Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2001-02-21
2001-12-11
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S004000, C435S007100, C435S007920, C435S013000, C435S174000, C435S283100, C435S285200, C435S286700, C435S287200, C435S287900, C435S288500, C435S808000, C436S018000, C436S063000, C436S147000, C436S164000, C436S172000, C436S518000, C436S543000, C436S544000, C436S545000, C436S546000, C436S805000, C436S807000, C204S400000, C204S403060, C204S408000, C204S409000, C204S422000, C422S044000, C422S530000, C548S335500
Reexamination Certificate
active
06329194
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to a histamine measuring apparatus and a histamine measuring method for quantitatively measuring histamine present in blood or mast cells suspensions.
2. Description of the Prior Art
A histamine releasing test is a quantitative analysis of histamine extracellularly released by stimulating leukocytes (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 (Tabe, kazuaki: histamine release test—diagnosis of allergies, SRL HOKAN, vol 21, pp. 17-22 (1997)
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 (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 (Japanese Patent Laid-Open No. H
10-170514
, Japanese Patent Laid-Open No.
10-62415
), competitive immunoassays commercially available from ICN Pharmaceuticals or Immunotech, and an ELISA.
SUMMARY OF THE INVENTION
In general, the measurement of histamine may require:
(1) direct analysis of histamine without using labeling thereof;
(2) direct quantitative measurement of histamine without pretreatment;
(3) quick delivery of results; and
(4) specificity to the histamine.
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 histamine analysis.
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.
In accordance with the histamine quantitative analyzing apparatus and method of the present invention, histamine quantitative analysis will be achieved by:
providing cells with histamine receptor being expressed;
adding a sample having been stimulated by an antigen to the cells expressing the receptor; and
detecting the resulting electrical response of cells.
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° 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.
Examples of sample include, whole blood specimen, leukocytes in the blood, and mast cell suspensions in which the mast cells of mucosal tissue are cultured.
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.”
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.
The ratio of histamine release &agr; (%) may be determined by
&agr;=100×(A−C)/B
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.
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”.
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.
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 he 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.
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 or reagents may be reduced to minimum and the cost and the time needed for the measurement may be significantly saved.
In the following description an oocyte of Xenopus lavis (African clawed frog) will be used for the cell expressing the histamine receptors, by way of example. It should be understood that the present invention may be applicable by using any other types of cell.
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 membra
Otomo Jun
Takeshita Tomoko
Hitachi , Ltd.
Le Long V.
Mattingly Stanger & Malur, P.C.
Padmanabhan Kartic
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