pre-treatment of the erythrocytes
with perlodate, or any other chemical agent such as chromic
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chloride or carbodiimide may not be required.
Certain micro-organisms will not à`dhere directly to
the erythrocytes and in such cases it may be possible to couple
the organism to the red cell indirectly, using compounds having
an affinity for red cells, such as lectins or lipo-polysaccharide.
Complement, which in one particular type of the system
is already incorporated into the gel, comprises a complex~group
of serum proteins, most of which have the characteristic of
interacting with antibody molecules once these have combined with
an antigen, the effect of such combination being to bring about
lysis where the antigen concerned is a cell such as a red cell.
The complement required for the purpose of the present invention
is conveniently vertebrate complement, which may preferably be
obtained commercially from Wellcome Reagents Ltd. or prepared by
` allowing blood to clot, removing the serum and storing it at
4~C or below, preferably at -20C. It is incorporated into the
system in an amount sufficient to ensure lysis.
The complement may also be pro~ided for incorporation
in the system, for instance in a sealed glass or plastic vial,
together with appropriate instructions for use with the gelO In
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addition it is convenient to provide two fu~ther sealed and
et~rilized vials containlng reference sera, one of which contains
antibodies to the microbe which has been incorporated into the
system, and the other being free from such antibodies. All these
essential and optional components of the system for testing body `~
fluids for specific microbial antibodies may be presented and
pacXaged in a box or container, together with instruction$ for
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useO It can therefore be emphasised that such a presentation of
the system, comprising the gel composition, the complement and
instructiohs as well as other optional
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components and aids, is considered to fall withln the scope
of the present invention and accordingly provide a testing kit.
The system can be adapted to detect a class of anti-
body, for example IgG, by using, in addition to a gel
incorporating the specific microbe, a gel incorporating anti
IgG serum. The zone produced by IgG can thereby be prevented
by incorporating the anti-immunoglobulin serum into the system4
Alternatively a well is cut in the centre of the
gel, into which the test fluid is placed and at a
distance from it another well is cut into which anti IgG
serum is deposited~ The gel is incubated to allow a zone
of lysis to develop and where a zone of no haemolysis occurs,
at the edge of the haemolysis zone near the adjacent well, -~
this shows that IgG antibodies were present in the test
fluid and have therefore been neutralized.
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In a further particular aspect therefore the system
may comprise a composition as hereinbefore defined, and also
an effective amount of an anti-immunoglobulin serum.
- Such anti-immunoglobulin sera, may be obtained,
commercially from Wellcome Reagents L~d, or by purifyiny sera
from myeloma patients, for instance on DEAE cellulose in order
to remove unwanted immunoglobulins, adding adjuvent and inject-
ing into animals. The animals are later bled and the sera is
removed and can be usad for incorporation into the gel.
The system may also ~e adapted to detect particular
haemagglutinin and neuraminidase antigens on influenza virus
particles, by using various recombinant strains of infIuenza
virus.
The vessel into which the gel is poured conveniently
comprises a plastic or glass plate which has a plain rectangular
recess suitable for containing the gel layer, and a flat, trans-
parent rectangular glass or plastic plate which can be placed
over the plate with the recess, in order to keep the gel moist,
sterile and prevent evaporation. Alternatively a Petri dish or
any suitable container can be used as long as the thickness of
the system is not less than about 0.lmm and not greater than
5mmO In addition the gel must have an even thickness O ''
The vessels used are normally packed under sterile
conditions and therefore require no further sterilizing treat-
ment~ In order to prevent the unwanted growth of bacteria or
fungi on the finished gel a bacteriocidal and fungicidal agent
such as sodium azide or Methiolate
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is incorporated into the gelO Therefore the sodium
azide has the property of prolonying the storage life of
the complement if it is incorporated into the gel.
The sample of body fluid for testing with the system
as hereinbefore defined, may be obtained from the patient
or animal by several different methods depending on the
particular fluid being tested. For instance blood can be
obtained by venopuncture. If serum is to be used for
the test, it is preferably heated to 56~C and held at that
temperature for 30 minutes in order to remove any complement
present. The samples can be applied to the gel either by
deposition in a well cut out of the gel, or adsorption onto
a circle of filter paper having a diameter of 4-5mm, which
in turn is placed on the surface of the gel.
Where spécific microbial antibodies are present in the
sample of fluid, a circular and visible zone of lysis is pro-
duced, whenever the complement is ,already present in the
gel or introduced subsequent to the fluid, the size of which is
positively related to the logarithm of the antibody titrP.
