Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ENDOTOXIN-DETECTING DEVICE
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to an endotoxin-detecting deviceO
Prior Art
It is believed that endotoxin is lipopolysaccharide
present in a cell wall of Gram-negative bacteria. Even a
very small amount of endotoxin causes various physiologic
activities such as pyrexia. The pyrexia of a living organism
caused by endotoxin has been a serious problem in the
medical, pharmaceutical and sanitary fields. In recent
years, it has been proposed to carry out an endotoxin
detection using an endotoxin reagent composed of either
amebocyte lysate extracted from blood cells of a horseshoe
crab or a mixture of a color producing agent and proenzyme
separated from the amebocyte lysate. This method is commonly
referred to as "limulus test" in the trade. This method
enables endotoxin to be detected much more quickly in
comparison with a conventional endotoxin-detecting method in
which a small amount of a specimen liquid is applied to a
rabbit to see whether the rabbit is subjected to pyrexia.
Various endotoxin-detecting devices for carrying out the
above-mentioned limulus test have heretofore been proposed.
Such detection devices comprise a reaction container such as
a tube holding the above-mentioned endotoxin reagent. For
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carrying out the endotoxin detection, a specimen liquid to be
examined must be transferred to the reaction container by the use
of an endotoxin-free thief tube such as a pipet and a syringe.
Therefore, the operation of the conventional endotoxin-detecting
devices is rather cumbersome, and in addition there is the risk
that the specimen liquid may be contaminated by endotoxin or the
like during the transfer of the specimen liquid to the reaction
oontainer.
SUMMARY OF THE rNVENTION
It is therefore an object of this invention to provide an
endotoxin-detecting devioe which obviates the need for a separate
thief tube for transferring a specimen liquid to a reaction tube,
thereby overcoming the above difficulties of the prior art.
According to the present invention, there is provided an
endotoxin-detecting devi oe which comprises: (a) a hermetically
sealed transparent reaction tube having a diameter of between
akout 0.1 and 5 mm; and (b) a freeze-dried endotoxin reagent
2a received in said tube.
The reaction tube is sealed at opposite ends thereof by
either a heat sealing or closure members such as caps and plugs.
The opposite ends of the reaction tube, when in use,
are opened, and one of the open ends is dipped in a specimen
liquid so that the specimen liquid is drawn into the reaction
tube by the capillary action. The specimen liquid so intro-
duced dissolves the endotoxin reagent to form a mixture
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which is heated to a predetermined temperature to react the
reagent with the specimen liquid. The reaction mixture in
the reaction tube is gelated, becomes turbid or is colored in
proportion to the concentration of the endotoxin contained in
the specimep liquid. Thus, the presence of endotoxin in the
specimen liquid can be easily detected. The reaction tube is
of sigh a diameter that the specimen liquid introduced into
the reaction tube is not caused to flow therefrom when it is
held horizontally. Preferably, the diameter of the reaction
tube should be 0.1 to 5 mm. The reaction tube may be
provided at one end thereof with a suction means for
positively drawing the specimen liquid into the reaction
tube. The reaction tube can be made of any material so long
as it has such a transparency that the inside of the reaction
tube can be inspected from outside it.
The endotoxin reagent is composed of either amebocy~e
lysate extracted from blood cells of a horseshoe crab or a
mixture of a color producing agent and proenzyme separated
from the amebocyte lysate. In view of preservation
~0 stability, the endotoxin reagent is preferably in the
freeze-dried form. A stabilizing agent may be added to the
endotoxin agent
BRIEF DESCRIPTION OF THE DRAWINGS
. .
FIG. 1 is a cross-sectional view of an endotoxin-
detecting device provided in accordance with the present
invention;
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FIG. 2 to 4 are views similar to FIG. 1 but showing
modified endotoxin-detecting devices, respectively;
FIG. 5 iS a cross-sectional view of another modified
endotoxin-detecting device having a membrane container;
FIG. 6 is a cross-sectional view of the endotoxin-
detecting device of FIG. 5 taken along the line VI-VI of FIG.
5; and
FIG. 7 is a view similar to FIG. 5 but showing a
plurality of reaction tubes sealed in the membrane container.
DESCRIPTION OF THE PREFBRRED EMBODIMENTS OF THE INVENTION
The invention will now be described with reference to
the drawings in which like reference numerals designate
corresponding parts in several views.
An endotoxin-detecting device shown in FIG. 1 comprises
a straight reaction tube 1 of a uniform diameter having
opposite closed ends, and an endoto~in reagent 2 sealed in
the reaction tube 1 and disposed intermediate the opposite
ends thereof. The tube 1 is made of a transparent material
such as glass, polymethyl methacrylate and polystyrene.
