JP2002506981A - Method and apparatus for measuring protein - Google Patents

Abstract

(57) Abstract: An apparatus and method for measuring plasma protein immunoturbidity using an apparatus for measuring an interference substance between plasma and serum. SOLUTION: An immunoturbidity measurement is performed on a sample in a disposable dispenser chip serving as a cuvette and a reaction chamber. This feature enables tests that are not possible with common chemical analysis instruments. The device of the present invention can also include a serum and plasma interferer screening system.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to immunoturbidimetry and spectrophotometric analysis of plasma proteins.

[0002]

[Prior art]

Clinical tests are routinely performed on whole blood serum or plasma. In a usual assay, red blood cells are separated from plasma by centrifugation, or centrifugation is performed after red blood cells and various plasma proteins are separated from serum by coagulation.

[0003] Hemoglobin (Hb), bilirubin (BR), biliverdin (BV), lipid particles and the like are typical light scattering substances that interfere with and affect spectrophotometry and blood analysis. These substances are commonly (and also here) referred to as interferennts. Increases in BR and BV, known as bilirubinemia and biliverdinemia, can be due to disease, and are increases in blood lipid particles, known as lipemia, due to disease? Haemoglobinemia, in which the Hb in the blood rises due to dietary conditions, may be due to the disease or may depend on the sample handling method.

[0004] Many tests performed on serum or plasma samples use a series of reactions, which ultimately develop color and facilitate detection by spectrophotometry at one or two wavelengths. It is important to determine the amount of interfering substance in the sample before the test in order to obtain significant and accurate test results. That is, if the sample is heavily contaminated by interfering substances, the test will not be performed properly and the results will be unreliable.

[0005] The current method used to detect hemoglobinemia, bilirubinemia and lipemia or turbidity is to use a color chart (or not) to visually inspect the sample. If there is a discrepancy between the test results and the patient's clinical condition, this visual inspection may be performed retrospectively to explain the discrepancy.

[0006] Preliminary inspection of samples by visual inspection is at best semi-quantitative, extremely subjective and cannot adequately guarantee the quality required for the test. In addition, visual inspection of plasma samples is a time consuming and rate-limited method. Thus, there is no visual inspection of the sample on a fully automated or semi-automated laboratory blood analyzer.

Another method for assessing sample contamination (ie, sample integrity) involves direct spectrophotometric measurement of a sample diluted in a special cuvette. In this method, a sample tube is capped to measure a plasma or serum sample, a portion of the sample is collected,
Must be measured after dilution. Each of these steps is time consuming and requires disposable cuvettes.

[0008] Plasma proteins such as immunoglobulin A (IgA
), Β2-microglobulin and C-reactive protein (CRP) can also be measured. This requires one antibody reagent for each protein and a 37 ° C. culture chamber. This assay is called immunoturbidimetry because the specific antibody reagent forms an immune complex with the corresponding protein present in the sample. Immune complexes scatter light in various directions. The direction depends on the size distribution of the immune complex or particle. The turbidity of the sample is the result of scattered light, and the absorbance increases inversely with wavelength. The term absorbance includes "true absorbance" and the effect of light loss by any other means. The detector of the spectrometer measures the light transmitted through the sample and the absorbance is calculated as the negative log of the transmittance. Thus, any light that is scattered by turbidity and does not reach the detector is interpreted as absorbed light.

[0009] At low concentrations (eg, about mg / L) of protein, the turbidity produced by the immune complex is very small and is generally measured by the following two methods. 1) A method of measuring light scattered in the forward direction using a device called a nephelometer, which is a spectrophotometer that measures light propagating at an acute angle with respect to incident light. This method requires an independent device and has a high test cost per test. 2) A method of measuring "absorbance" at 340 nm with a spectrophotometer. In the conventional method using absorbance measurement, after culturing at about 37 ° C for about 2 minutes, the absorbance at 340 nm at zero is subtracted from the absorbance at 340 nm to eliminate the influence of the sample interferer. This conventional method cannot be used for near-infrared (NIR) and visible wavelengths adjacent thereto, where the scattering of light due to the immune complex is very low.

[0010]

[Problems to be solved by the invention]

In order to perform immunoturbidity measurement, it is desirable to use an apparatus for measuring an interference substance between plasma and serum. By doing so, it becomes possible to perform a test that cannot be performed with a general chemical analysis instrument. This device can also be used as a screening system for serum and plasma interferers.

The present invention uses a novel wavelength range and uses a method to eliminate endogenous sample turbidity and the effects of other interferers. The present invention provides a new method of using disposable dispenser tips as reaction and culture chambers and even cuvettes. By using a disposable dispenser chip as a reaction chamber and cuvette, the present invention can be integrated with a chemical analysis instrument, and can be manufactured as a separate instrument for measuring serum / plasma interferents and plasma proteins. The present invention is particularly useful for analytical instruments that do not have the optical device of the present invention for facilitating the measurement of serum / plasma interferers and plasma proteins. Incorporating this optical capability into chemical analysis instruments and measuring immunoturbidity can further advance current testing methods.

[0012]

[Means for Solving the Problems]

The present invention provides an apparatus for measuring the concentration of a plasma protein in a sample by measuring an immunoturbidity comprising the following (1) to (10): (1) a blood analyzer, (2) a disposable dispenser chip, (3) means for sealing the first end of the disposable dispenser tip; (4) a second chip insertable into the second open end of the disposable dispenser tip for adding a reagent to the disposable dispenser tip; A heating space for accommodating the sample in the disposable dispenser tip, (6) means for putting the disposable dispenser tip into and out of the heating space, (7) a light source for emitting light, (8) means for directing light to the sample in the disposable dispenser chip, (9) a sensor responsive to light, and (10) means for correlating the concentration of one or more proteins in the sample with a response signal of the sensor from the sample. The sealing means is a vise and the light source, the means for directing the light beam to the sample and the sensor are preferably housed inside a spectrophotometer. The rays are near infrared and adjacent visible rays,
Preferably, the wavelength is between about 475 and about 910 nm.

