U.S. patent application number 09/788481 was filed with the patent office on 2001-06-28 for histamine measuring apparatus and a histamine measuring method.
Invention is credited to Otomo, Jun, Takeshita, Tomoko.
Application Number | 20010005580 09/788481 |
Document ID | / |
Family ID | 16158535 |
Filed Date | 2001-06-28 |
United States Patent
Application |
20010005580 |
Kind Code |
A1 |
Takeshita, Tomoko ; et
al. |
June 28, 2001 |
Histamine measuring apparatus and a histamine measuring method
Abstract
Histamine may be quantitatively measured by providing the steps
of: holding in a recess formed at the bottom of a vessel an oocyte
that expresses histamine receptors, inserting first and second
electrodes into the oocyte, measuring the membrane potential of the
oocyte by means of said first electrode to stabilize the membrane
potential of said oocyte at a predetermined level by driving a
current through second electrode inserted into said oocyte by means
of a circuitry for clamping membrane potential of oocyte, infusing
a sample into a fine reacting tube having an antigen immobilized on
the inner surface thereof together with some buffer solution to
promote a histamine releasing reaction, transferring the solution
containing histamine released in the fine reacting tube to the
vessel to make contact with said oocyte in the vessel, detecting an
electric response of oocyte caused by the contact with said
solution, and determining the concentration of histamine released
by said histamine releasing reaction in said fine reacting tube.
The whole blood or mast cell suspension may be used as a sample
without pretreatment.
Inventors: |
Takeshita, Tomoko;
(Higashimatsuyama, JP) ; Otomo, Jun; (Tokyo,
JP) |
Correspondence
Address: |
MATTINGLY, STANGER & MALUR, P.C.
104 East Hume Avenue
Alexandria
VA
22301
US
|
Family ID: |
16158535 |
Appl. No.: |
09/788481 |
Filed: |
February 21, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09788481 |
Feb 21, 2001 |
|
|
|
09604512 |
Jun 27, 2000 |
|
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Current U.S.
Class: |
435/4 ;
205/780.5 |
Current CPC
Class: |
Y10S 435/808 20130101;
Y10S 436/807 20130101; Y10S 436/806 20130101; Y10T 436/108331
20150115; Y10S 436/805 20130101; Y10S 436/826 20130101; G01F 1/00
20130101; G01N 2333/726 20130101 |
Class at
Publication: |
435/4 ;
205/780.5 |
International
Class: |
G01F 001/64; G01N
017/00; G01N 027/26 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 30, 1999 |
JP |
11-184740 |
Claims
What is claimed is:
1. A histamine measuring apparatus for quantitatively analyzing the
concentration of histamine, comprising: a vessel having at the
bottom thereof a recess for holding an oocyte which expresses
histamine receptors; first and second electrodes for inserting into
said oocyte; a circuitry for measuring the potential of membrane of
said oocyte by means of said first electrode and for flowing a
current through said second electrode to said oocyte to maintain
the membrane potential of said oocyte at a predetermined constant
level; a fine reacting tube with an antigen immobilized onto the
inner surface thereof; and flowing tubes for flowing a sample
together with some buffer solution into said fine reacting tube to
promote a histamine releasing reaction therein and for transferring
the solution containing released histamine into said vessel to make
contact with said oocyte in said vessel; wherein said circuitry
detects the electric response from said oocyte caused by the
contact with said solution in order to determine the concentration
of histamine released by the histamine releasing reaction occurred
in said fine reacting tube.
2. A histamine measuring apparatus according to claim 1, in which:
said sample is a whole blood sample or mast cell suspension without
pretreatment.
3. A histamine measuring apparatus according to claim 1, further
comprising: a temperature controller device for controlling the
temperature of said fine reacting tube to a predetermined
temperature level.
4. A histamine measuring apparatus according to claim 1, further
comprising: a temperature controller device for controlling the
temperature of said fine reacting tube to a temperature ranging
from 30.degree. C. to 45.degree. C.
5. A histamine measuring apparatus according to claim 1, further
comprising: a tubing connected to said vessel for infusing buffer
solution thereto and a tubing for purging the content of said
vessel.
6. A histamine measuring apparatus for quantitatively analyzing the
concentration of histamine, comprising: a vessel having at the
bottom thereof a recess for holding an oocyte, which expresses
histamine receptors; a fine reacting tube with an antigen
immobilized onto the inner surface thereof; flowing tubes for
flowing a sample together with some buffer solution into said fine
reacting tube to promote a histamine releasing reaction therein and
for transferring the solution containing released histamine into
said vessel to make contact with said oocyte in said vessel; and a
circuitry for detecting electric response of said oocyte caused by
the contact with said solution; wherein said apparatus determines
the concentration of histamine released by the histamine releasing
reaction occurred in said fine reacting tube.
7. A histamine measuring apparatus according to claim 6, in which:
said sample is a whole blood sample or mast cell suspension without
pretreatment.
