JPS6238346A - Fluorescent polarized light measuring instrument - Google Patents

Abstract

PURPOSE:To eliminate the influence of background fluorescence, to simplify an operation for measurement and to shorten the time for measurement by simultaneously measuring the vertically polarized light and horizontally polarized light of the fluorescent light with two wavelengths. CONSTITUTION:Excitation light from a light source 1 is horizontally and perpendicularly polarized by a polarizer 4a and is irradiated to a sample 5.The vertically and vertically polarized light are alternately taken out of the fluorescence from the sample 5 by a polarizer 4b. The light part the polarizer 4b is bisected by a half mirror 2b and the bisected light are respectively converted to monochromatic light by interference filters 3b, 3c. Such light are detected by photomultipliers 15a, 15b. The plane of polarization of the polarizers 4a, 4b are alternately moved 90 deg. vertically and horizontally by the control of a computer 8 and while the excitation light is the vertically polarized light, the vertically and horizontally polarized light are successively selected by the fluorescent side polarizer 4b and two wavelength photometry is executed with the respective polarized light. The electric signal proportional to the fluorescent intensity detected by the photomultipliers 15a, 15b and the electric signal proportional to the light source intensity detected by the photomultiplier 15c are inputted to the computer 8 and are displayed 10 after the prescribed operation.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to a fluorescence polarization measurement device, and more particularly to a fluorescence polarization measurement device suitable for measuring the state of antigen-antibody reaction based on changes in the degree of fluorescence depolarization.

[Background of the invention]

A method for measuring the state of antigen-antibody reaction from changes in the degree of fluorescence depolarization was described in 1970 by Dan and Liker et al. in "Immunochemistry, voQ.
,7. Such a fluorescence polarization immunoassay (F 1uorescanca P
olarization I mmunoassay
.

An example of an apparatus for executing FPIA (hereinafter sometimes abbreviated as FPIA) is shown in Japanese Patent Laid-Open No. 136142/1983.

Next, the measurement principle of FPIA will be briefly explained.

The molecules to be measured are molecules with a molecular weight of 5,000 or less, preferably 1,000 or less, and these are immunologically determined.

called hapten. Fluorescent dyes are called tracers.1
The molecular weight is preferably 1000 or less. A combination of a hapten and a tracer is called a fluorescent vsm, while a protein that can cause an antigen-antibody reaction with a hapten is called an antibody.

In FPIA, reagents include a fluorescent label and an antibody, which are mixed with a sample containing an unknown amount of hapten to cause competitive antigen-antibody reactions between the fluorescent label and the antibody and between the hapten and the antibody. In general, fluorescent labels that do not react with antigens and antibodies have small molecular weights and undergo rapid rotational molecular motion, but fluorescent labels that have undergone antigen-antibody reactions combine with antibodies, which are proteins, resulting in slow rotational molecular motion. The change in fluorescence polarization derived from the rotation speed of the molecule is correlated with the presence or absence of an antigen-antibody reaction.

In this way, FPIA identifies the reaction between an antigen labeled with a fluorescent dye and an antibody based on the degree of depolarization of fluorescence. More specifically, a fluorescent dye is excited by light of a specific wavelength polarized in a single direction, and the degree of polarization of the emitted fluorescence is detected as the light intensity of an orthogonal vector of the specific wavelength.6 However, coexistence interference with the measurement target If there is a background fluorescent substance as a substance, the background fluorescence will synergize with the label fluorescence, making accurate measurement impossible. Therefore, when quantifying serum components using this method, the main interfering components, such as bilirubin and riboflavin, cannot be measured. To eliminate the influence, measure the background fluorescence of the serum sample before adding the labeled fluorescent substance, and then add the labeled fluorescent substance to cause an antigen-antibody reaction and measure fluorescence polarization. The true amount of fluorescence polarization had to be determined by subtracting the background fluorescence of the serum present.

In order to subtract the background fluorescence, the reaction is divided into two steps, the first step is to measure the background fluorescence, the second step is to measure the synergistic value of the background fluorescence and the labeled fluorophore, and the second step is to measure the background fluorescence and the labeled fluorophore. Take steps to subtract the fluorescence intensity of the steps. Namely.