In the device of the present invention, the above-mentioned correlating means includes IgA, β2-microglobulin and C
-Use the following calibration algorithms (a) to (c) for reactive protein (CRP): (a) mg / L IgA = -a (Xnm) + b (Ynm) -c [where a , B, c are the coefficients of the first derivative of the absorbance measured at wavelengths X and Y, (Xnm) is the first derivative of the absorbance measured at wavelength X, and (Ynm) is the coefficient of the absorbance measured at wavelength Y. The first derivative, preferably a = 3327100-3327120, b = 484250-4842
90, c = 70-85, more preferably a = 3327114.33, b = 484270.80, c = 77.3, X is about 780-800 nm, Y is about 820-830 nm, preferably X is about 789 nm And Y is about 825 nm) (b) mg / Lβ2-microglobulin = a (Xnm) + b (Ynm) + c (where a, b and c are the absorbances measured at wavelengths X and Y) Is the coefficient of the first derivative, (Xnm) is the first derivative of the absorbance measured at wavelength X, (Ynm) is the first derivative of the absorbance measured at wavelength Y, preferably a = -33640 to 33660 , B = 36550-36560, c
= 2-3, more preferably a = -33648.79, b = 36556.81, c = 2.3, X is about 545-550 nm, Y is about 825-835 nm, preferably X is about 548 nm, Y is about 8
(C) mg / L CRP = a (Xnm) + b (Ynm) + c (where a, b, and c are the coefficients of the first derivative of the absorbance measured at wavelengths X and Y, (Xnm) is the first derivative of the absorbance measured at wavelength X, (Ynm) is the first derivative of the absorbance measured at wavelength Y, preferably a = (-1813675) to (-1813685), b = 18
08670-1808680, c = 9.5-10, more preferably a = -1813682.71, b = 1808677.58,
c = 9.8, X is about 655-665 nm, Y is about 675-685 nm, preferably X is about 661 nm and Y is about 679 nm.)

According to another aspect of the present invention, the present invention measures the concentration of one or more plasma proteins in a sample by measuring immunoturbidity in a blood analyzer comprising the following (1) to (7): The method provides: (1) filling the disposable dispenser tip with a sample, (2) sealing the first end of the tip with sealing means, (3) inserting a second insertable tip into the second open end of the disposable dispenser tip. A reagent was added to this opening using a chip, (4) the disposable dispenser tip was placed in the heating space, (5) light from the light source was emitted to the sample in the disposable dispenser chip, and (6) the sample was transmitted. Sensing the light beam and (7) correlating the concentration of one or more proteins in the sample with the response of the sensor from the sample. The disposable dispenser tip containing the reagents and sample may be removed from the heated space before exposure to the light beam. The preferred sealing means is a vise. In the method of the present invention, the light is near infrared and adjacent visible light, and the wavelength of near infrared and adjacent visible light is preferably from about 475 to about 910 nm.

In the method of the present invention, the correlating means uses the following calibration algorithms (a) to (c) for IgA, β2-microglobulin and C-reactive protein (CRP), respectively: mg / L IgA = -a (Xnm) + b (Ynm) -c [where a, b, and c are the coefficients of the first derivative of the absorbance measured at wavelengths X and Y, and (Xnm) is the wavelength X Is the first derivative of the absorbance measured at, and (Ynm) is the first derivative of the absorbance measured at the wavelength Y, preferably a = 3327100-3327120, b = 484250-4842
90, c = 70-85, more preferably a = 3327114.33, b = 484270.80, c = 77.3, X is about 780-800 nm, Y is about 820-830 nm, preferably X is about 789 nm And Y is about 825 nm) (b) mg / Lβ2-microglobulin = a (Xnm) + b (Ynm) + c (where a, b and c are the absorbances measured at wavelengths X and Y) Is the coefficient of the first derivative, (Xnm) is the first derivative of the absorbance measured at wavelength X, (Ynm) is the first derivative of the absorbance measured at wavelength Y, preferably a = -33640 to 33660 , B = 36550-36560, c
= 2-3, more preferably a = -33648.79, b = 36556.81, c = 2.3, X is about 545-550 nm, Y is about 825-835 nm, preferably X is about 548 nm, Y is about 8
(C) mg / L CRP = a (Xnm) + b (Ynm) + c (where a, b, and c are the coefficients of the first derivative of the absorbance measured at wavelengths X and Y, (Xnm) is the first derivative of the absorbance measured at wavelength X, (Ynm) is the first derivative of the absorbance measured at wavelength Y, preferably a = (-1813675) to (-1813685), b = 18
08670-1808680, c = 9.5-10, more preferably a = -1813682.71, b = 1808677.58,
c = 9.8, X is about 655-665 nm, Y is about 675-685 nm, preferably X is about 661 nm and Y is about 679 nm.)

The present invention further provides a blood analyzer comprising the following (1) to (7) for measuring the turbidity of the immunoassay to determine the concentration of plasma protein IgA, β2-microglobulin or C-reactive protein in the plasma sample. Provides a method to measure: (1) aspirate a small amount of plasma into a disposable dispenser tip, (2) further aspirate this small amount of sample in the sample tip, pull the sample away from the lower end of the tip, (3) Without trapping air below the sample in the chip, seal the lower end of the chip with sealing means, and (4) dispose the antibody reagent using the second dispenser tip that can be inserted into the open end of the disposable dispenser tip. (5) Heat the disposable dispenser tip in the heating space, (6) irradiate the sample in the disposable dispenser tip with a spectrophotometer, (7) IgA, β2- B globulin or C- concentration of reactive protein, the step of correlating the response of the sensor from the sample. Preferably, the temperature of the heating space is 37 ° C. and the chips are kept in the heating space for 2 minutes. Preferably, the plasma sample is 5 μl. In a preferred embodiment of the invention, the reagent is I
Antibody reacting against gA, antibody reacting against β2-microglobulin and C-
About 60 μl of the antibody selected from the group consisting of antibodies that react with the reactive protein.