8. A histamine measuring apparatus according to claim 6, further
comprising: a temperature controller device for controlling the
temperature of said fine reacting tube to a temperature ranging
from 30.degree. C. to 45.degree. C.
9. A histamine measuring method for determining the concentration
of histamine, comprising the steps of: holding in a recess at the
bottom of a vessel an oocyte that expresses histamine receptors;
measuring the potential of membrane of said oocyte by means of
first electrode inserted into said oocyte to stabilize the membrane
potential of said oocyte at a predetermined level by driving a
current through second electrode inserted into said oocyte;
infusing a sample into a fine reacting tube having an antigen
immobilized on the inner surface thereof to promote a histamine
releasing reaction; transferring the solution containing histamine
released in said fine reacting tube to said vessel to make contact
with said oocyte in said vessel; detecting an electric response of
said oocyte caused by the contact with said solution; and
determining the concentration of histamine released by said
histamine releasing reaction in said fine reacting tube.
10. A histamine measuring method according to claim 9, in which:
said sample is a whole blood sample or mast cell suspension without
pretreatment.
11. A histamine measuring method according to claim 9, further
comprising the step of: controlling the temperature of said fine
reacting tube at a predetermined temperature level.
12. A histamine measuring method according to claim 9, further
comprising the step of: controlling the temperature of said fine
reacting tube to a temperature ranging from 30.degree. C. to
45.degree. C.
13. A histamine measuring method according to claim 9, further
comprising the step of: determine the concentration (A) of
histamine released by said histamine releasing reaction in said
fine reacting tube by means of a predefined calibration curve
obtained by stimulating said oocyte with a plurality of known
concentrations of histamine to detect the electric response of said
oocyte to predefine the correlation between said electric responses
and said plurality of known concentrations of histamine.
14. A histamine measuring method according to claim 9, further
comprising the step of: determining the histamine releasing rate
given by (A-C)/B where B is a histamine concentration given by the
quantitative measurement of histamine contained in the cells in
said sample and released by freezing and thawing the sample
stimulated by the antigen, C is a concentration of free histamine
released without stimulation after adding some buffer solution
instead of the antigen into the sample.
15. A histamine measuring method for determining the concentration
of histamine comprising the steps of: holding in a recess at the
bottom of a vessel an oocyte that expresses histamine receptors;
infusing a sample into a fine reacting tube having an antigen
immobilized on the inner surface thereof to promote a histamine
releasing reaction; transferring the solution containing histamine
released in said fine reacting tube to said vessel to make contact
with said oocyte in said vessel; detecting an electric response of
said oocyte caused by the contact with said solution; and
determining the concentration of histamine released by said
histamine releasing reaction in said fine reacting tube.
16. A histamine measuring method according to claim 15, in which:
said sample is a whole blood sample or mast cell suspension without
pretreatment.
17. A histamine measuring method according to claim 15, further
comprising the step of: controlling the temperature of said fine
reacting tube to a temperature ranging from 30.degree. C. to
45.degree. C.
18. A histamine measuring method according to claim 15, further
comprising the step of: determine the concentration (A) of
histamine released by said histamine releasing reaction in said
fine reacting tube by means of a predefined calibration curve
obtained by stimulating said oocyte with a plurality of known
concentrations of histamine to detect the electric response of said
oocyte to predefine the correlation between said electric responses
and said plurality of known concentrations of histamine.
19. A histamine measuring method according to claim 15, further
comprising the step of: determining the histamine releasing rate
given by (A-C)/B where B is a histamine concentration given by the
quantitative measurement of histamine contained in the cells in
said sample and released by freezing and thawing the sample
stimulated by the antigen, C is a concentration of free histamine
released without stimulation after adding some buffer solution
instead of the antigen into the sample.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention is directed to a histamine measuring
apparatus and a histamine measuring method for quantitatively
measuring histamine present in bloods or mast cells
suspensions.
[0003] 2. Description of the Prior Art
[0004] Histamine releasing test is a quantitative analysis of
histamine extracellularly released by stimulating leucocytes (white
blood cells) in the blood or mast cells in the mucosa and promoting
releasing histamine contained therein outside the cells. The
histamine release test has been reported to be a method useful in
identifying some allergens in some allergic disorders and diseases
(c.f., Prior Art 1: Tabe, kazuaki: histamine release
test--diagnosis of allergies, SRL HOKAN, vol 21, pp. 17-22
(1997).
[0005] The known measurements of freed histamine (histamine
released in free state) includes, for example, a method using
fluorescent HPLC (fluorescent high performance liquid
chromatography) to purify the histamine to react it with a
fluorescent reagent in order to measure the amount of fluorescence
emitted from the fluorescent reagent reacted with the histamine
(c.f. , Prior Art 2: Japanese Patent Laid-Open No. H6-331619), a
method using glass fibers to purify the freed histamine to react it
with a fluorescent reagent in order to measure the amount of
fluorescence emitted from the fluorescent reagent coupled with the
free histamine (c.f., Prior Art 3: Japanese Patent Laid-Open No.