The degree of polarization p is expressed as p= (technique v - engineering h)/(technique, +rh),
Iv and Iゎ are the vertical and horizontal components of the polarized fluorescence intensity, respectively. When the labeled fluorescence is expressed by the superscript of t in tracer and the background is expressed by the superscript of S in sample, the apparent fluorescence intensity is expressed as a.

I- is respectively.

Iv1 = engineering vt juku v S I h & = I ht + I h', and the corrected polarization degree P that corrected the background fluorescence
c is (Iv"I-'Ih"+Ih")(Iv"
Iv'+Ih"Ih").

In this conventional technique, in order to obtain the corrected polarization degree Pc,
Two reaction vessels were prepared for each sample, and background fluorescence was measured in one, and apparent fluorescence intensity was measured in the other. For this reason, the measurement time required was twice that of the method without background measurement, and the amount of reagents was also twice as necessary.

[Purpose of the invention]

An object of the present invention is to provide a fluorescence polarization measuring device that can easily eliminate the influence of background fluorescence and can perform rapid measurements.

[Summary of the invention]

The present invention provides a sample container containing a fluorescent liquid sample, excitation side polarization means for polarizing excitation light to be irradiated onto the sample, and fluorescence side light means for alternately polarizing fluorescence from the sample into vertically polarized light and horizontally polarized light. a wavelength extracting means for extracting the fluorescence maximum wavelength for each of the vertically polarized fluorescence and the horizontally polarized fluorescence, and for extracting a wavelength having a background fluorescence equivalent to the amount of background fluorescence at the maximum fluorescence wavelength; It is characterized by comprising a plurality of photodetectors that detect fluorescence polarization corresponding to each wavelength.

In the present invention, only the fluorescence polarization of the fluorescent label is extracted by optical detection means from fluorescence polarization data in which the fluorescence intensity of the fluorescent label and the background fluorescence of the sample are superimposed, and accurate fluorescence polarization is determined by data calculation. You can find the degree.

[Embodiments of the invention]

Prior to a detailed explanation of the present invention, how to determine the degree of polarization will be explained with reference to FIG.

The absorption curve in Figure 2 shows that a reagent containing DTAF, a tracer with a maximum fluorescence wavelength of 525 nm, was added to a bilirubin concentration of
This is a fluorescence spectrum obtained by diluting with 5 mg/dQ patient serum and irradiating monochromatic light with an excitation wavelength of 480 nm.

The two wavelengths to be detected are the maximum fluorescence wavelength of the fluorescent tracer (first wavelength) and the wavelength (second wavelength) at which the fluorescence amount of the fluorescent pack Gland is equivalent to the fluorescence amount of the background fluorescence at that wavelength. The difference between the 6 first wavelength and the second wavelength in which the combination is used is preferably 30 to 40 r+m. In the example of FIG. 2, the first wavelength is 525 nm and the second wavelength is 56 nm.
It is 0 nm.

Here, the first wavelength and the second wavelength are each (1).

Expressed in (2), regarding the polarization of fluorescence emitted from the sample, a total of four types of fluorescence polarization can be detected by detecting wavelengths (1) and (2) corresponding to vertical polarization and horizontal polarization, respectively. Provides strength.

These polarized light intensities are Ih<tc Iv(1)+Ih
When expressed as c2+Ivcz+, the relationship between the fluorescence intensity of a sample containing an interfering component can be expressed as linear.

I ha<z) = I ht (11+
I hS (1) I v a (1 = I
v' (I + I v s (x) I h
”(2,1= I h″L2) + I hS(z)I
v” (z) = I v’ (zn
+ I v”t, z) Therefore, the apparent degree of polarization P
a is, and the true degree of polarization Pt is Iv' (l+ -Iv' L21 +Ih''(1+
-Ih' tuIn the above formula, Ivs(12"p I v"(
21+I%(1): Ih5(Q),
P a ” P t.

Therefore, by using the method shown in FIG. 2, it is possible to obtain a degree of polarization in which the interference of background fluorescence has been eliminated, without performing the conventional complicated procedures.

Next, an embodiment of the present invention will be described with reference to FIG.

The light emitted from the light source lamp 1 is divided into two parts by a half mirror 2a, and one of the lights is filtered by an interference filter 38 at 480r+m.
The excitation wavelength light is turned into horizontally polarized light or vertically polarized light by the polarizer 4a, and is irradiated onto the reaction container containing the sample 5 containing the tracer and serum.