[0017]

Embodiment

The present invention provides an apparatus and a method for measuring immunoturbidity using a plasma and serum interference measuring apparatus. This feature enables tests that are not possible with general chemical analysis instruments. The device of the present invention can also include a serum and plasma interferent testing system. The apparatus for measuring serum and plasma interference includes a housing for housing a sample, a light source, a sensor, and a means for optically connecting the light source to the sensor along a sample optical path passing through the housing and a reference optical path bypassing the sample. Means for selectively introducing light from the sample light path and light from the reference light path to the sensor; and correlating the response of the sensor from the sample light path to the response of the sensor from the reference light path to an amount of a known substance in the sample. Means. As shown in FIG. 1, the sample container can be part of a conveyor system. Also, as shown in FIG. 1, a cavity for accommodating the sample and a lid for selectively opening and closing the cavity may be used. In automated systems, the dispenser base (st
em, stem) into the dispenser tip to obtain sufficient light-shielding properties,
In addition, a lid may not be necessary because the placement of the shutter can subtract the dark current of both the sample light measurement and the reference light measurement to effectively eliminate the effects of room lighting. The light source emits a light beam, and the sensor is responsive to the light beam. When measuring the immunoturbidity using an existing device, a means for sealing the lower end of the dispenser tip is required. The preferred sealing means is a small vise. A preferred example of a dispenser tip is Johnson & Johnson.
Johnson) is a disposable tip used in Vitros ™.

The apparatus of the present invention comprises: a quartz tungsten halogen lamp capable of emitting near infrared rays and adjacent visible light having a wavelength of 475 nm to 910 nm; a sample optical path for transmitting light from the quartz tungsten halogen lamp along the sample optical path; A bifurcated optical fiber cable for bifurcation with the reference optical path propagating along the reference optical path. Further, a shutter for selectively blocking a sample optical path propagating along the sample optical path and a reference optical path propagating along the reference optical path, and a shutter for propagating the sample optical path passing through the sample accommodated in the accommodation unit. An optical fiber bundle and an optical fiber bundle for propagating a sample optical path from the sample to another bifurcated optical fiber cable. The light from the sample path and the light from the reference path are collected on a single fiber optic cable. It will be appreciated that the means for eliminating light from sources other than the light source of the device are optional and all fall within the scope of the present invention. Also, if the dark current, that is, the response of the sensor when the light of the device does not enter, is subtracted from both the reference measurement value and the sample measurement value, the room light hitting the sensor should be effectively subtracted without substantially impairing the device performance. Can be.

After the sample is sucked into the dispenser tip, it is preferable that the lower end of the tip is flattened and sealed between the small vice clamping parts. Preferably, the dispenser tip is disposable. The dispenser chip is used as a reaction chamber and a culture chamber after sealing the sample inside. The sealed dispenser tip can be further used as a cuvette.

Samples such as proteins, for example, immunoglobulin A (IgA), β2-microglobulin, and C-reactive protein (CRP) are measured by the present invention using a reagent such as an antibody in an immunoturbidity measurement process. Can be. Each plasma protein requires a specific antibody.
The specificity of each test can be increased by subtracting the first derivative of the absorbance at zero from the first derivative of the absorbance at single or multiple wavelengths after standing at 37 ° C. for approximately 2 minutes. It will be appreciated that the optimal incubation time and temperature can vary for each plasma protein. 5 μL of sample and 60 μL of antibody reagent are required. It will be appreciated that the optimal amounts of sample and reagent can vary for each protein.

In another aspect of the invention, the lower end of the same dispenser tip used to aspirate 5 μL of sample is sealed by depressurizing the tip by an equivalent of 4 μL. When other disposable chips are used, deviation from this capacity is also included in the scope of the present invention. 4
An additional vacuum equivalent to an aspirate volume of μL was sufficient to separate the fluid from the lower end of the dispenser tip, which lower end did not trap air below the 5 μL sample and was sealed below this seal. The sample is gripped by a small vice clamp without trapping the sample. In the preferred embodiment of the present invention, the clamp is not parallel, as shown in FIG. 3, so that any residual fluid gripped by the clamp is pushed up. This feature of the invention can reduce sample loss.

In an embodiment of the present invention, a mixture of a sample obtained by injecting 60 μL of the antibody reagent into 5 μL of the sample is used. Preferably, 60 μL of the antibody reagent is placed in a thin pipette tip as shown, for example, in FIG. 3, 4 and this pipette tip is inserted up to the bottom of a sealed dispenser tip, and the antibody reagent is properly distributed with sufficient space. . A thin pipette tip as shown in Figure 3-4 is a 960Enviroti made by Eppendorf.
Preferably it is p®, but any similar pipette tip can be used. The ratio of the volume of the antibody reagent to the volume of the sample is desirably a ratio that facilitates sufficient mixing of the sample and the reagent. In an embodiment of the present invention, it is preferable that only the fluid is discharged when the antibody reagent is released, and that air is not introduced into the reaction chamber.

After dispensing the antibody reagent into the sealed dispenser tip of the present invention, a zero absorbance measurement is started at the dispenser base attached to the tip. In one embodiment of the invention, the tip holder has a sliding lid that closes after dispensing the antibody reagent.

In another aspect of the invention, a shutter is located on the lamp assembly and the dark current of both the sample light measurement and the reference light measurement is subtracted to effectively eliminate the effects of room lighting. it can. The sample chamber preferably blocks light, but need not be completely light-tight. In an automated system, inserting the dispenser base into the dispenser tip would provide sufficient light blocking without having to subtract dark current, and would not require a lid.