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In yet another aspect of the invention there is
pro~ided a method o~ detecting the presence of specific viral
antibodies in body fluids which compris~s applying a sample
of body -fluid, for example blood, serum, plasma, nasal washings,
cerebrospinal fluid, saliva ox milk from a patient or animal,
to a diagnostic system, of the invention, as herein~efore defined,
allowing the fluid to diffuse into the gel and interact with
the components of the system and introducing complement onto the
gel, to provide a visible zone of erythrocyte lysis correspond-
ing to the amount of viral antibody in the body fluid.
The method of detecting specific viral antibodies
described may be used for diagnosing many dis~ases, for example
viral diseases such as influenza, both A & B, rubella, measles,
parainfluenza, cytomegalovirus and louping ill. ~he test is
especially useful for detecting those people or animals who
have no immunity or low levels of immunity to a particular
disease and therefore are at risk and require vaccination.
The test can also be used for diagnosing virus diseases in
plants. A preparation of diseased plant tissue is injected into
an animal and after a period of time serum, which contains
antibodies to the plant virus, is removed from the animal and
used in the test. The test can also be used for detecting
antibodies to psittacosis, Mycoplasma as well as bacteria.
The advantages of this method in the viral field over
previously used methods are that it is quick, simple and
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easy to carry out, and it is very specific since it
can distinguish between antisera to closely r~lated
strains of a virus, particularly strains of influenza
virus, and may even differentiate between primary and
secondary infection. The advantage of the system provided
by the invention is that, compared with the schild technique,
no purification or concentration of the virus may be
needed, indeed with influenza virus crude allantoic fluid may
be used. In addition the visible area of the lysis
provides antibody levels within an accuracy of 5 to 2~/o.
A Hyland plate suitable for containing the gel accord- -
ing to the invention will now be described with reference to
the accompanying drawing. The Hyland plate 2 comprises
a plain rectangular base 4 and a lid 6, both made from
a transparent plastic material. The base 4 has a rectàngular
recess 8 intoiwhich a gel 10 is poured. Wells 12 are cut
` out of the gel 10 in order to receive body fluid samples.
; The lid 6 fits tightly over the base 4'in order to protect
the gel from drying and physical damage~ The lid 6 is
surrounded by a rim 14 which engages a lip which surrounds the
base 4.
The invention will now be described with reference
to the following Examples but is in no way to be considered
limited by the aame.
Example 1 ~ -~
Equal volumes of washed packed sheep red blood cells
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and a 5.5 x 10 M solution of potassium periodate in-
saline were mixed and allowed to stand at room temperature
for 10-minutes. Allantoic fluid, from chicken eggs, --
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containing influenza virus strain A2 (haemagglutination titre
approximately 4 x 10 /ml) was added, the volume of the fluid
being ten times that of the packed erythrocytes. The mixture
was left at room temperature for 10 minutes, the coated erythro-
cytes were then washed three times in Barbitone buffered saline
~- i.e. saline containing barbitone and buffered at pH 7.2, to re-
move free virus not attached to the erythrocyte, and finally
made up to a 5~/O v/v suspension in buffered saline.
1% Agarose was made up in Barbitone buffered saline,
and an aliquot (1.5 ml) of this was poured into a Hyland plate
to form a base layer (l mm thick). A sample (loO~V`of the 50~/0
erythrocyte suspension was mixed with agarose (l.S ml) at 45~C
and the ~esultant suspension was poured on top of the base layer
and allowed to solidify to form a test system having an even
thicXness (2 mm~.
Example 2
The 50~/O v/v virus coated erythrocyte suspension was
prepared a~ in Example 1. A portion (100~1) of this wa~ added
to agarose (3 ml) and to this was added guinea pig complement
solution (100~1 of 1:160 haemolytic titre) obtainable from
Wellcome Reagents I,td. The resultant suspension was poured
into a Hyland plate and allowed to solidfy to give an even
layer (2 mm thick).
A 1% solution of Agarose was made up in Barbitone
buffered saline and to this was added sufficient sodium azide
to give a 0.1% solution. A suspension containing virus coated
red blood cells and guinea pig complement was then prepared as
in Example 2, using the agarose solution and an aliquot ~3 ml~
- 30 was then poured into a Hyland plate. ~-
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Example 4
A virus coated red cell suspension was prepared as in
Example 1. After leaving at room temperature for 10 minutes
the cells were washed once, then 1 ml of the packed cells were
treated with 0.5 ml of 1% gluteraldehyde solution for 7 1/2
mi-nutes. The cells were then washed four times to remo~e excess
gluteraldehyde and a gel plate was then prepared as described
in Example 1.
Example 5
A gel was prepared as in Example l,- except that the
virus used was rùbella, BHK grown Thomas strain (haemagglutina-
tion titre approximately 1024/ml). The red cells and rubella
virus were allowed to stand for 1 hour at 4C to allow for
adsorption. In addition 0.1% sodium azide was incorporated into
the agarose gel.