H10-170514, Japanese Patent Laid-Open No.10-62415), competitive
immunoassays commercially available from ICN Pharmaceuticals or
Immunotech, and an ELISA (c.f., Prior Art 4).
SUMMARY OF THE INVENTION
[0006] In general, the measurement of histamine may require:
[0007] (1) direct analysis of histamine without using labeling
thereof;
[0008] (2) direct quantitative measurement of histamine without
pretreatment;
[0009] (3) quick delivery of results; and
[0010] (4) specificity to the histamine.
[0011] Prior Art methodologies as have been described above are all
indirect, quantitative measurement methods of histamine, which
isolate the histamine from a sample, and label the isolated
histamine with a fluorescent reagent to measure the labeled
histamine reacted with a fluorescent reagent. All of the Prior Art
methodologies requires complex pretreatment such as purification of
samples, isolation of histamine, and labeling of histamine with a
fluorescent reagent, and also requires for hours to obtain a
quantitative results of histamine analysis.
[0012] The primary object of the present invention is in general to
provide a histamine measuring apparatus and a histamine measuring
method, which may satisfy the requirements listed above of
histamine measurement.
[0013] In accordance with the histamine quantitative analyzing
apparatus and method of the present invention, histamine
quantitative analysis will be achieved by:
[0014] providing cells with histamine receptor being expressed;
[0015] adding a sample having been stimulated by an antigen to the
cells expressing the receptor; and
[0016] detecting the resulting electrical response of cells.
[0017] For example, a predetermined amount of antigen will be added
to a sample of collected whole blood, the sample with antigen will
be incubated for 10 to 30 minutes at 37.degree. C. to promote an
allergic reaction (antigen challenge) therebetween to release the
histamine into the plasma. It may be preferable to gently shake the
blood sample while promoting the allergic reaction. The period of
time of reaction described here is indicated by way of example. The
reaction time may vary depending on the optimized allergic
reaction.
[0018] Examples of sample include, whole blood specimen, leucocytes
in the blood, and mast cell suspensions in which the mast cells of
mucosal tissue are cultured.
[0019] An allergic reaction may be invoked in general by the
binding of an allergen with a corresponding IgE present in the cell
membrane of cells contributing to the allergic reaction such as
mast cells, basophils, in the sample blood. The binding may trigger
a reaction of releasing a relevant chemical mediator (chemical
transmitter) such as histamine in the cells contributing to the
allergy reaction. In the description hereinbelow, the reaction of
releasing histamine will be referred to as "histamine release
(releasing) reaction."
[0020] The concentration (A) of histamine extracellularly released
by the histamine releasing reaction may be determined by using a
calibration curve thereof. The calibration curve may be obtained by
stimulating cells with the histamine receptor preliminary expressed
by means of a plurality of known concentrations of histamine to
detect the cellular electric responses at respective concentration
of histamine and may be expressed as a relation between a plurality
of known concentrations of histamine and detected electric
responses.
[0021] The ratio of histamine release .alpha. (%) may be determined
by
.alpha.=100.times.(A-C)/B
[0022] where B is a histamine concentration given by the
quantitative measurement of histamine released from the sample
after freezing and thawing the sample having stimulated by the
antigen, C is a concentration of free histamine released in non
stimulated state by adding some buffer solution instead of the
antigen to the sample.
[0023] The concentration of released histamine (C) designates to a
released histamine concentration by the stimulation with the
buffer, which is so-called "concentration for the correction of
background level".
[0024] The apparatus and method in accordance with the present
invention allows the histamine released in the sample to be
directly and quantitatively analyzed by using cells with histamine
receptors expressed. The cells expressing histamine receptors may
identify specifically and directly the histamine, even in case in
which derivatives of histamine or histamine-like compounds are
present in the sample, or in case in which there is a trace of
histamine contained in the sample. The apparatus and method in
accordance with the present invention, accordingly, may allow
direct quantitative analysis of histamine to be performed in a
shorter time, with no complex pretreatment of samples which may
require long time.
[0025] An exemplary configuration of the present invention will be
summarized below. The oocyte expressing the histamine receptors is
held in the recess formed at the bottom of a vessel. A first
electrode and a second electrode will be inserted into the oocyte
to determine the membrane potential of oocyte by the first
electrode, and then maintain the potential of oocyte membrane to a
predetermined level by flowing electric current through the second
electrode, by means of a circuitry for maintaining the potential of
oocyte membrane. A sample will be flew through together with some
buffer into a fine reacting tube with an antigen fixed on the inner
surface of tube wall to promote the histamine releasing reaction.
Thereafter the solution containing free histamine will be flew
through a flowing tube into the vessel to make contact with the
oocyte in the vessel. The electric response of the oocyte caused by
the contact with the solution will be detected by the potential
maintainer (clamping) circuit in order to determine the
concentration of free histamine released by the histamine releasing
reaction. The whole blood, or suspension may be used as the sample,
without the need for any pretreatment.