In another embodiment of the invention, the spectrometer can operate in a single beam mode. In another aspect of the invention, zero measurements are used as reference scans when the spectrometer operates in single beam mode. Preferably, the rate of change of the first derivative of the absorbance during the first 15 seconds is monitored to predict if a high concentration hook effect will occur. Immunoturbidity measurements are performed using multiple wavelengths of visible light and NIR electromagnetic radiation.

The method of the present invention measures the concentration of a series of proteins in a sample by recording the absorbance spectra of the sample before and after culturing with an antibody specific to each protein. The effect of interferers in the sample can be minimized by the wavelength range used, ie, NIR and adjacent visible light. Preferably, the first-order derivative of the absorbance at zero is subtracted from the first derivative of the absorbance after culturing at 37 ° C. for 2 minutes to substantially eliminate residual effects of the interferent. It will be appreciated that other times and culture temperatures may be used. The effect of small bubbles on absorbance is minimized using the first derivative of absorbance.
It will be appreciated that higher derivatives of the absorbance, for example the second derivative of the absorbance, can also be used.

A system including the device 10 of the present invention is shown generally in FIG. The apparatus 10 generally comprises a spectrometer 14 optically connected or coupled to the sample via fiber optic bundles 44, 46.
It has. The sample is held on a conveyor 94 that passes through a cover 92 or is placed in a sample holder 98 having a cover 100. The apparatus 10 of the present invention is mounted or installed adjacent to an automatic conveyor 94 that supports a plurality of sample tubes, for example, 86,88. Since the sample is in test tubes of different dimensions, a gap may be created between the test tube wall and the ends of the optical fibers 44,46. Focusing lens 96 at the end of the fiber
Is installed. Sample holder 98 is designed for a disposable dispenser tip. The cover 92 serves as a light shield and prevents the movement of the optical fibers 44 and 46. Figure
The first cover 100 also serves to shield the device from light.

[0028] The dispenser tip holder is designed to be deep enough to accommodate the dispenser base (stem) of the analytical instrument, such that the dispenser base and dispenser tip retainer of the analytical instrument serve as a light shield. You can also. The dispenser tip holder, cover 100 and cover 92 do not form a light-tight sample chamber. The sample on the conveyor line 94 shown in FIG. 1 is only for analyzing the sample integrity function of the spectrometer, and in the present invention the sample is measured by optics in the chip holder 98 of FIG. Is done.

For protein measurement by immunoturbidity measurement, a separate sample holder (
98) is required. This sample holder 98 is embedded in a heated block. In a preferred embodiment, 5 μL of plasma is aspirated into a dispenser tip as shown in FIG. The sample is additionally depressurized by an amount corresponding to the suction of 4 μL, and the fluid is pulled away from the lower end of the dispenser tip held by the holding portion as shown in FIG. Chips of different capacities can also be used. It will be appreciated that the purpose is to sufficiently pull the sample away from the dispenser tip so that the tip can be sealed. This additional vacuum must be sufficient to separate the fluid from the lower end of the dispenser tip with no air trapped below the 5 μL sample. After aspirating the sample, seal the same dispenser tip used to aspirate 5 μL of the sample. End of dispenser tip is 5
Below the μL sample, it is crushed and sealed between the small vise clamps shown in 5 of FIG. FIG. 3-2 shows a sealed dispenser tip having a flattened lower end. While FIGS. 1 and 2 show Vitros ™ chips, other disposable tips can be used and it will be appreciated that deviations from 4 μL are within the scope of the present invention. The holding portion 5 in FIG. 3 is not parallel, and pushes up the residual fluid gripped by the holding portion. This aspect of the invention prevents the loss of 5 μL of sample.

In the present invention, a sample can be measured by the present apparatus using a reagent by an immunoturbidity measurement process. Each protein requires an antibody specific to it, and the specificity of each test is determined from the first derivative of the absorbance at single or multiple wavelengths after holding at 37 ° C. for about 2 minutes. It can be increased by subtracting the first derivative. It will be appreciated that optimal incubation times and temperatures may vary for each protein.

Aspirate 60 μL of the antibody reagent from the bottle into a thin pipette tip shown in FIG. In the preferred embodiment, the thin pipette tip shown in FIG.
pendorf), but other similar pipette tips that can reach the bottom of a sealed dispenser tip may be used.
The Eppendorf chip or its equivalent must be able to reach the bottom of the Vitros chip or its equivalent, and the sufficient space between its outlet and 5 μL of sample will allow the antibody reagent to be removed. It can be easily distributed correctly. Inject 60 μL of antibody reagent into 5 μL of sample to make a mixture with the sample. Keep air out of the sample. This can be achieved by injecting this volume in cultures that allow accurate distribution of less than 60 μL of antibody reagent. It will be appreciated that the reaction mixture may be re-aspirated and redistributed for further mixing.

After the sample is sealed inside, the disposable dispenser tip of the analytical instrument is used as a reaction room and a culture room. This sealed dispenser tip is also used as a cuvette. Although only one tip holder 98 is shown in FIG. 1, in the preferred embodiment, two tip holders 98 are used for interferometer measurement and protein measurement. One tip holder can do both tasks, and the downtime for interferometric measurements is only 1 second, so even if heated to favor protein measurements, a single tip holder It can be understood that this does not affect the measurement of the interferer. When two separate chip holders are mounted, they are connected to the same optical fiber 44 of FIG. 1 via a bifurcated optical fiber. Two new shutters must be installed outside the lamp assembly 20 of FIGS. This new shutter allows light to only hit the functioning chip holder.

