JPH10319018A - Chromatographic device with passage cutting region - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prevent the bleeding and the aging change of a detecting band, which are resulted from an excessive expanding solvent or liquid sample, by providing a region for automatically cutting a passage after a fixed time when an analysis matter is detectable stream from a detecting region. SOLUTION: A region (connecting part 10) where a passage is cut after a fixed time from the start of expansion is provided up stream from a detecting region 8. The means for cutting the passage include dissolution after a fixed time after the contact with an expanding solvent, the loss of the capillary activity of the passage. For example, a water-soluble paper is cut to 5 mm×10 mm to form the connoting part 10, a chemical paste is applied to four corners thereof to stick the water-soluble paper onto a sample receiving part 5 and a nitrocellulose film 6. The water-soluble paper is broken after a fixed time to cut the passage. Thus, the water-soluble paper is melted a little before the judgment of the result to cut the passage, and the liquid sample quantity expanded on a chromatographic medium never exceeds a fixed quantity even if an excessive sample is applied to the sample receiving part 5.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chromatographic apparatus for avoiding an abnormal measurement value caused by an excessive amount of applied sample.

[0002]

2. Description of the Related Art Chromatography is an important technique widely used for analyzing various substances. In the field of clinical chemistry, high performance liquid chromatography is extremely useful for analyzing trace components in urine, serum, and blood. In particular, immunochromatography using a sample itself as a developing solvent is used for measurement of various items such as pregnancy diagnosis because of its simplicity, and is rapidly spreading. Most of them are devices in which a porous film such as nitrocellulose is used as a chromatographic medium, a movable labeled first antibody is disposed upstream thereof, and a second antibody is immobilized downstream thereof. The analyte and the conjugate of the analyte and the labeled first antibody are captured by the immobilized second antibody, and the amount of label is identified.

[0003] In Japanese Patent Publication No. 7-13640, a first antibody labeled with an insoluble vesicle marker (colloidal gold or colored latex) is supported on a first portion of a chromatograph comprising a first portion and a second portion. A device having a second antibody immobilized on a second portion is disclosed. When the liquid sample is dropped on the first portion, the sample and the components of the first portion move to the second portion by capillary flow, and the component reacting with the second antibody, that is, the antigen of the labeled first antibody and the antigen in the sample Antibody reactants are captured and remain in the immobilization region, and a colored band appears depending on the amount of antigen. Japanese Patent Publication No. 7-46107 discloses an immunochromatographic apparatus in which a chromatographic medium other than a sample receiving portion is protected by a hollow casing. An observation window is provided at the detection site of the casing where the second antibody is immobilized and at the downstream side of the deployment confirmation site.

[0004]

The above-mentioned method of applying a sample to a chromatograph generally involves dropping a liquid sample from a dropper-shaped sample collection container onto a sample application section of the chromatograph. If the dropping amount is too small, the sample does not spread to the detection area and no measurement result can be obtained. As a means for confirming that the development has been completed normally, a water-soluble dye disposed downstream of the detection area is eluted (JP-A-61-142).
463), and a technique of immobilizing an antibody against the first antibody downstream of the detection area and capturing the colored and labeled first antibody that has moved with the developing solution (Japanese Patent Publication No. 7-46107). On the other hand, if the applied liquid sample is too large, the sample solution developed to the end of the chromatographic medium (hereinafter referred to as chromatographic medium) flows back to the detection area, and the colored band to be detected bleeds, making it difficult to discriminate. Further, when an excessive amount of the liquid sample is supplied, even after the measurement is completed, the excessive amount of the movable labeled first antibody upstream of the chromatographic medium is gently pushed out to the detection area, and the color is further increased after the determination. That is, if a chromatogram determined to be negative is observed several hours later, it may show false positive or positive coloration. Applying an excess liquid sample to an immunochromatograph may lead to a prozone phenomenon due to an excess of antigen, which may cause a false negative.

