JPH01124749A - Method for measuring multi-sample easily, speedily and high sensitively - Google Patents

Abstract

PURPOSE:To easily and speedily inspect many samples by using a microplate or strip cell and combining a fluorophotometer for the high-sensitivity microplate and an absorptiometer. CONSTITUTION:Hemoglobin is adsorbed by special filter paper as blood filter paper, and dried and then punched by a punch into a disk of proper size, which is put in a test tube or microplate container. Then hemoglobin denaturation fixation is carried out by a formate vapor method, a steam denaturation fixing method, or a denaturation fixing method by a special combination system of plural kinds of organic solvents. Thus, many samples are processed in a short time at the same time and measured by the microplate type fluorophotometer or absorptiometer as measuring equipment to establish a fine amount determining method for various fluorescent materials and fluorescent coloring matter such as absorbing material, thereby measuring various materials having them as reaction systems.

Description

[Detailed Description of the Invention] [Field of Application] The present invention aims to reduce the amount of target samples in clinical tests, to minimize the amount, to simultaneously process multiple samples, to simplify, speed up, and save labor. By changing the method and performing the reaction method in a microplate container, and measuring with a microplate fluorometer or spectrophotometer as a measuring device, various fluorescent substances and absorbing substances such as reduced nicotine adenine dinucleotide and reduced type nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NAD (P)
II), p-nitrophenol, umbelliferone, HPPA-H20□ condensate, tyramine-H,0,
Condensate, FITCSDTAF, XRITC.

TRITC, S ITC, Cover Spheres (Blue.

The establishment of a microquantitative method for fluorescent dyes (red, green) has made it possible to measure various substances that have these in reaction systems, such as enzyme activities and substrates. In addition, blood samples can be easily used by using a blood filter paper disk and introducing a hemochromatic fixation process. Furthermore, it is now possible to process large numbers of samples in a short time without diluting them with only a small amount of material, making it ideal for mass screening tests for neonatal and adult diseases, as well as mass tests for cancer, which have recently been widely implemented around the world. It is.

[Prior art]

Conventional measurement methods involve reaction operations in test tubes, cuvette cells, flow cells, and test tube cuvettes, which require a large volume of liquid and require large amounts of samples and reagents due to dilution, and require measurements using a highly sensitive fluorometer. However, it was not suitable for measuring trace concentrations of reagents or samples. In addition, protein removal operations are complicated, such as addition of large amounts of acids, alkalis, salts, heavy metals, and organic solvents, and centrifugation operations, and mass processing that requires processing a large number of samples.
Screening requires a long time for both operation and measurement, and requires a lot of effort.

[Problem that the invention seeks to solve]

The disadvantages of the conventional test tube method are the large volumes of reaction and measurement solutions, the complexity of protein removal and centrifugation operations, and the time and labor required for the measurement operations themselves. Future tests that require high sensitivity, speed, simplicity, and labor saving, especially newborn mass screening, adult disease, and cancer tests, will require some ingenuity.

[Means for solving the problems and their effects] In order to save valuable samples, reduce costs, and measure trace amounts, it is necessary to reduce the amount of liquid in the reaction system and measurement system. In addition, microplate containers are very advantageous for processing multiple samples at the same time in a short time (pretreatment, reaction operation, washing operation, measurement operation), as well as for being simple, quick, and labor-saving. be. The inventors devised and improved the reaction system to suit these purposes, and were particularly successful in reducing the amount. In addition, when using blood samples, protein removal operations (addition of large amounts of acids, alkalis, salts, heavy metals, and organic solvents) and centrifugation operations are cumbersome and time-consuming, especially in the case of multi-sample tests such as mass screening. Since it takes a lot of force, new innovations are required. In other words, blood pigments are adsorbed onto a special filter paper, such as blood filter paper used in neonatal mass screening, in order to interfere with fluorescence measurements. Or place it in a microplate container and perform hemochromatic fixation first. There are several methods for this. For example, the formic acid vapor method, the steam denaturation fixation method, and the denaturation fixation method using a special combination of several types of organic solvents can process many samples at the same time in a short time, and they do not require protein removal or centrifugation like conventional methods. This will instantly solve the huge amount of effort and complexity that was involved in the past.

a) Formic acid vapor method A beaker containing 98% formic acid is placed in a sealed container, a microplate containing a sample is placed in the container, the container is sealed, and the container is left in saturated vapor of formic acid at room temperature for 1 hour to denature and fix hemoglobin. In order to remove the acid attached to the removed microplate, it was placed in a desiccator with a stopcock and suctioned with a water pump.
Turn off the cook and put it in a 37°C thermostat for several hours, or blow it away with a hair dryer.

b) Steam denaturation fixation method The hemoglobin is denatured and fixed by keeping the steam temperature at 80-85°C and exposing the sample to the steam for 10 minutes. The moist blood filter paper is 3
Dry in a constant temperature oven at 7°C or dry with a hair dryer.