After dispensing the antibody reagent, a zero absorbance measurement is performed at the dispenser base attached to the dispenser tip. In another embodiment of the invention, the tip holder comprises a sliding lid, which dispenses the antibody reagent and closes after the dispenser base has opened the dispenser tip. Subtracting the first derivative of the absorbance at zero from the first derivative of the absorbance after incubation at 37 ° C for 2 minutes can almost eliminate the influence of the interferent. It will be appreciated that other times and culture temperatures can be used. In this design, the sample holder acts as both an incubator and an optical reader. The culturing can be performed in a separate room, the cultured sample can be aspirated into a disposable dispenser tip, and the dispenser tip can be placed in a tip holder 98 as shown in FIG. If a separate culture chamber is used, the same reader (or chip holder) 98 can be used for both interfering and protein measurements, as shown in FIG. When using an incubator and reading unit in combination, a separate chip holder is required for measuring the interfering substance, and it is necessary to supply a light beam to the “incubator-reading unit” and to receive a light beam from it. A set of optical fibers and a shutter are required for the body. If you want to keep the dispenser base on the dispenser tip,
Another dispenser base can be added to the device.

The optical fibers 44 and 46 guide the light from the light source and the sample, and can be used to place the entire device far away from the sample. Multiplex optical fiber 46, 48
Is a bifurcated optical fiber cable coupled to a composite optical fiber 54 connected to the spectrometer 14. The reference fiber 66 is connected to the bifurcated fiber cable 48 via the coupling 52.
Joined to. The coupling 52 can be selected to sufficiently attenuate the reference beam so that the detector can be optimally integrated in a short period of time. The fiber 66 is a single fiber and the fiber 44 can be single or multiple fibers depending on the amount of light desired.

Referring to FIG. 1, the apparatus 10 of the present invention includes a spectrometer 14, a central processing unit 16, a power supply 18, a lamp assembly module 20, and a sample holder 92, 94 or 98. .

Referring to FIG. 2, the lamp assembly module 20 uses a light source 62. The light source is preferably a quartz tungsten halogen 20 watt lamp, although other wattage lamps may be used. The input power is AC, but the output to the light source is a stabilized DC. The lamp is fitted with a photodiode 80 to monitor the lamp output. The spectral output from light source 62 is broadband including the visible and NIR regions. Although the NIR region of the electromagnetic spectrum is generally considered to be in the range of 650 nm to 2700 nm, the preferred embodiment has a nominal wavelength range of 475 to 910 nm, which is referred to herein as the `` near infrared and adjacent visible region. It is called. Rays of radiation from light source 62 are directed through bandpass filter 64 and shaped filter 69 in spectrometer 14. Bandpass filters are needed to reduce unwanted radiation outside 475-910 nm. Shaping filter 69 is necessary to "flatten" the optical response of the detection system. The light beam from the filter 64 propagates through the bifurcated plurality of fiber bundles 60 and is provided as a sample light beam and a reference light beam. The bifurcated fiber bundle 60 randomly extracts the lamp radiation and provides a sample beam and a reference beam via two arms 60, 80 and 82, respectively. In the preferred embodiment, a photodiode array (PDA) detector 78 balances 99% and 1% of the emitted radiation from both the sample and reference paths 80 and 82, respectively. When the shutter 58 closes and the shutter 56 opens, the emitted light is directed to the sample through the optical fiber 44, and the emitted light is transmitted through the sample in a multi-labeled test tube or plastic dispenser tip. The light received at 46 is returned to the spectrometer 14.

The sample beam and the reference beam enter the arms 46 and 48 of the bifurcated optical fiber bundle, respectively, are coupled by the fiber 54, and are alternately focused on the slit 70 by the focusing lens 68 and the shaping filter 69. The outgoing radiation is collimated by a lens 72 and the light is directed to a diffraction grating 74, which is a dispersion element that separates component wavelengths. In a preferred embodiment, dichromated gelatin is used as the diffraction grating material. The component wavelength is collected on the PDA 78 by the lens 76. Each element or pixel of the PDA is set to receive and collect a predetermined wavelength. In the preferred embodiment, PDA 78 is 256 pixels. This pixel is rectangular in order to optimize the amount of radiation detected.

The spectrometer 14 is preferably a dual-beam-in-time spectrometer with a fixed integration time for the reference beam and a selectable integration time for the sample beam. Since the sample is only shielded and not in the light-tight cage, the dark scan of the sample and reference light must be subtracted from the sample light scan and reference light scan, respectively. The sample dark scan and the reference dark scan are performed with the same integration time as each optical scan. In the preferred embodiment, the reference scan is performed in 13 ms, the sample scan is performed in 20 ms, and the maximum ADC value obtained in 20 ms for a particular sample determines the new integration time. The new integration time is less than 2600 milliseconds and is determined not to saturate any pixels of the detector. The maximum time allowed for all samples is determined by the required speed of sample inspection. Also, noise can be minimized by averaging multiple scans, but the average scan should not take more than one second.

In use, each pixel or wavelength portion is measured almost simultaneously at each scan time. The radiation entering each photosensitive element is integrated for a specific time and the individual pixels or wavelengths are sampled sequentially by a 16-bit analog-to-digital converter or ADC.

Although the method of using a PDA is described in detail in the embodiments of the present invention, another arbitrary means for obtaining the same result is included in the scope of the present invention. For example, a filter-wheel device can be used. At the time of measurement, 1 to 3 wavelengths or pixels are used for each sample. If the first derivative of the absorbance measurement at the PDA is the difference in absorbance at two adjacent pixels, then the first derivative of the absorbance at one wavelength in the filter-wheel device will contain two different narrow bandpass filters. Requires the absorbance measured in. One skilled in the art will readily recognize that any structure that does not require the assembly of a filter on the rotating wheel, but that results in narrow bandpass filtering of the absorbed light, is within the scope of the present invention.

A PDA integrates light for a specific time and converts the optical signal into a time multiplexed analog electronic signal (called scanning). The absorbance is calculated as follows: Absorbance = log (reference measurement _i / sample measurement _i ) + log (ITM / ITR) (where reference measurement _i = readout of reference pixel i sample measurement _i = sample measurement Pixel i reading ITM = integration time measurement ITR = integration time reference i = specific pixel of PDA.