As a means for adjusting the flow rate of the excess sample, Japanese Patent Application Laid-Open No. 4-507146 discloses a "sink" made of a microporous material.
A working sample application section is disclosed. "sink"
It acts as a dam for storing liquid samples and can control the flow rate of the chromatograph by storing excess samples. However, if a large excess sample is used, which is larger than the capacity of the dam, it is not useful, and the phenomenon that the labeled antibody is pushed out onto the chromatographic medium even after the determination is enhanced. Tokuhei 6
In -27738, the liquid sample is sucked into the water-absorbing pad provided downstream of the medium, thereby speeding up the capillary flow and preventing the excess liquid sample from flowing backward. Also in this device, since all of the applied liquid sample passes through the detection solution on which the antibody is immobilized, there is no effect in increasing the coloring of the colored band and taking measures against excess antigen such as prozone. 6-14
In 037, the liquid swelling substance provided between the two flow paths absorbs the liquid flow and swells, and a mechanism for contacting the two flow paths and the pressure of the liquid swelling substance provided at the end of the other flow path. A switch mechanism for cutting off communication between two flow paths has been devised. It's a clever mechanism, but its structure is complicated,
Since the liquid swelling substance is provided downstream of the detection area, it is not very effective for countermeasures against antigen excess, as in JP-B-6-27738.

[0006] The immunochromatographic apparatus characterized by its simplicity is often used not only by skilled technicians but also in ordinary households as represented by pregnancy diagnosis.
Therefore, the instruction manual attached to the device clearly describes standard usage and precautions. However, there are many cases where a user who has fully understood the contents applies a large excess of the sample in use. If the amount is insufficient, the user may be worried that correct measurement values will not be obtained and will apply the excess amount. It is very difficult to eradicate misuse based on such psychological factors, and it cannot be prevented only with the care of the instruction manual. Therefore, there is a demand for the development of a device that gives a correct measurement value by automatically using the required amount of sample if a relatively large amount of sample is applied instead of a device that forces the user to adhere to the amount of applied sample. Was.

[0007]

SUMMARY OF THE INVENTION A solution to this problem is to provide an apparatus having means for supplying no excess of a liquid sample or excess of a developing solvent applied to a chromatograph to a chromatographic medium. That is. Specifically, this is an apparatus provided with a portion (hereinafter, referred to as a flow channel cutting system) in which a flow channel is cut after a certain period of time from the start of development, upstream of the chromatography medium. Means for cutting the flow path include dropping a flow path made of a substance that dissolves or loses physical strength after a certain period of contact with the developing solvent, or that loses the capillary action ability of the flow path. The most suitable substance that causes the channel to be missing in the aqueous developing solvent is water-soluble paper. The term "water-soluble paper" used herein refers to a paper made of a fiber component and a water-soluble binder such as carboxymethyl cellulose or a paper made mainly of carboxymethyl cellulose. A product obtained by coating one side of the water-soluble paper with a polyvinyl alcohol resin, a polyvinylpyrrolidone resin, or the like to slow the dissolution rate in water may be used.

[0008] These water-soluble papers have almost the same water absorption as filter paper, and support capillary action. The development time of the chromatograph varies depending on the object to be measured, but if the water-soluble paper dissolves shortly before the result is determined and the flow path is cut off, the development on the chromatographic medium will be possible even if excess sample is applied to the receiving part. The amount of liquid sample to be performed does not exceed a certain amount.

The channel cutting system is also achieved by gradually losing the capillarity of the cutting site due to the developing solvent. For example, if a porous polyester sheet impregnated with a microcapsule soluble in a developing solvent containing an adhesive substance is used, the adhesive substance flowing out of the microcapsules broken by contact with the developing solvent fills the porous material and Flow is stopped. In a thin-layer chromatograph or a liquid chromatograph using many organic solvents, microcapsules soluble in an organic solvent may be used, and microcapsules soluble in water may be used if the developing solvent is an aqueous solution. The cut portion may be formed by using a filter paper impregnated with a substance which is solidified or hardened by a developing solvent. Filter paper impregnated with a small amount of α-cyanoacrylate hardens upon contact with moisture and loses capillary action. In addition, if the bisepoxy compound and the bifunctional substance as the polymerization initiator are arranged at different positions of the cleavage site, both the reagents come into contact with the developing solvent and are cured to cut the flow path. By arranging these flow path cutting portions upstream of the detection region of the chromatographic medium, it is possible to avoid all the above-mentioned problems caused by the excessive supply of the sample.