~0 Preferably, the tube 1 has a small diameter, for example, of
0.1 to 5 mm.
A notch or a line of cut may be formed circumferentially
in each of the opposite end portions of the tube 1 to
facilitate the removal of these end portions from the tube 1.
The tube 1 may have opposite open ends which can be closed by
closure members such as plugs and caps, respectively. One or
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both of the opposite end portions of the tube 1 may have
either smaller or larger diameter than the major portion
extendinq between these opposite end portions. The reaction
tube 1 may be formed into a U-shape.
Although the endotoxin reagent 2 is received only in a
part of the tube 1, the reagent may be filled in the tube 1
along the entire length thereof.
For determining the concentration of endotoxin contained
in a specimen liquid to be examined, the opposite ends of the
tube 1 are first removed or cut of so that the tube has
opposite open ends. Then, one of these open ends is dipped
in a specimen liquid so that the specimen liquid is
introduced into the tube 1 by the capillary action. The
specimen liquid introduced into the reaction tube 1 dissolves
the endotoxin reagent 2 to form a mixture. Then, the tube 1
holding the mixture is placed in a dry warming device to heat
the mixture to a predetermined temperature so that the
endotoxin reagent 2 is caused to react with the specimen
liquid. Then, the tube 1 is tilted to see whether the
reaction mixture in the tube 1 is subjected to gelation.
Rt the same time, the reaction tube 1 is observed to
determine whether the reaction mixture in the tube 1 is
subjected to turbidity or coloring. The degree of gelation
and turbidity ox the reaction mixture are proportional to the
concentration of endotoxin in the specimen liquid. Thus, the
concentration of the endotoxin can be easily determined.
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As described above, the tube 1 serves as both a thief
tubQ and a reaction tube. Thus, the endotoxin-detectinq
device according to the present invention obviates the need
for a separate eDdotoxin-free thief tube for taking the
specimen liquid. Therefore, with this endotoxin-detecting
device, the specimen liquid does not need to be transferred
from a thief tube Jo a reaction tube, so that the detection
of endotoxin can be carried out easily. In addition, the
risk of contamination of the specimen liquid by endotoxin or
the like can be substantially reduced. Further, the reaction
tube 1 is of such a small diameter that it holds a relatively
small amount of specimen liquid. Therefore, the amount of
the reagent for endotoxin in the tube can be small.
FIG. 2 shows a modified endotoxin-detecting device which
differs from the endotoxin-detecting device of FIG. 1 in that
a transparent tube 1 has a colored portion 3 extending along
the length thereof. The colored portion 3 is formed by a
color coating applied to a half of the outer circumferential
surface of the tube 1 and extending substantially along the
entire length thereof. The colored portion has a white
color. The colored portion 3 may be provided on one fourths
to three fourths of the outer circumferential surface of the
tube 1. The colored portion 3 does not necessarily be
provided substantially along the entire length thereof, and
it may be provided only at the area of the reaction tube 1
where an endotoxin reagent 2 is positioned. Also, the
colored portion 3 may be formed by a colored film adhesively
bonded to the outer circumferential surface of the reaction
tube 1. Further, the colored coating or the colored film may
be applied to the inner circumferential surface of the tube
1. The color of the colored portion 3 may be one other than
a white color so long as it is opaque.
A specimen liquid is introduced into the reaction tube 1
in the same manner as described above for the endotoxin-
detecting device of FIG. 1. By virtue of the provision of
the colored portion 3, the gelation and coloring of the
reaction mixture of the reagent 2 and the specimen liquid can
be easily observed with naked eyes if they develop.
FIG. 3 shows another modified endotoxin-detectin~ deYice
which differs from the endotoxin detecting device of FIG. 1
in that a reaction tube 1 has an open end to which a suction
member 4 is attached. The suction member 4 is in the form of
a bulb and is made of an elastic material such as rubber and
a synthetic resin. The suction member 4 is snugly fitted on
the open end of the reaction tube 1 in an air-tight manner
and is fixed thereto. The suction member 4 may be formed
integrally with the reaction tube l
In operation, the closed end of the reaction tube 1 is
first removed, and the suction member 4 is squeezed by
fingers. Then, the end of the tube 1 remote from the suction
member 4 is dipped in a specimen liquid, and the pressure on
the suction member 4 is reduced or released so thaw the
specimen liquid is drawn into the reaction tube 1 by suction.
Then, the gelation and coloring of the reaction mixture of an
endotoxin reagent 2 and the specimen liquid are observed in
the same manner as described above for the endotoxin-
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detectinq device of FIG. 1 to determine the concentration ofendotoxin contained in the specimen liquid. sy virtue of the
provision of the suction member 4~ the liquid specimen can be
introduced into the tube 1 easily and positively. The
suction member 4 may be of any shape so long as it can draw
the specimen liquid into the reaction tube 1 by suction.