The above equation can also be expressed as absorbance = log {reference dark measurement value−reference dark measurement value} / {sample measurement value−sample dark measurement value} + log (ITM / ITR)}. The measured values of the reference darkness and the sample darkness may or may not be taken depending on the degree of light shielding and the timing accuracy of the device. The electronic signal is proportional to the time during which the sensor integrates the optical signal.
This electronic signal is amplified by an analog electronic amplifier and converted into a digital signal by an analog-to-digital converter ADC. The digital information from this converter is analyzed and data analyzed by a microprocessor connected to a computer 84 via an RS232 connector. The data analysis result can be displayed on an output device such as a display and a printer.

The first step in the process of creating a calibration curve is to accumulate spectral data for a calibration set. The calibration algorithm for each protein must be set in the microprocessor so that when testing unknown samples for a particular protein the results are rapid. Any of a number of methods can be used to calculate the abundance of any protein, all of which are within the scope of the present invention.

A preferred method is to calculate the first derivative of a particular part of the spectrum for the protein to be measured. It is also possible to calculate the second or third derivatives, which are within the scope of the invention, but calculating these derivatives can be time consuming for each step of taking the difference, and can reduce noise. Increase. In practice, the optimal combination of the first derivatives of at least two parts of the spectrum obtained by scanning a particular protein is used for calculating the protein concentration. The actual method used depends on the protein to be measured.

[0045]

【Example】

To obtain a calibration curve for IgA, 5 μL of each standard was aspirated into a Vitros dispenser tip using an Eppendorf pipet. Pipette setting 5μ
The volume is changed from L to 9 μL, and this additional decompression allows the sample to be pulled away from the lower end of the dispenser tip held by the clamp of the vise shown in FIG. The lower end of the dispenser tip was crushed and sealed with a pair of pliers to prevent fluid leakage. The dispenser tip containing the fluid was placed in a heated tip holder as shown at 98 in FIG. Using a second pipette, 60 μL of the antibody reagent was added to the sample such that the lower end of the Eppendorf pipette tip was almost in contact with the sample as shown in 3 of FIG. The Eppendorf pipette tip should reach as low as possible without restricting the flow of the antibody reagent. The absorbance spectrum immediately after addition of the antibody reagent was recorded as the measured value at zero. After 2 minutes, a second absorbance spectrum was recorded. This procedure was repeated for four standards and five individual samples used for validation of the calibration algorithm created. The absorbance spectra of the standard substance and the confirmation sample set are shown in FIGS. 4 and 5, respectively. The linear regression fits for the standard and the validation sample set are shown in FIGS. 6 and 7, respectively.

Similarly, a calibration algorithm was created for β2-microglobulin and C-reactive protein. These linear regression fits are shown in FIGS. 8 and 9, respectively. β
Antibodies used for 2-microglobulin are covalently bound to polystyrene beads, and antibodies used for CRP are not as enhanced as IgA antibodies. These antibodies are also commercially available. It will be appreciated that specific antibodies are available in the present invention and that any protein having a concentration sufficient to generate a detectable immune complex can be measured. In the case of a relatively low concentration of protein, polystyrene beads can be bound to the antibody to enhance the signal.

Due to the low expected absorbance at the wavelength used, the absorbance spectra at zero obtained for IgA were observed in an irregular order as shown in FIG. This is probably due to the presence of small air bubbles in the fluid and uneven wall of the dispenser tip. However, both the absorbance after 2 minutes at 37 ° C. and the first derivative of the absorbance are proportional to the concentration of β2-microglobulin, as shown in FIG. In the conventional method, from the absorbance at 340 nm after culturing at about 37 ° C. for about 5 minutes to eliminate the influence of the interferent in the sample, about 340 n
The absorbance at zero at m was subtracted. One skilled in the art will appreciate that the use of a dispenser tip in a conventional manner to eliminate the effects of interferers is not possible in the wavelength range specified in the present invention. The present invention uses a novel method,
Subtract the first derivative of absorbance from the first derivative of absorbance at all wavelengths after 2 minutes at ° C, process this difference using stepwise linear regression statistics, and then apply Eliminate the effects of interferers. The preferred embodiment requires the raw absorbance at 9 pixels or wavelengths to calculate the first derivative of each absorbance, and if a filter is used instead of the PDA used in the present invention, the first order of each absorbance will be used. It can be seen that two narrow bandpass filters are needed to obtain the derivatives.
Therefore, multiple wavelengths are required even if the calibration algorithm uses only one first derivative of absorbance.

For IgA, optimal results are obtained by calculating the first derivative of the absorbance at wavelengths between about 789 and 825 nm. For β2-microglobulin the optimal result is about 548-8
Obtained by calculating the first derivative of the absorbance at a wavelength of 29 nm. Optimal results for CRP can be obtained by calculating the first derivative of the absorbance at wavelengths of about 661-679 nm.

The calibration algorithm for IgA made based on the four standards is as follows: mgIL IgA = -3327114.33 (789 nm) +484270.80 (825 nm) -77.3 [where (Xnm) is the specific wavelength The calibration algorithm for β2-microglobulin based on the seven standards is as follows: mg / L β2-microglobulin = -33648.79 (548 nm) +36556.81 ( 829nm) +2.3 [where (Xnm) is the first derivative of absorbance at a specific wavelength] The calibration algorithm for CRP based on 9 standards is as follows: mgL CRP = -1813682.71 (661nm) +1808677.58 (679nm) +9.8 [where (Xnm) is the first derivative of absorbance at a specific wavelength]

It will be appreciated that a plurality of calibration algorithms can be created for each protein using the above-described apparatus for determining the integrity of an analyte. Protein measurement is based on the principle of immunoturbidity measurement, ie, the formation of an antibody-antigen complex or immune complex that causes turbidity. Since "absorbance" results from the scattering of light due to the immune complex, intrinsic turbidity or true absorbance due to interferents in the sample will apparently increase the signal. To determine the contribution of the interferers, a 2 g / L aqueous solution of IgA was mixed with IL to produce four different samples containing 1 g / L IgA and various amounts (0-4 g / L) of IL. Separate algorithms were created for IgA with and without zero correction.