The purpose can be achieved by arranging the flow path cutting portion at any position upstream of the detection area of the above-mentioned chromatographic apparatus. However, when the cut portion is arranged in the middle of the chromatographic medium, the chromatographic liquid flow may be disturbed and the image of the detection area may be blurred. Therefore, it is preferable that the cleavage site is located on the upstream side of the chromatographic medium, especially between the sample receiving portion and the chromatographic medium, avoiding the chromatographic medium. The simplest structure is one in which the sample receiving portion of the above-mentioned chromatograph and the chromatographic medium are arranged with an interval of about 5 mm, and the sample receiving portion and the chromatographic medium are communicated with each other by a water-soluble paper as a cutting portion.
The sample receiving section is a sheet or block made of a porous resin or nonwoven fabric, and may contain a first antibody that has been labeled with a colored latex or the like in advance. The chromatographic medium is, for example, a material in which a polyester sheet or the like is lined with a nitrocellulose membrane, and the second antibody is immobilized in the detection area. When the labeled first antibody is not contained in the sample receiving portion, the labeled first antibody is movably arranged several cm upstream of the detection area on the chromatography medium. The technique disclosed in Japanese Patent Publication No. 7-46017 can be used for labeling the antibody, arranging the antibody on a chromatography medium, and immobilizing the antibody.

The above-mentioned components such as the sample receiving portion and the chromatographic medium are arranged at predetermined positions, and water-soluble paper, which is approximately the same width as the chromatographic medium and cut to a length of about 1 cm, is placed in each of the sample receiving portion and the chromatographic medium. Overlap. It is even better to glue both ends of the water-soluble paper to complete the contact.
When water-soluble paper coated with a hydrophobic substance such as polyvinylpyrrolidone resin on one side is used, the sheets must be overlapped with the coated surface facing upward.
This is because not only the movement of the liquid sample is incomplete, but also the adhesion is difficult. The gap between the sample receiving portion and the chromatographic medium is necessary to such an extent that the water-soluble paper which collapses after dissolution does not remain in the flow path.
When the chromatograph is placed vertically and deployed upward, the flow path can be completely shut off even if the gap is 2 mm. When developing the chromatographic medium horizontally, it is safer to secure a gap of about 5 mm. Furthermore, it is more preferable to arrange an empty room for receiving the collapsed fiber mass or an empty room including a water absorbing pad immediately below the communication portion.

One end of the water-soluble paper may be laid under the sample receiving portion, and the other end may be overlaid on the chromatographic medium. In this case, since the liquid sample flows from the front surface to the back surface, water-soluble paper coated on one side cannot be used. Alternatively, a cut may be provided in the sample receiving block in advance, one end of the water-soluble paper may be inserted into the cut, and the other end may be overlapped with the chromatographic medium. What is necessary is just to select the arrangement method of these communication parts according to a casing so that an existing casing can be used as it is.

Since the chromatographic medium is lined with a transparent sheet (polyester), it has sufficient elasticity. By utilizing this elasticity, a flow path cutting system can be assembled. After fixing the downstream end of the chromatographic medium having a length that overlaps with the sample receiving portion, it is joined to the sample receiving portion so as to bend (with a physical load) through the water-soluble paper. When the water-soluble paper is dissolved by the liquid sample, the bent chromatographic medium is separated from the sample receiving portion by elasticity, and the flow path is cut. Due to the limited thickness of the casingd chromatographic device, the sample receiving part is arranged so that the chromatographic medium is lower than the parallel extended height. Alternatively, arranging a spacer in the casing on the side facing the backing sheet of the chromatographic medium facilitates flexing. The position of the spacer is most effective at a portion corresponding to the center of the chromatographic medium.