Also, the tube 1 may have a colored portion for facilitating
the observation of the reaction mixture in the tube 1, as
described above for the endotoxin-detecting device of FIG. 2.
FIG. 4 shows a further modified endotoxin-detecting
device which differs from the endotoxin-detecting device of
FIG. 1 in that a reaction tube 1 is provided with a graduated
scale 5. A notch or a line 6 of cut is formed
circumferentially in each of the opposite end portions of the
tube 1 to facilitate the removal of the opposite end portions
from the tube 1. The graduated scale 5 comprises a pair of
lines each formed circumferentially around the reaction tube
1 and disposed intermediate an endotoxin reagent 2 and a
respective one of the notches 6. A plurality of scale lines
5 may be provided on the tube 1 between the reagent 2 and a
respective one of the notches 6. Also, the scale lines 5 may
be replaced by dots. Alternatively, the reaction tube 1 may
have regions of a transparent color which replace the scale
lines S. Further, although the reagent 2 is received in the
tube 1 at a central portion thereof, the reagent may be
disposed at any position between the opposite scale lines 6.
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In operation, the opposite ends of the -tube 1 are broken
of from the reaction tube 1 at the respective notches 3.
Then, one of the opened ends of the reaction tube 1 is dipped
in a specimen liquid to draw it into the tube 1 up to the
scale line 5 by the capillary action. Then, the gelation and
turbidity of a reaction mixture of the xeagent 2 and the
specimen liquid are observed to determine the concentration
of endotoxin in the specimen liquid. By virtue of the
provision of the graduated scale 5, a constant amount of the
specimen liquid is always introduced into the reaction tube
1, so that the concentration of endotoxin in the specimen
liquid can be accurately determined with reproducible
results.
The reaction tube 1 may have a colored portion for
facilitating the observation of the reaction mixture in the
tube 1, as described above for the endotoxin-detecting device
of FIG. 2. Also, the reaction tube 1 may be provided at one
end with a suction member for positively drawing the specimen
liquid into the tube 1, as described above for the
endotoxin-detecting device of FIG. 3.
FIG. 5 shows a still further modified endotoxin-
detecting device which differs from the endotoxin-detecting
device of FIG. 1 in that a reaction tube 1 has opposite open
ends and in that the reaction tube 1 is hermetically sealed
in a membrane container 7.
The membrane container 7 comprises a pair of identical
rectangular films 7a and 7b between which the tube 1 is
sandwiched, the films 7a and 7b being hermetically sealed or
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bonded together along their entire peripheral margins by
either heat-sealing or a suitable adhesive, thereby providing
a peripheral sealed portion 8. A triangular notch 9 is
formed in the peripheral sealed portion 8 for facilitating
the tearing of the membrane container 7 when in use. The
notch 9 may be replaced by a slit. Each of the films 7a and
7b comprises a film impermeable to moisture, such as a
laminated aluminum film incorporating a polymer, a
polyvinylidene film, a polyolefin film and an ethylene vinyl
chloride copolymer film.
The membrane container 7 may be made of a single square
film folded in two, with three open sides of the folded film
being sealed. Alternatively, the membrane container 7 may be
made of a film tube with opposite open ends being sealed, in
which case a fused portion is formed on the membrane
container in which fused portion the notch 9 is provided.
Also, the membrane container 7 may be vacuum sealed. A
desiccant may be contained in the membrane container 7
together with the reaction tube 1. A plurality of reaction
tubes 1 may be sealed in the membrane container 7 as shown in
FIG. 7.
The provision of the membrane container 7 obviates the
need for directly closing the opposite open ends of the
reaction tube 1 by either heat-sealing or closure members
such as caps and plugs after an endotoxin reagent 2 is filled
in the tube 1. Therefore, the risk of contamination of the
reaction tube 1 by endotoxin or the like when sealing the
tube 1 can be substantially reduced. In addition, the innar
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and outer surfaces of the membrane container 7 can be
preserved in an endotoxin-free condition. And, in the case
where a plurality of reaction tubes 1 are sealed, much time
and labor can be saved.
The reaction tube 1 may have a colored portion for
facilitating the observation of the reaction mixture in the
tube 1, as described above for the endotoxin-detecting device
of FIX. 2. Also, the reaction tube 1 may be provided at one
end with a suction member for positively drawing the specimen
liquid into the tube 1, as described above for the
endotoxin-detecting device of FIG. 3. Further, the tube 1
may be provided with a graduated scale for introducing a
constant amount of the specimen liquid into the tube 1, as
described above for the endotoxin-detecting device of FIG. 4.