The other light split by the half mirror 2a is detected by a photomultiplier tube 15c and taken into the digital computer 8 via the amplifier circuit 9.

Fluorescence is emitted from the sample 5. In FIG. 1, due to space constraints, the extraction direction of fluorescence is shown as being in the same direction as the excitation light, but normally the fluorescence is extracted from a direction different from the excitation light, for example, in a direction perpendicular to the excitation light. The fluorescence from the sample 5 is alternately extracted into vertically polarized light and horizontally polarized light by the polarizer 4b. The light that passes through the polarizer.

The polarized light is divided into two parts by the half mirror 2b, and one polarized light is made monochromatic by the first wavelength interference filter 3b and sent to the photomultiplier tube 15.
Detected at a. The other polarized light divided into two passes through a reflecting mirror 6, is made monochromatic by a second wavelength interference filter 3c, and is detected by a photomultiplier tube 15b. The first wavelength is 525n
m, and the second wavelength is 560r++++.

Polarizers 4a and 4b are driven by polarization plane drive circuits 7a and 7b to move their planes of polarization 90" vertically and horizontally, and their operation is controlled by computer 8. The excitation light is vertically polarized light. In the meantime, vertically polarized light and horizontally polarized light are sequentially selected by the fluorescent side polarizer 4b, and two-wavelength photometry is performed for each polarized light.Of course, while the excitation light is horizontally polarized light, the fluorescent side polarizer 4b selects vertically polarized light and horizontally polarized light. Polarized light can be extracted sequentially.

The electric signals proportional to the fluorescence intensity detected by the photomultiplier tubes 15a and 15b are input to the amplifier circuit 9, and are divided by the electric signals proportional to the light source lamp intensity detected by the photomultiplier tube 15c, and then The signal is input to a digital computer 8 which passes through a subtraction circuit (not shown) for a signal originating from the radiation light of the wavelength of the tube 1 and a signal originating from the radiation light of the second wavelength. The computer 8 calculates the concentration of the target component in the sample based on the polarization plane conditions and the emitted light intensity, and outputs it to the printer 10.

FIG. 3 shows an example of measuring the drug concentration in 6 blood, showing the results of an experiment to confirm the effects of the present invention. A representative example of a blood drug is gentamicin, an aminoglycoside antibiotic. A constant concentration of gentamicin (approximately 2.
Standard bilirubin (Sigma) was added to the sample containing 0.5゜10.15.20mg/m
Q was added, and the results measured by conventional FPIA using one wavelength and FPIA using two wavelengths based on the present invention were compared. With the conventional single-wavelength method, as the bilirubin concentration increases, a negative error occurs in the measured value of gentamicin.

In the two-wavelength method, this tendency was not observed up to bilirubin of 20 μm/m12.

〔Effect of the invention〕

According to the present invention, by simultaneously measuring vertically polarized light and horizontally polarized light of fluorescence at two wavelengths,
Since the influence of background fluorescence can be removed,
Measurement operations are simplified and measurement time is also shortened.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a schematic configuration of an embodiment of the present invention. FIG. 2 is a diagram for explaining how to determine the degree of polarization using the two-wavelength method, and FIG. 3 is a diagram showing the results of an experiment examining the influence of bilirubin. 2a, 2b-half mirror-13a, 3b, 3
c = interference filter, 4a, 4b... polarizer,
5...Sample. 8...Digital computer, 15a, 15b. 15c...Photomultiplier tube.

Claims (1)

[Claims] 1. A sample container containing a fluorescent liquid sample, an excitation side polarization means for polarizing excitation light to be irradiated onto the sample, and a fluorescence side polarization means for alternately polarizing fluorescence from the sample into vertically polarized light and horizontally polarized light. a wavelength extraction means for extracting the fluorescence maximum wavelength for each of the vertically polarized fluorescence and the horizontally polarized fluorescence, and extracts other wavelengths having an amount of background fluorescence equivalent to the amount of background fluorescence at the maximum fluorescence wavelength; A fluorescence polarization measurement device comprising a plurality of photodetectors that detect fluorescence polarization corresponding to each wavelength.