The overlapped sheets are pushed down to the sample receiving portion, and the sample receiving portion and the chromatographic medium are joined by water-soluble paper having both ends glued. By adhering at a position slightly away from both ends of the water-soluble paper using a strong adhesive, the water-soluble paper is pressed against the sample receiving portion and the chromatographic medium, and the liquid sample can move without being affected by the adhesive. If the sample receiving portion and the leading end of the chromatographic medium are sufficiently separated, the residue of the water-soluble paper remaining in the sample receiving portion after the dissolving of the water-soluble paper does not come into contact, and the flow path cutting does not become insufficient. It is necessary to keep pressing the joint until the adhesion is complete because it is bonded to the sample receiving part against elasticity, but the operation is completed in seconds if an instant adhesive is used.

A channel cutting system utilizing the elasticity of a chromatographic medium can be realized without using a substance soluble in a developing solvent. A flow path cutting system can also be achieved by locally directly bonding the sample receiving portion to the nitrocellulose side of the deformed chromatographic medium with an adhesive such as a normal glue that loses adhesive strength due to penetration of moisture. However, in order to fix the sagging chromatographic medium by such local adhesion of the adhesive, it is troublesome to maintain the pressure bonding until the adhesive strength increases. If a commercially available adhesive tape (cellophane tape, gum tape) is used, a cutting system can be obtained more easily. The upstream end of the chromatographic medium is overlapped with the sample receiving portion while being bent, and an adhesive tape cut to an appropriate size is stuck thereon and fixed. Since the adhesive tape does not hinder the movement of the developing solvent at all, it can be made large enough to obtain a sufficient adhesive force instantaneously. Upon contact with the developing solvent, the adhesive tape rapidly loses its adhesive strength, and after a certain period of time, the flow path is cut by the elasticity of the chromatographic medium.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0017]

Example 1 OC-Hemocatch 'Eiken' (Eiken Chemical Co., Ltd.) which is a commercially available immunochromatography apparatus for fecal occult blood test
Hereinafter, it is called hemo catch. ) Was provided with a communicating part made of the water-soluble paper of the present invention. The hemocatch is a 0.8 μm thick nitrocellulose film (0.7 cm × 5 cm) lined in a hollow casing with a water absorbing sheet (1 mm thick) of 0.7 cm × 2 cm as a sample receiving portion and a transparent sheet as a chromatographic medium. ) Are stored with their ends in contact, and the block contains blue colored latex sensitized with the mouse primary antibody against human hemoglobin A0. An anti-human hemoglobin A0 mouse second antibody was immobilized at a position 1.5 cm downstream (detection area) from the upstream end of nitrocellulose, and at a position 1 cm downstream of the detection area as a control area to confirm the spread of the liquid sample. An anti-mouse IgG antibody is immobilized. In the upper part of the casing, a hole (sample well) for applying a sample is provided just above the sample receiving part, and an observation window is provided just above the detection area and the control area.

The measurement is started by dropping two drops of the filtrate of the stool suspension from the dedicated stool collection device into the sample well. The dedicated stool collection device contains 2 ml of buffer solution, and the stool collected with the stool collection stick attached to the device is 1 /
It is diluted to 200 and filtered for use with a filter provided in the dropping part. If hemoglobin is present in the sample dropped into the sample receiving part, the sample binds to the mouse latex-sensitized blue latex. The sample solution is developed into the chromatographic medium by capillary action, and hemoglobin and blue latex bound to hemoglobin are captured by the mouse second antibody immobilized in the detection area to form a blue band. Furthermore, the sample liquid developed on the chromatographic medium causes a blue line to appear in the control area 5 minutes after the sample droplet is dropped, but if the sample liquid is insufficient, the control liquid does not reach the control area. No blue line appears.