FIG. 10 shows an error rate in the prediction result when the subtraction at the time of zero is performed and when the subtraction is not performed. The present invention differs from the prior art in that it uses a plurality of long wavelengths and that the immunocomplex reduces the absorbance at these wavelengths so that endogenous interferers must be compensated. Due to the effects of small bubbles and the inaccuracy of the absorbance of the disposable dispenser tip, this compensation cannot be done using the raw absorbance, but can be done effectively using the first derivative of the absorbance. When using the first derivative of absorbance, multiple wavelengths are required even if the calibration algorithm uses only one first derivative of absorbance. While the present invention has been described with reference to particular embodiments, it will be understood by those skilled in the art that various modifications can be made without departing from the scope of the invention.

[Brief description of the drawings]

FIG. 1 is a perspective view of a system including the apparatus of the present invention for analyzing sample integrity and measuring various proteins.

FIG. 2 is a conceptual diagram of each element used in the apparatus of FIG.

FIG. 3 is a perspective view of two disposable dispenser tips and a small vice clamp for crushing and sealing the lower end of the tip.

FIG. 4 is a graph showing absorbance spectra of various amounts of IgA in a dispenser chip of an analyzer, which were measured immediately after culturing at 37 ° C. with an antibody against IgA (at zero). IgA concentrations are indicated in the figure.

FIG. 5 is a graph showing absorbance spectra of various amounts of IgA in a dispenser chip of an analytical instrument, measured after culturing with an antibody against IgA at 37 ° C. for 2 minutes. I
The gA concentration is shown in the figure.

FIG. 6 is a graph showing the linear regression fit of the data used to generate the algorithm for calibrating the IgA of the sample in the dispenser tip of the analytical instrument in milligrams / liter on the horizontal and vertical axes.

FIG. 7 is a graph showing the linear regression fit of data on the expected concentration of IgA in samples not used in the calibration process in milligrams / liter on the horizontal and vertical axes.

FIG. 8 is a graph showing the linear regression fit of data used to generate a calibration algorithm for β2-microglobulin of a sample in a dispenser tip of an analytical instrument in milligrams / liter on the horizontal and vertical axes.

FIG. 9 is a graph showing the linear regression fit of data used to generate an algorithm for calibrating the C-reactive protein of a sample in a dispenser tip of an analytical instrument in milligrams / liter on the horizontal and vertical axes.

FIG. 10 is a graph showing the error rate of the predicted IgA value due to intrinsic turbidity caused by intralipid when the first derivative of absorbance at zero is subtracted and when it is not subtracted.

Claims (20)