C) Modification fixation method using organic solvent The ratio of water: acetone: methanol (20:20:60%) mixed thread and ether or methylene chloride is 1:3 to 1:3.
Add 10-20μ2 of normal solvent solution at 5 to 2 blood filter paper disks. Ordinary microplates are sensitive to solvents, so plates made of vinyl chloride are used. Leave at 37°C for 130 minutes.

Furthermore, the advantage of the present invention is that since the sample is not diluted, the measurement sensitivity is significantly increased and it is possible to reduce the amount of the sample. or,
We have developed a hemoglobin fixation method that fixes only the hemoglobin protein on filter paper so that enzyme activity in living organisms, especially blood, can be processed and measured simultaneously in multiple samples without using isotopes, etc., but the enzyme protein can be easily extracted (Example 6).

The ESISA method has been devised to date for the use of microplates, but the method currently used is based on an absorbance meter, which has its limitations, especially when measuring trace amounts in vivo, where highly sensitive fluorescence measurements are required. Rapid multi-sample measurement of substances such as trace hormones and blood metabolites requires a microplate-based highly sensitive fluorescence needle.

Most of the substance concentrations in blood are around 10-6 to 10-1 °M. The detection limit of conventional measuring instruments, for example Corona Electric's MTP22F and Titertic's Fluoroscan, is 10-h to 10-''M, but the recently developed Corona Electric's MTP32F and 100F can be used to reach 10 Since it has become possible to measure -6 to 10-''M, and highly sensitive measurements can be expected, the fields and objects of use of the substance to be measured have expanded. If a measuring instrument with improved sensitivity to such small amounts is used, the microplate method of the present invention, which can complete pretreatment, reaction operations, washing operations, and measurement operations for many samples at once in a short time, will become a mass market.・Ideal for screening tests.

The present invention can be applied, for example, as a newborn mass screening test, for the determination of galactose and galactose-1-P in the blood to detect galactosemia, galactokinase, uridine diphosphate-galactose-4-epimerase, and uridyltransferase. Measurement of TSH and T4 for hypothyroidism and hypothyroidism (fretinism), 17α-hydroxyprogesterone (17α-hydroxyprogesterone) for adrenal cortical hyperplasia
7α-0HP), testing methods for urea cycle metabolic disorders such as ornithineemia, argininemia, and arginosuccinic aciduria, clinical tests for metabolites with NAD(P)H as a coenzyme, insulin and fetal , Fetoprotein measurement, newborn and adult hemoglobin and cholesterol measurements, the list is endless.

Next, the present invention will be described with reference to examples, but the present invention is not limited thereto.

Example 1. Rapid quantification of umbelliferone Rapid quantification of umbelliferone is achieved by changing the voltage applied to the photomultiplier of a Corona Electric MTP32F or 22F microplate type fluorometer to adjust the concentration from high to low. It can be measured. Figure 1 shows the measurement range of each photometer. The numbers in the figure indicate each concentration of umbelliferone, 100 μr, and the load voltage.

Corona Electric's MTP22F type, Titertic's Fluoroscan is 10-6 ~ 10-7 ~ (10-1 l) M,
Corona Electric MTP32F is 10-h~10-9~(1
0-”)M, Corona Electric test tube insertion type FPMII is 1
Suitable for measurement of 0-6 to 10-''' to (10-'')M.

When the voltage is increased, the fluorescence intensity increases.

Example 2. Mass screening method using highly sensitive and rapid measurement of TSH Standard blood filter paper or general sample blood disk 4III + 1 (blood 4
~6 μl), β-galactose dehydrogenase labeled anti-human T
Mix 100 μl of SH rabbit IgG serum, put a seal on the plate, react overnight at 37°C, remove the contents, and add 0.01M phosphate buffer (pH 7.4)-0.I.
M NaC1-1mM MgC1z (Tween 2
Wash 3 times with 300 μl (containing 0). During this time, remove the filter paper disc. 4-Methylumbelliferyl-β-
After adding 100 μi of D-galactoside and reacting at 37°C for 11 hours, add 200 μl of 0.1 M glycine-NaOH buffer (pH 10,3) as a reaction stop solution and mix.
Measured with X365nn+ Ets 450nta. Figure 2 shows the TSH calibration curve.