FIG. 1 is a side view showing a state in which a bottom cover of a casing of a hemocatch is removed and a communicating portion of water-soluble paper is attached. 1 is a lower half of a casing, 2 is a sample application well provided in the casing, 3 is a detection area observation window, and 4 is a control area observation window. Reference numeral 5 denotes a polyester water-absorbing sheet of a sample receiving part containing a blue sensitized latex, 6 denotes a nitrocellulose film lined with a transparent sheet, 7 denotes a transparent sheet, 8, 9 denotes detection areas provided on the nitrocellulose film, And a control area, and 10 indicates a water-soluble paper of a connecting portion. The actual work was done with the top lid of the casing down. The lid of the casing of the hemocatch is removed, and 1 cm of the chromatographic media 6, 7 overlapping the sample receiving portion 5 is cut off. Since this part does not contain any components related to the measurement, even if it is cut out, it does not interfere with the measurement. A water-soluble paper (made by Mishima Paper, basis weight 30 g / m ₂ ) is cut into 5 mm × 10 mm and the communicating portion 5
Chemical glue splits (manufactured by KOKUYO Co., Ltd.) were lightly applied to the four corners, and were applied to the sample receiving portion 5 and the nitrocellulose film 6 in an overlapping manner. After leaving in a desiccator for 1 hour to completely dry the glue, the bottom lid of the casing was set.

The sample to be used for the measurement was prepared as follows. Human fresh blood was lysed, and 1 ml of the lysate was added to 100 ml of 10 mM phosphate buffer (pH 7.4) to obtain a high concentration hemoglobin solution. Commercially available hemoglobin measuring reagent OC-
Using Hemodia Auto 'Eiken' (trade name, manufactured by Eiken Chemical Co., Ltd.), the above high-concentration hemoglobin solution was treated with the above phosphate buffer.
300 μl of reagent in 50 μl of measurement sample diluted 1,000 times
Was added and reacted at 37 ° C. for 3 minutes. The hemoglobin concentration was determined from the change in absorbance at a wavelength of 585 nm based on the immunological agglutination during this time. As a standard, human hemolyzed blood (phosphate buffer containing 2 μg / ml human hemoglobin) attached to the reagent was used. Based on the measured values obtained, a high-concentration hemoglobin solution was added to a 10 mM phosphate buffer (pH
7.4), appropriately dilute, and hemoglobin concentration 100 ng /
Samples for measurement of 5, 5, and 500 μg / ml were obtained. Using a dropper, 2 drops and 5 drops of the sample of each concentration were placed on the apparatus prepared in this example using hemocatch as a control.
Drops were dropped on the sample application well 5 of each device. After leaving for 5 minutes with the downstream side of the device raised about 2 cm and tilted, the detection area 3
Was visually determined. The criterion was-, ±, +, ++ according to the density of the blue band.
Table 1 shows the results.

[0021]

[Table 1]

In the case of two drops, which is the standard sample application amount for hemocatch, there is no discrepancy in the measured values between the present invention and the control hemocatch. This suggests that the communication section made of the water-soluble paper does not affect the capillary flow of the chromatography. On the other hand, as a result of dropping five drops which was twice or more of the standard application amount, dissociation was observed in the high concentration sample. The measurement range of hemocatch is 50 ng / ml to 500 μg / ml,
The ± value of the 500 μg / ml sample measured by hemocatch was ± due to prozone in excess of antigen. Although not shown in the table, the blue band in the detection area of hemocatch where 5 drops were applied was blurred and difficult to determine. After the measurement, the upper lid of the apparatus of the present invention was removed, and the communicating portion of the water-soluble paper was observed. In each case, the water-soluble paper collapsed and the flow path was completely cut.