A sample of patients bla,od was placed on an area of
filter paper (whatmarl) in an amount sufficient that discs (4 mm
diameter) cut out of it each con~ained 2.3 x 10 2ml of blood
adsorbed thereon~ The blood soaked discs ~ere then placed on
~ top o~ the gel left at 4C overnight andthen incubated at 37C
for 3 hours, at the end of which time any zone of lysis under
each filter paper disc indicated that the patient had
antibodies in their blood to rubella.
Example 6 - -
A gel was prepared as in Example 1 except that the
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virus used was louping ill (haemagglutination titre 1 256)o
The red cells were treated with an equal volume of 10 3M
potassium periodate before being left to stand with the virus
for 45 minutes at 4~C to allow for adsorption. The standardprocedure for preparation of the plate, as described in
Example 1, was then carried out~
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Example 7
A gel was prepared as in Example 1 except that the red
cells used were Patas Monkey cells and the virus us~d was
measles virus (haemagglutination titre 1:1024). The Patas
Monkey erythrocytes are treated with an equal volume of 10 3M
potassium period~te before being left to stand with the virus
for 15 minutes at 37C. The standard procedure for preparation
of the plate, as described in Example 1, was then carried out. -~
Example ~
A gel was prepared as in Example 1 except that the
micro-organism used was Psittacosis (complement fixation titre
1:64). The red cells and Psittacosis organisms were allowed to
stand for 1 hour to allow for adsorption. The standard procedure
for preparation of the plate, as described in Example 1, was
then carried out.
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A gel was prepared as in Example 1 except that the
micro-organism used was Mycoplasma ~allisepticum (haemagglutina-
tion titre 1:64). The periodate treated erythrocytes were mixed
with 2.0 ml of a sonicated 1 in 5 dilution of the Mycoplasma
preparation and left to stand for 20 minutes. The standard
procedure for preparation of the plate, as descri~ed in
Example 1, was then carried out.
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Example 10
A gel was prepared as in Example 1 except that the
micro-organism used was Bordetella pertussis. The red cells
and Bordetella organisms were allowed to stand for an hour
at 4C, with intermittent shaking, to allow for adsorption.
In addition 0.1% sodium azide was incorporated into the
agarose gel. The standard procedure for preparation of the
plate, as described in Example 1, was then carried out.
Exam~le 11
A gel was prepared as in Example 10 except that the
micro-organism used was Vibrio chlolerae Inaba
Example 12 -~
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A patient suspected of sufering from influenza strain
A2 provided samples of blood. -
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A test system was prepared as in Examples 1, 2 and
3 and wells (3 mm diameter) were punched out. To each well
was added an aliquot (10~1) of the test body fluid e.g. serum,
~' which had previously been heated to 56C and held at that
-l~ temperature for 30 minutes in order to inactivate it. The
system was kept overnight in a moist box at a reduced
temperature ~4VC) in order to allow the completion of diffusion.
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After diffusion the system was covered with a solution of guinea
pig complement (1 ml of a freeze dried preparation obtainable
from Wellcome Reagents Ltd., and reconstituted with distilled
water according to the instructions on the container) and
then incubated (37C) until zones of lysis developed (2-3 hours)
which indicated that the patient has at some time suffered
from type A influenza. The system was then washed to
remove complement and released haemoglobin and it was finally
fixed in Formal Saline ( l~/o) for storage and reference purposesO
The diameter of the zones of lysis produced were then measured,
and compared with calibrated reference zones.
Example 13
A gel was prepared as in Example 2, except that the
virus used was Rubella, and a well (3 mm diameter) was cut
out of the centre. Two further we:lls were cut out at equal
distances from the central well. Xnto the centre well an
aliquot (10~1) of the test fluid was put, and into the
other wells a volume (10~1) of anti- IgG serum was deposited.
The gel was incubated until zones of lysis developed.
The zones of lysis were examined and in the part of the
` zone nearest the IgG well and half moon portion pointing
towards the test fluid well, of no haemolysis was visible,
which showed that IgG immunoglobulin molecules were present
in the test fluid and had been neutralized.
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Example 14
A kit for use in diagnosing influenza A2 or for . -
detecting the presence of influenza A2 antibodies is composed
of a ~ial containing a freeze dried preparation of reference
serum, one serum containing specific influenza A2 antibodies and
the other not. Also contained in the kit are Hyland plates
containing the system as prepared in either Example 1 or 4
together with instructions for use as set out in Example 5.
Example 15 ~:
A kit for use in diagnosing influenza A2 strain or for
detecting the presence of influenza A2 antibodies is composed
of two vials each containing a freeze preparation of reference
serum, one containing specific influenza A2 antibodies and the
other not, together with Hyland plates containing the system
as prepared in any of Example 2, 3 or 4, together with
instructions for use.
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