[0026] In accordance with the present invention, the concentration
of histamine may be determined in a shorter time, without the need
for any pretreatment, so that the process steps for quantitative
analysis, the number and amount of reagents may be reduced to
minimum and the cost and the time needed for the measurement may be
significantly saved.
[0027] In the following description an oocyte of Xenopus lavis
(African clawed frog) will be used for the cell expressing the
histamine receptors, byway of example. It should be understood that
the present invention may be applicable by using any other types of
cell.
[0028] Also in the following description of preferred embodiments,
an exemplary case will be described in which the electric response
of the cell with histamine receptors expressed will be detected by
identifying histamine with the histamine receptors expressed on the
cell membrane and by measuring the change in the membrane potential
along with the open and close of chloride ion channel caused by the
intracellular signal transduction. It is to be understood that the
preferred embodiments described herein are presented and described
for the purpose of illustrating the principle of the present
invention and are not intended to be limiting. For example, it
should be recognized that the present invention may be achieved by
using the cellular response derived from the receptor stimulation
of any other kinds such as those described in the Japanese Patent
Laid-Open No. H11-083785 (Prior Art 5), or by using any other
detecting methods.
[0029] Additional objects and advantages of the invention will be
set forth in part in the description which follows and in part will
be obvious from the description, or may be learned by practice of
the invention. The objects and advantages of the invention may be
realized and attained by means of the instrumentalities and
combinations particularly pointed out in the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 shows a schematic diagram illustrating an apparatus
in accordance with one preferred embodiment of the present
invention, which is a histamine measuring apparatus for
quantitatively analyzing the histamine by using the oocyte with
histamine receptors expressed;
[0031] FIG. 2 shows a schematic diagram illustrating an exemplary
analysis of histamine concentration and electric response of the
oocyte both obtained by the apparatus shown in FIG. 1;
[0032] FIG. 3A shows a schematic diagram illustrating an exemplary
analysis of electric response of oocyte, induced by the blood
sample containing histamine, and obtained by using the apparatus
shown in FIG. 1;
[0033] FIG. 3B shows a schematic diagram illustrating an exemplary
analysis of electric response of oocyte, induced by the blood
sample containing no histamine, and obtained by using the apparatus
shown in FIG. 1;
[0034] FIG. 4 shows a protocol of histamine analysis method in
accordance with a preferred embodiment of the present
invention;
[0035] FIG. 5 shows a result of histamine analysis using a whole
blood sample, obtained by using the apparatus shown in FIG. 1;
and
[0036] FIG. 6 shows a schematic diagram of an apparatus for
measuring histamine in accordance with another preferred embodiment
of the present invention, illustrating the overview of apparatus
for quantitatively analyzing histamine by using the oocyte with
histamine receptors expressed and a whole blood sample.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0037] In the following description of the preferred embodiments,
the histamine measuring apparatus and the histamine measuring
method in accordance with the present invention will be described
in greater details with reference to the quantitative analysis of
histamine in the whole blood sample by using the oocyte with
histamine receptors expressed.
[0038] A whole blood sample stimulated by the antigen will be added
to the oocyte with histamine receptor expressed to detect the
resulting electric response of the oocyte. A predetermined amount
of antigen will be added to the collected whole blood sample (some
buffer may be introduced if the whole blood sample is too viscous),
and the whole blood sample and the antigen will be incubated at a
temperature ranging from 30.degree. C. to 45.degree. C. for 10 to
30 minutes for promoting the allergy reaction therebetween in order
to release histamine into the plasma.
[0039] The concentration of histamine (A) released into the plasma
from the blood cells in the sample caused by the histamine
releasing reaction may be determined by means of a calibration
curve. The calibration curve may be obtained by stimulating cells
with the histamine receptor preliminary expressed by means of a
plurality of known concentrations of histamine to detect the
cellular electric responses at respective concentration of
histamine and may be expressed as a relation between a plurality of
known concentrations of histamine and detected electric
responses.
[0040] The ratio of histamine release .alpha.(%) may be determined
by
.alpha.=100.times.(A-C)/B
[0041] where B is a histamine concentration given by the
quantitative measurement of histamine released from the cells in
the sample blood after freezing and thawing the sample stimulated
by the antigen, C is a concentration of released histamine in non
stimulated state by adding some buffer instead of the antigen into
the blood sample.
[0042] The concentration of released histamine in non-stimulation C
designates to the concentration of free histamine released by the
stimulation of buffer, i.e., so-called "concentration of correction
of background".
[0043] The histamine analysis as have been described above may be
carried out within approximately 20 to 50 minutes, including the
calibration measurement for determining the calibration curve to be
used. In case in which the calibration curve has been determined in
advance, then the histamine measurement will be completed at most
in 30 seconds for each sample.