[0023]

Example 2 The chromatographic media 6, 7 were removed from the casing 1 of the hemocatch. A 5 mm × 5 mm, 2 mm thick cardboard was pasted as a spacer 11 on a casing at an intermediate portion between the detection area observation window 3 and the control observation window.
The chromatographic media 6 and 7 were returned to the casing 1 again, and the downstream end was fixed. Water-soluble paper (made by Mishima Paper, basis weight 60g /
m ₂ ) was cut into 5 mm × 10 mm, and a portion of 5 mm was placed on the upstream end of the nitrocellulose film 6 and a part thereof was adhered with Aron Alpha (trade name of adhesive, Toa Gosei). Two minutes later, Alon Alpha was thinly applied to the four corners of the unbonded portion of the connecting portion 10, and the chromatographic media 6, 7 existing apart from the sample receiving portion were pressed down by the spacer 11, and quickly pressed to the sample receiving portion 5. The chromatographic media 6 and 7 were fixed in a gentle curve with the spacer 11 at the top.

Using this apparatus, human hemoglobin was measured in the same manner as in Example 1, and after 5 hours, the colored band of each apparatus was observed. Table 2 shows the results.

[0025]

[Table 2]

The determination value after 5 minutes has almost no difference from the hemocatch as in the first embodiment. This suggests that the water-soluble paper connection by the apparatus of Example 2 also did not affect the capillary flow of the chromatographic medium. However, in the re-evaluation after 5 hours, the colored band of hemocatch increased by almost one rank, whereas no change was observed in the colored band of the present invention. In the apparatus of the present invention in which the flow path is cut off about 5 minutes after the sample is applied, this is because there is no supply of the excess liquid sample and the labeled antibody, and the effect of the connection part by the water-soluble paper is remarkable.

[0027]

As described above, by incorporating the cut portion of the present invention, which cuts the flow path after a certain period of time from the start of development, in a chromatographic apparatus, various problems of the chromatograph caused by a large excess of the developing solvent and liquid sample can be solved. You. Specific effects include prevention of bleeding of the detection band, prevention of the change over time of the detection band, and avoidance of the prozone phenomenon in immunochromatography.

[Brief description of the drawings]

FIG. 1 is a side view of an example of a basic embodiment of the present invention.

FIG. 2 is a modified example of a connecting portion.

[Explanation of symbols]

1. Top lid of casing 7. Polyester sheet 2. 7. Sample application well Detection area 3. 8. Detection area observation window Control area 4. Control area observation window 10. Connecting part 5. Sample receiving section 11. Spacer 6. Nitrocellulose film

Claims (12)

[Claims] 1. A chromatograph using a developing solvent, wherein a chromatograph apparatus is provided upstream of a site for detecting an analyte and has a site for automatically cutting off a flow path after a predetermined time after the analyte can be detected. 2. The apparatus according to claim 1, wherein the channel cutting portion is made of a substance that dissolves in a developing solvent and / or loses physical strength. 3. The apparatus according to claim 2, wherein the substance that dissolves in the developing solvent and / or loses physical strength is water-soluble paper. 4. The apparatus according to claim 3, wherein water-soluble paper is used which dissolves within 30 seconds to 5 minutes after being hydrated. 5. The apparatus according to claim 2, further comprising a channel cutting system in which the chromatographic medium is immobilized under a physical load by a substance that loses physical strength due to the developing solvent. 6. The apparatus according to claim 2, wherein after the substance dissolved in the developing solvent is dissolved in the developing solvent, the flow path is blocked by the natural fall of the substance dissolved in the developing solvent. 7. The apparatus according to claim 6, further comprising a space for accommodating a substance dissolved in a developing solvent. 8. The apparatus according to claim 7, wherein a water absorbing pad is provided in a space for containing a substance dissolved in the developing solvent. 9. The apparatus according to claim 1, wherein the flow channel cutting portion is made of a substance which is solidified by the developing solvent and loses the capillary action. 10. The apparatus according to claim 1, wherein the measuring system is an immunochromatograph. 11. The apparatus according to claim 1, which is an immunochromatograph using an insoluble carrier as a marker. 12. A chromatographic apparatus having a communication part made of a water-soluble substance upstream of a chromatographic medium, wherein the communication part made of a water-soluble substance is dissolved by a developing solvent containing a liquid sample and a flow path is cut off. Measurement method used