Example 3. Rapid quantification of NAD(P)H For rapid quantification of NAD(P)H, place 100μ2 of NADH solution in each cell of a microplate and use Corona Electric's M
By changing the photomal load voltage with TP32F,
Concentrations suitable for purposes can be measured from low to high concentrations. Figure 3 shows the NADH measurement range when the photomultiplier voltage is 300V.At sensitivity 1, NADH
~40 nmol/100 ul in O at sensitivity 2
High sensitivity measurement of ~2nmol/100μi is possible.

Example 4. Blood galactose (Gal) and galactose-1-P (Gal-1-P) quantitative reaction system 1:
(Gal+Gal-1-P) is 13mM NAD 10
ul.

1M Tris-HCl (pH 8.0) 10 μl, alkaline phosphatase (AP, Boehringer) 10.0 OOU
/d1μ! , β-galactose dehydrogenase (β-Gal
-DH) 9μ2 and blood sample or standard galactose-containing blood filter paper (0-3mM) (1 piece of filter paper disk 3+@+m diameter with hemoglobin fixation treatment) or Gal aqueous solution 0-4 r
tmol.

Total 30μ! Reaction system 2: For (Gal), remove AP from reaction system 1 and add distilled water.

Reaction system 3: (Blank) is β-Gal from reaction 1
- Remove only DH and add distilled water.

Perform each reaction system in a microplate at 37°C for 1 hour, add 100 μl of distilled water or buffer, mix well, and transfer the 100 μl to another microplate.

Figure 4 shows the calibration curve of Gal and Gal-1-P.

The amount of Gal-1-P is calculated from reaction system 1-2, and the amount of Gal is calculated from reaction system 2-3, and determined from the calibration curve. Furthermore, the fluorescence intensity varies greatly depending on the type of microplate, so a plate with little variation among wells should be selected. Figure 5 shows the correlation between the final method and the conventional method, r ~ 0.959
It shows that there is a good correlation with

Example 5. Mass screening method for urea cycle metabolic disorder, trace amount determination of blood ornithine In each well of a microplate, 5 μmol of Tris-HCl (pH 8,0), 0.25 μmol of α-ketoglutaric acid, 0 dithiothreitol was added as a reaction system. .5n
mol, pyrroline-5-carboxylic acid reductase (manufactured by Amano Pharmaceutical) 3.7ml+, ornithine transferase (manufactured by Amano Pharmaceutical) 3.7ml, NADH 3-5nmol, ornithine O-2nmol or 0-2mM blood filter paper disk or test blood Filter paper disk (3 after hemochromatic fixation treatment)
1 mm diameter), total 50μ! The control is a reaction system in which no sample is added. The reaction was carried out at 37°C for 11 hours, then with distilled water at 80°C.
Add μ2, mix, and transfer 100 μl to another microplate for measurement. Figure-6 shows the calibration curve, and CV
3.1 to 6.0%, 71 mM (7) One 3 mm blood disc corresponds to 2.1 nmol.

Example 6. The hemoglobin fixative used in the new hemoglobin fixation method that does not deactivate enzyme activity is first of all solution A [water: acetone:
Add 10 μl of methanol (30:35:35%) to each sample, dry and fix at 37°C for 30 minutes, and then add solution B [
Mixture system of water:acetone:methanol (20:20:60%) and ether or methylene chloride in a ratio of 3 to 5:110 to
If 20 μl is added and left at 37°C for 30 minutes, most of the hemoglobin will be fixed, but the enzyme protein will not be deactivated and can be extracted.

The following systems can be used as liquid B.

For example (10:10:80) 115 (20:1
0 near 0) 115~1/1 (20: 6
0:20) 3/1~1/1(2
0:10 near 0) l10 (10:80:
10) 1. /1~1/4 (30:10:
60) 1/4 (30: 35: 35
) 1/1 to 1/4 Example 7. Rapid fecal measurement method for trace hemoglobin Place one 3fflIIIφ blood disk (2.5μ) into a microplate and add 200μ2 of hemoglobin measurement solution.Use 200μ2 of the stock solution and 1/2 diluted solution of acute globin as the standard solution.
After reacting at 37°C for 130 minutes, 100μ2 of the solution was dispensed into another measurement plate, and the A 55° absorption was measured using Corona Electric Microplate Absorption Photometer MTP32. Figure 7 shows a calibration curve, and Figure 8 shows dried blood filter paper disks and quantitative properties of hemoglobin amount.