[0044] In this preferred embodiment of the apparatus and method in
accordance with the present invention, quantitative measurement of
histamine released from the cells in the whole blood to the plasma
may be directly carried out. If the histamine derivatives or
histamine-like compounds are present in the cells or plasma of
whole blood sample, or if the amount of histamine to be released
into the plasma from the cells of whole blood is very low, the
oocyte with histamine receptors expressed will specifically and
directly identify the histamine. Accordingly the apparatus and
method for measuring histamine in accordance with the present
invention may directly and quantitatively analyze the released
histamine from the whole blood cells to the plasma without the need
for any pretreatment of whole blood sample.
[0045] Since the apparatus and method of the preferred embodiment
in accordance with the present invention may detect the histamine
by using the oocyte with histamine receptors expressed, the
electric response of the oocyte caused by the physiological
reaction induced at the time when the oocyte identifies the
presence of histamine may be directly detected as an electric
signal. The physiological reaction of oocyte is known to be
occurred within milliseconds, thus the time required for detecting
the histamine may be within one second.
[0046] The apparatus and method for measuring histamine of the
preferred embodiment in accordance with the present invention may
perform a quantitative measurement in significantly shorter time
than any other Prior Art techniques requiring at least one hour for
the analysis.
[0047] A detailed description of some preferred embodiments
embodying the present invention will now be given referring to the
accompanying drawings.
[0048] FIG. 1 shows a schematic diagram illustrating an apparatus
in accordance with one preferred embodiment of the present
invention, which is a histamine measuring apparatus for
quantitatively analyzing the histamine by using the oocyte with
histamine receptors expressed;
[0049] In this embodiment the oocyte of Xenopus lavis (African
Clawed Frog) will be used. The unfertilized oocytes (eggs) of
Xenopus lavis are subjected to be injected mRNA of histamine
receptor. These oocytes will be incubated for two or three days in
a culture medium so as to cause histamine receptors to be
expressed. The histamine receptors will be active within the cell
membrane of oocyte. When histamine binds to the histamine
receptors, the information will be transferred inside the oocyte
through the mediator (second messenger) already present in the
oocyte.
[0050] As the result of a plurality of chemical mediation inside
the oocyte, intracellular calcium ions are raised in the oocyte,
opening the chloride ion channel of the oocyte cell membrane. This
reaction will cause the change in potential of cell membrane inside
and outside the oocyte.
[0051] In the present preferred embodiment of the present invention
the change in the electric response of oocyte before and after the
binding of histamine to the histamine receptors, namely, the change
in potentials between the inside and the outside of oocyte cell
membrane, will be detected as the change in electric current
response.
[0052] In the present preferred embodiment of the present invention
a method of whole cell clamp will be used in which a feedback
circuitry will be used, which may flow current through the oocyte
in the direction of suppressing the difference of membrane
potential at the moment of change in potential of membrane of
oocyte to maintain the potential to a predetermined constant
holding potential.
[0053] As shown in FIG. 1, at the recess 16 (not shown) at the
bottom of a vessel 1 fulfilled with buffer solution 23 for the
oocyte, an oocyte 2 with the histamine receptors expressed will be
held. The fine tips of glass electrodes 3 and 4, filled with KCl of
3 mole. and containing an Ag wire coated with AgCl will be inserted
into the oocyte 2. After the insertion, each of the tips of glass
electrodes 3 and 4 will be fixedly held by an apparatus for holding
electrodes, which is not shown in FIG. 1, but is provided
correspondingly for respective electrode.
[0054] The potential of the glass electrode 3 will be transmitted
to the differential amplifier 6 and the recorder 7 and recorded
therein as the difference in potential to an external electrode 5.
The glass electrode 3 may be an electrode for measuring the
potential in the membrane of oocyte, while the glass electrode 4
may be used for maintaining the membrane potential of the oocyte to
a predetermined constant level of -60 mV.
[0055] The differential amplifier 6 will apply the differential
current between the signal transferred from the glass electrode 3
to the differential amplifier 6 and the holding potential in the
whole cell clamping method to the oocyte 2 through the glass
electrode 4. In this manner, when the histamine 8 will be dropped
onto the oocyte 2 with the potential of cell membrane held at a
constant level of -60 mV, by means of for example a micro-syringe
or a pipette 9, the electric response (current response 10) of the
oocyte 2 may be obtained. The current response 10 will be described
in greater details below.
[0056] FIG. 2 shows a schematic diagram illustrating an exemplary
analysis of histamine concentration and electric response of the
oocyte both obtained by the apparatus shown in FIG. 1. The abscissa
is the known concentration of histamine (nM) of the samples. The
ordinate is the amount of change in potential between the inside
and outside of the oocyte before and after the binding of histamine
to the histamine receptors, measured as the change of current
(.mu.A).
[0057] As shown in FIG. 2, it is clear that the current change is
in positive correlation with the concentration of histamine in the
range of histamine concentration from 20 nM to 200 nM. This
correlation (calibration curve) may be used for measuring a sample
containing histamine at an unknown concentration by using the
apparatus of the present invention shown in FIG. 1 so as to
determine the unknown concentration of histamine.