[Claims] An apparatus for measuring the concentration of a plasma protein in a sample by measuring an immunoturbidity comprising the following (1) to (10): (1) a blood analyzer, (2) a disposable dispenser chip, 3) means for sealing the first end of the disposable dispenser tip; (4) a second tip insertable into the second open end of the disposable dispenser tip for adding reagents to the disposable dispenser tip; (6) a heating space for accommodating the sample in the disposable dispenser tip, (6) a means for putting the disposable dispenser tip into and out of the heating space, (7) a light source for emitting light, (8) a means for directing a light beam to the sample in the disposable dispenser tip, ( 9) a sensor responsive to light, and (10) means for correlating the concentration of one or more proteins in the sample with the response signal of the sensor from the sample. 2. Apparatus according to claim 1, wherein the sealing means is a vise. 3. The apparatus according to claim 1, wherein the light source, the means for directing the light beam to the sample and the sensor are contained in a spectrophotometer. 4. The device according to claim 1, wherein the light beams are near-infrared light and adjacent visible light. 5. The device according to claim 1, wherein the wavelength of near-infrared light and adjacent visible light is about 475 to about 910 nm. 6. The method according to claim 1, wherein the correlating means includes the following calibration algorithms (a) to (c) for IgA, β2-microglobulin and C-reactive protein (CRP). (A) mg / L IgA = -a (Xnm) + b (Ynm) -c [where a, b and c are coefficients of the first derivative of absorbance measured at wavelengths X and Y. Yes, (Xnm) is the first derivative of the absorbance measured at wavelength X, (Ynm) is the first derivative of the absorbance measured at wavelength Y, X is about 780-800 nm, and Y is about 820- (B) mg / Lβ2-microglobulin = a (Xnm) + b (Ynm) + c (where a, b, and c are the coefficients of the first derivative of the absorbance measured at wavelengths X and Y. Yes, (Xnm) is the first derivative of the absorbance measured at wavelength X, (Ynm) is the first derivative of the absorbance measured at wavelength Y, X is about 545-550 nm, and Y is about 825- (C) mg / L CRP = a (Xnm) + b (Ynm) + c (where a, b c is the coefficient of the first derivative of the absorbance measured at wavelengths X and Y, (Xnm) is the first derivative of the absorbance measured at wavelength X, and (Ynm) is the first derivative of the absorbance measured at wavelength Y. X is about 655-665 nm and Y is about 675-685 nm.) 7. The calibration algorithm for IgA is a = 3327100-3327120, b = 484250-484290, c = 70-85, and the calibration algorithm for β2-microglobulin is a = -33640-33660, b = 36550-36560 , C = 2-3, and the calibration algorithm for C-reactive protein is a = (-1813675)-(-1813685), b = 1808670-1808680, c = 9.5-10. . 8. The apparatus according to claim 7, wherein the calibration algorithms for IgA, β2-microglobulin and C-reactive protein are (a) to (c) respectively: (a) mg / L IgA = -a (Xnm) + b (Ynm) -c (where a = 3327114.33, b = 484270.80, c = 77.3, (Xnm) is the first derivative of the absorbance measured at wavelength X, and (Ynm) is The first derivative of the absorbance measured at wavelength Y,
(X is about 789 nm, Y is about 825 nm) (b) mg / Lβ2-microglobulin = a (Xnm) + b (Ynm) + c (where a = −33648.79, b = 36556.81, c = 2.3, (Xnm) is the first derivative of the absorbance measured at wavelength X, (Ynm) is the first derivative of the absorbance measured at wavelength Y, X is about 548 nm, and Y is about 829 nm.) (c) mg / L CRP = a (Xnm) + b (Ynm) + c (where a = -1813682.71, b = 1808677.58, c = 9.8, (Xnm) is the first derivative of the absorbance measured at wavelength X) Where (Ynm) is the first derivative of the absorbance measured at wavelength Y,
X is about 661 nm and Y is about 679 nm.) 9. A method for measuring the concentration of plasma protein in a sample by measuring immunoturbidity using a blood analyzer comprising the following steps (1) to (7): (1) Filling a sample into a disposable dispenser chip (2) sealing the first end of the chip with a sealing means; (3) adding a reagent to the opening using a second chip insertable into the second open end of the disposable dispenser chip; Put the disposable dispenser tip into the heating space, (5) emit light from the light source to the sample in the disposable dispenser tip, (6) sense the light transmitted through the sample, (7) remove one or more proteins in the sample. Correlate the concentration with the sensor response from the sample. 10. The method according to claim 9, wherein the sealing means is a vise. 11. The light of claim 9 or 1 wherein the light is near infrared and adjacent visible light.
The apparatus according to claim 0. 12. The method according to any one of claims 9 to 11, wherein the wavelength of near infrared and adjacent visible light is from about 475 to about 910 nm. 13. The method according to claim 9, wherein the correlating means applies the following calibration algorithms (a) to (c) to IgA, β2-microglobulin and C-reactive protein (CRP), respectively. Method according to one paragraph: (a) mg / L IgA = -a (Xnm) + b (Ynm) -c [where a, b, and c are the first derivatives of the absorbance measured at wavelengths X and Y. (Xnm) is the first derivative of the absorbance measured at wavelength X, (Ynm) is the first derivative of the absorbance measured at wavelength Y, X is about 780-800 nm, and Y is about (B) mg / Lβ2-microglobulin = a (Xnm) + b (Ynm) + c (where a, b and c are the first derivatives of the absorbance measured at wavelengths X and Y) Where (Xnm) is the first derivative of the absorbance measured at wavelength X, (Ynm) is the first derivative of the absorbance measured at wavelength Y, X is about 545-550 nm, and Y is about (C) mg / L CRP = a (Xnm) + b (Y where a, b, and c are the coefficients of the first derivative of the absorbance measured at wavelengths X and Y, (Xnm) is the first derivative of the absorbance measured at wavelength X, and (Ynm ) Is the first derivative of the absorbance measured at wavelength Y, where X is about 655-665 nm and Y is about 675-685 nm.) 14. The calibration algorithm for IgA is a = 3327100-3327120, b = 484250-484290, c = 70-85, and the calibration algorithm for β2-microglobulin is a = -33640-33660, b = 36550-36560 , C = 2-3, and the calibration algorithm for C-reactive protein is a = (-1813675)-(-1813685), b = 1808670-1808680, c = 9.5-10. . 15. An IgA, β2-microglobulin and C-reactive protein (CRP).
The method according to claim 14, wherein the calibration algorithms for (a) to (c) are as follows: (a) mg / L IgA = -a (Xnm) + b (Ynm) -c [where a = 3327114.33, b = 484270.80, c = 77.3, (Xnm) is the first derivative of the absorbance measured at wavelength X, (Ynm) is the first derivative of the absorbance measured at wavelength Y,
(X is about 789 nm, Y is about 825 nm) (b) mg / Lβ2-microglobulin = a (Xnm) + b (Ynm) + c (where a = −33648.79, b = 36556.81, c = 2.3, (Xnm) is the first derivative of the absorbance measured at wavelength X, (Ynm) is the first derivative of the absorbance measured at wavelength Y, X is about 548 nm, and Y is about 829 nm.) (c) mg / L CRP = a (Xnm) + b (Ynm) + c (where a = -1813682.71, b = 1808677.58, c = 9.8, (Xnm) is the first derivative of the absorbance measured at wavelength X) Where (Ynm) is the first derivative of the absorbance measured at wavelength Y,
X is about 661 nm and Y is about 679 nm.) 16. A method for determining the concentration of plasma protein IgA, β2-microglobulin or C-reactive protein in a plasma sample by measuring immunoturbidity using a blood analyzer, including the following steps (1) to (7): How to: (1) aspirate a small amount of plasma into the disposable dispenser tip, (2) further aspirate this small amount of sample in the sample tip, pull the sample away from the lower end of the tip, (3) sample in the tip Without trapping air below the tip, seal the lower end of the tip with tip sealing means. (4) Use the second dispenser tip that can be inserted into the open end of the disposable tip to transfer the antibody reagent to the disposable tip. (5) Heat the disposable dispenser tip in the heating space, (6) emit light in the spectrophotometer to the sample in the disposable dispenser tip, (7) IgA, β2-microglobulin in the sample Or C- concentration of reactive protein is correlated with the response of the sensor from the sample. 17. The method according to claim 16, wherein the temperature of the heating space is 37 ° C. 18. The method according to claim 17, wherein the chip is maintained in the heating space for 2 minutes. 19. The method according to claim 17, wherein the amount of the plasma sample is reduced to 5 μl. 20. The method according to any one of claims 16 to 19, wherein the antibody reagent is about 60 μl of an antibody selected from the following group: an antibody that reacts with IgA, β2-microglobulin Antibodies reactive against C-reactive protein.