Example 8. New mass screening method for galactokinase deficiency (simple and rapid measurement method for galactokinase) Gal 1mM, NaF 1mM, MgC
Iz 8mM++ DTT 10℃ starvation 8TP
60-120mM, saponin 0.01%, hydrogen peroxide 0
．． 01%, Tris-HCI (pH 7,4) 200
mM, 100μ2 of reaction solution per disk at 37°C16
0 minutes to 90 minutes, NAD IOIIIM 1
0,1/L β-Gal-DH stock solution 25U/mj!
Add 10 μ2 of a diluted solution of
Measured at PM 250V, sensitivity 1 at Em 450nm.

In this case, as a pretreatment, hemoglobin charcoal fixation of the blood disk is performed. At this time, a fixative solution is used that can extract galactokinase activity without inactivating it. First, place a 3mm-1 blood sample disk on a Falcon thin disposable plate.
and 10 al of organic solvent mixed fixative solution A solution each.
Leave it at 37°C for 15-30 minutes, and add 10μ of the same fixative B and a solvent with an ether ratio of 3/1! Add and heat to 37°C13
After processing for 0 minutes, add the reaction solution. In addition, blank discs are fixed with formic acid vapor or water vapor (120'C11o).
minutes).

-Results As shown in Figure 9, it was confirmed that a linear relationship was established between the amount of galactokinase and the fluorescence intensity in the fluorescence intensity range of 0 to 200, and that quantification was possible.

Example 9. Mass screening for hypercholesterolemia Following the denaturation and fixation treatment of hemoglobin according to the method of Example 5, extraction was performed and the cholesterol measurement was performed using a cholesterol measurement kit (Sterozyme 545, manufactured by Fujirebio). As a result, good linearity was obtained in the cholesterol concentration range of 0 to 800 μ/d1, and the addition recovery rate was 95 to 10.
It was a good 5%.

〔Effect of the invention〕

According to the present invention, many samples can be tested more easily and quickly than ever before, and mass screening can now be performed with high sensitivity.

[Brief explanation of the drawing]

Figure-1 shows the quantitative results of umbelliferone, and -Kenichi, -1-1, and - respectively indicate Corona FPM11.
These are the results for load voltages of 750■, 550■, and 440■, respectively, -0- is the result for Corona MTP22F, and -0- is the result for Corona MTP32F. Figure 2 shows the TSH calibration curve. Figure 3 shows the relationship between NADH and fluorescence intensity, where -1-0- are the results for sensitivity 1 and sensitivity 2, respectively. Figure-4 is Gal, Gal-
It shows the relationship between 1-P and fluorescence intensity, -・-1-
0- is the result of sensitivity 1 and sensitivity 2, respectively. Figure 5 shows the correlation between the nature and the conventional method. Figure 6 shows the calibration curve for ornithine. Figure 7 shows the calibration curve of acute globin. Figure 8 shows the relationship between the number of blood discs and the amount of hemoglobin. Figure 9 shows the relationship between galactokinase and fluorescence intensity.

Claims (1)

[Claims] 1) A simple and rapid multi-sample high-sensitivity measurement method characterized by using a microplate or a strip cell in combination with a high-sensitivity microplate fluorometer or spectrophotometer. 2) A simple, rapid, and highly sensitive multi-sample measurement method according to claim 1, which uses dried blood filter paper as a measurement sample. 3) Reduced nicotine adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate, umbelliferone or 4-methylumbelliferone, 3-p-hydroxyphenylpropionic acid (HPPA)-hydrogen peroxide condensate, and tyramine-peroxide condensate. The simple, rapid, multi-sample, high-sensitivity measurement method according to claims 1 and 2, which comprises a hydrogen oxide condensate and nitrophenol. 4) The fluorescent dye is fluorescein isothiocyanate (F
ITC), dechlorotriazinylfluorescein (DT
AF), rhodamine isocyanate (XRITC), tetramethyl rhodamine isocyanate (TRITC),
4-acetamido-4″-isothiocyanostiben-2
, 2'-desulfonic acid (SITC), and Cobaspheres (blue, red, green). 5) Claim 1 in which the measurement target is hemoglobin
The simple, rapid, multi-sample, high-sensitivity measurement method described in Sections 1 and 2. 6) A simple, rapid, multi-sample, high-sensitivity measurement method according to claims 1 and 2, wherein the measurement target is cholesterol.