[0058] FIG. 3A and FIG. 3B show schematic diagrams illustrating an
exemplary analysis of electric current response of the oocyte,
induced by the whole blood sample containing histamine (FIG. 3A)
and by the blood sample containing no histamine (FIG. 3B), and
obtained by using the apparatus shown in FIG. 1.
[0059] As shown in FIG. 3A, when at the moment as shown by the
vertical arrow, the whole blood sample containing histamine is
added to the oocyte expressing histamine receptors, the current
response will be reached at the maximum value within one second,
then will decrease gradually together with the elapsed time to
disappear approximately 30 seconds after.
[0060] On the other hand, as shown in FIG. 3B, when the whole blood
sample containing no histamine is added to the oocyte expressing
histamine receptors at the moment as shown by the vertical arrow,
no change in current response will be observed. This indicates that
the change in current response as shown in FIG. 3A is based on the
reaction between the histamine and histamine receptors, rather than
the change of response caused by the impurity of whole blood
sample. When applying the maximum value of the current response
shown in FIG. 3A to the correlation (calibration curve) shown in
FIG. 2, it can be seen that this maximum value of the current
response may be caused by the histamine of concentration of
approximately 100 nM. The outlined vertical arrow shown in FIG. 3A
indicates the change in current before and after adding a whole
blood sample containing histamine to the oocyte expressing
histamine receptors.
[0061] As can be seen from the foregoing description, in accordance
with the present invention, a whole blood sample may be used for
quantitative analysis of histamine without needs for isolation,
generator and labeling. As have been described above, by measuring
the change in membrane potential as the change in current passing
therethrough, along with the opening and closing of chloride ion
channel caused by intracellular chemical mediator after identifying
histamine by an oocyte expressing histamine receptors, the
measurements for quantitative analysis of histamine may be obtained
within one second after the oocyte identifies histamine.
EMBODIMENTS
First Embodiment
[0062] By stimulating oocytes expressing histamine receptors with a
plurality of known concentrations of histamine to detect the change
in current as have been described above, the correlation
(calibration curve) between the change in current and the
concentrations of histamine as shown in FIG. 2 may be
predetermined.
[0063] FIG. 4 shows a protocol of histamine analysis method in
accordance with a preferred embodiment of the present invention.
The protocol will be described below in greater details with
reference to FIG. 4:
[0064] (1) A whole blood sample 11 will be collected from vein of a
human elbow, using heparin as anticoagulant.
[0065] (2) A predetermined amount of antigen 12 such as cedar
pollen will be added to the collected whole blood sample 11.
[0066] (3) The whole blood sample 11 will be incubated for
approximately 30 minutes at 37.degree. C. to promote histamine
releasing reaction between the whole blood and antigen. Due to the
allergy reaction basophils 13 in the whole blood sample will be
stimulated and will release histamine 8;
[0067] (4) The whole blood sample 11 having stimulated by the
antigen 12 will be sampled by means of a micro-syringe or a
pipette.
[0068] (5) The sample will be added to the oocyte expressing
histamine receptors.
[0069] (6) The change in electric response of the oocyte will be
measured by means of the apparatus shown in FIG. 1 to determine the
change in current as have been described above.
[0070] (7) From the current change value, the concentration of
histamine (A) in the whole blood sample 11 having stimulated by the
antigen 12 may be determined by referring to the predetermined
correlation (calibration curve).
[0071] The releasing rate of histamine .alpha. (%) may be
determined by
.alpha.=100.times.(A-C)/B
[0072] where B is a histamine concentration given by the
quantitative measurement of histamine released from the cells in
the sample whole blood after freezing and thawing the sample having
stimulated by the antigen, C is a concentration of free released
histamine in non stimulated state by adding the buffer solution 23
instead of the antigen into the blood sample.
[0073] The concentration of free histamine released in
non-stimulation C designates to the concentration of free histamine
released by the stimulation of buffer, i.e., so-called
"concentration of correction of background".
[0074] The time required for a quantitative analysis of histamine
in accordance with the first preferred embodiment will be
approximately 30 minutes, which is significantly faster when
compared with 3 hours needed for the method described in the Prior
Art 3 above and 4 hours needed for the method described in the
Prior Art 4 above, resulting in a saving of time.
[0075] The procedure of the method of measuring histamine in
accordance with the first preferred embodiment of the present
invention is characterized in that the collected sample may be used
for quantitative analysis without need of any pretreatment of whole
blood, with significantly fewer steps of processing as compared
with any of Prior Art. The procedure of the method in accordance
with the present invention requires only histamine of known
concentration at the time of making a calibration curve, and no
other reagents are needed, allowing the analytical cost to be
reduced.
Second Embodiment
[0076] FIG. 5 shows an example of results of histamine analysis
using whole blood samples (5 samples), obtained by using the
apparatus shown in FIG. 1. The cedar pollen was used as antigen.
FIG. 5 shows the results of measurement of histamine, A
(concentrations of histamine in the whole blood samples stimulated
by the cedar pollen), B (concentrations of histamine quantitatively
determined from the histamine released from the cells of whole
blood samples by freezing and thawing the whole blood samples
stimulated by the cedar pollen), C (concentrations of freely
released histamine in non stimulated state derived from the cells
in the whole blood sample by adding the buffer solution 23 thereto
instead of the cedar pollen antigen) and .alpha. (%) (histamine
releasing rate), each determined for respective sample in
accordance with the procedure similar to that of the first
embodiment as have been described above.
[0077] In the histamine-releasing test in general with respect to
blood samples, when the histamine-releasing rate of a sample is
higher than or equal to 10% when stimulating with a specific
allergen, the sample may be considered to be positive in the
allergy reaction. In the exemplary embodiment shown in FIG. 5, the
samples 1 and 3 are to be considered that the allergy reaction for
the cedar pollen has been occurred. These results were consistent
with the results of conventional HRT (Histamine Release Test)
method as well as the subjective symptom of examinees.
Third Embodiment
[0078] FIG. 6 shows a schematic diagram of an apparatus for
measuring histamine in accordance with another preferred embodiment
of the present invention, illustrating the overview of an apparatus
for quantitatively analyzing histamine by using the oocyte with
histamine receptors expressed and a whole blood sample.
[0079] The apparatus shown in FIG. 6 is consisted of, in addition
to the components of the apparatus shown in FIG. 1, a pouring tube
14 for pouring the buffer solution 23 into the vessel 1 with the
assistance of a pump and the like, a draining tube 15 for draining
excessive buffer solution 23 from the vessel 1 with the assistance
of a pump and the like, and a draining tube 20 for purging the
solution contained in the vessel 1 with the assistance of a pump
and the like each time when the histamine analysis of a sample is
completed. It should be noted that in FIG. 6 the external electrode
5, the differential amplifier 6, the grounding wire of differential
amplifier 6, the recorder 7, the electric response of oocyte 10 are
omitted for the sake of simplification.
[0080] The buffer solution 23 for oocyte 2 will be infused to the
vessel 1 through the pouring tube 14 in excess of the liquid level
detector portion of liquid level meter (not shown in FIG. 6)
disposed inside the vessel. Then the driving apparatus of drain
pump will be driven based on the output from the level detector
until the level of buffer 23 will reach to the level of the level
detector sensing portion in order to drain the excessive buffer
solution 23 from the vessel 1 through the draining tube 15.
[0081] Thereafter, if for some reason or another the level of
buffer solution 23 contained in the vessel 1 is decreased or
increased, the decrease of buffer solution 23 will be detected by
the level detector sensor portion and the driving apparatus of
drain pump will be driven in accordance with the output of the
level detector to automatically add an amount of buffer solution 23
through the pouring tube 14 into the vessel 1 in order to maintain
the level of buffer solution 23 at a predetermined level. In this
manner a predetermined constant amount of buffer solution 23
sufficient for dipping the oocyte 2 will be held in the vessel
1.
[0082] As shown in FIG. 6, in the recess 16 at the bottom of the
vessel 1 filled with the buffer solution 23 for oocyte 2, a Xenopus
oocyte expressing histamine receptors 2 will be immersed in a
static manner. As similar to the apparatus shown in FIG. 1, the
fine tips of glass electrode 3 and glass electrode 4 will be
inserted into the Xenopus oocyte 2. The potential appeared in the
glass electrode 3 will be transmitted to the differential amplifier
6 not shown in FIG. 6 as the differential potential from the
external electrode 5. The differential amplifier 6 receiving the
potential signaled from the glass electrode 3 will generate the
current difference between the glass electrode 3 signal and the
fixed potential in the whole cell clamping method and apply it to
the Xenopus oocyte 2 through the glass electrode 4. The membrane
potential of the oocyte will be accordingly held at a predetermined
constant level of -60 mV.
[0083] Then the whole blood sample 11 prepared in accordance with
the procedure shown in FIG. 4 will be added gently over the Xenopus
oocyte 2 the membrane potential of which is held at -60 mV by means
of an instrument such as micro-syringe or pipette 9. The whole
blood sample 11 added to the vessel over the oocyte contains the
histamine 8 released by the histamine releasing reaction, antigens
12, basophils 13, plasma and the like. The change of current before
and after the histamine 8 is bound to the histamine receptors will
be measured. As have been described in the foregoing discussion,
the concentration of histamine 8 released from the cells in the
whole blood may be determined from the change of current by
predetermining a calibration curve.
[0084] Another configuration of apparatus for adding the prepared
whole blood sample 11 over the Xenopus oocyte 2 without using a
micro-syringe or pipette 9 will be described below in greater
details. As shown in FIG. 6, a fine reacting tube (glass capillary)
17 may be provided to the apparatus for carrying out therein the
histamine releasing reaction in the whole blood sample, such that
step (3) of the procedure shown in FIG. 4 will be completed within
the fine reacting tube 17. On the inner surface of the fine
reacting tube 17 the antigen 12 such as cedar pollen and the like
are immobilized.
[0085] A temperature controlling device (heater) 18 configured so
as to surround the outer surface of tubing wall of the fine
reacting tube 17 will control the temperature of fine reacting tube
17 at a temperature ranging from 30.degree. C. to 45.degree. C. for
promoting the histamine releasing reaction. 5 to 20 mL of whole
blood sample will be directly poured into the fine reacting tube 17
held at 37.degree. C. via the tubing of left hand side of a cock 22
in the figure and the sample will be held therein for 30 minutes to
initiate and promote the histamine releasing reaction.
[0086] Once the histamine releasing reaction in the fine reacting
tube 17 has been completed, the temperature inside the fine
reacting tube 17 will be decreased back to the room temperature and
then a cock 19 will be opened so that the blood sample solution
containing the histamine 8 released by the histamine releasing
reaction, antigen 12, basophils 13, plasma and the like will be
transferred to the surface of the Xenopus oocyte 2 held at the
recess 16 of the bottom of vessel 1 through the cock 19 and flowing
tube 21. Through the fine tip of the flowing tube 21, the solution
containing histamine 8 and the like will be added over the Xenopus
oocyte 2. After transfer the cock 19 may be closed.
[0087] The cross-sectional form of the flowing tube 21 is round and
the tube 21 is bent at the proximity of its tip. The flowing tube
21 will be inserted to an opening provided on the sidewall of the
vessel 1 in round cross-sectional shape. The axis of the opening
aligns to the projection line extendingly projected to the bottom
of vessel 1, the axis passing through the center of recess 16. The
flowing tube 21 may be movable in rotative and translatory manner
within the opening. The relative position of the tip of flowing
tube 21 with the surface of oocyte held on the recess 16 may be
adjusted by moving the flowing tube 21 in rotative or translatory
direction as described above.
[0088] After the adjustment of relative position, the sample
solution containing the histamine 8, antigen 12, basophils 13,
plasma and the like will be gently add the Xenopus oocyte 2 having
the membrane potential held at -60 mV by controllably flowing the
buffer solution 23 or sterile gas from the tubing of left hand side
of the cock 22 with the flow rate being controlled.
[0089] As have been described above, by measuring the change in
current before and after the histamine 8 is bound to the histamine
receptors of oocyte and using the predefined calibration curve, the
concentration of histamine 8 released from the cells in the whole
blood sample may be determined.
[0090] When the measurement of change in current has been
completed, the glass electrodes 3 and 4 may be drawn from the
oocyte, and the solution in the vessel 1 together with the oocyte
may be drained through the draining tube 20 by for example suction
by means of a purge pump. Then the electrodes 3 and 4 may be moved
in one side of recess on which the oocyte will be held. Some
washing solution may be poured into the vessel 1 through the
pouring tube 14 to stir washing solution with a stirrer not shown
in the figure in the vessel 1 to wash and rinse the vessel and
thereafter the washing solution may be drained through the draining
tube 20. The above washing process will be repeated three to four
times to complete washing step of the vessel.
[0091] Then, by opening the cock 19 to flow washing solution
through the tubing of left hand side of the cock 22 for several
times to wash and rinse the inside of the fine reacting tube 17,
cock 19, and flowing tube 21. Finally, the above washing process
will be iterated for several times to complete washing step of the
apparatus. In the configuration using the fine reacting tube 17,
the quantitative analysis of the concentration of histamine
released from the cells in the whole blood sample along with the
histamine releasing reaction of whole blood may be continuously
carried out. More specifically, the sequential change of histamine
releasing process may be allowed to measure in a contiguous manner.
In such a case the relative position of the fine tip of flowing
tube 21 and the oocyte held in the recess 16 should be controlled
by the rotative and translative displacement as have been described
above prior to analysis.
[0092] Then the sample will be directly injected through the tubing
of left hand side of the cock 22 to the fine reacting tube 17
having the antigen 12 such as cedar pollen immobilized on the
surface of inner wall thereof and held by the temperature
controlling device (heater) 18 at a temperature in a range from
30.degree. C. to 45.degree. C. for promoting the histamine
releasing reaction. Immediately after that, the cock 19 will be
opened to flow the buffer solution 23 held at a temperature
approximately same to that of the fine reacting tube 17 at slower
rate through the tubing of left hand side of the cock 22.
[0093] A membrane having a number of micropores through which the
histamine 8 may be passed but the basophils 13 may not be provided
for example between the fine reacting tube 17 and the cock 19.
[0094] Although the present invention has been described in
conjunction with several preferred embodiments thereof, it should
be understood that these embodiments are disclosed by way of
examples and the present invention is not to be limited thereto. It
should be recognized that many changes and modifications may be
made by those skilled in the art without departing from the true
spirit and the scope of the present invention according to the
appended claims.
* * * * *