JPS6058826B2 - Nephelometry method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to nephelometry, which is one of the immunoassay methods for quantifying various components contained in body fluids such as blood and urine.

In the nephelometry method for immunoassay, in which the formation of antigen-antibody complexes is measured using a light scattering photometer, there is a conceptual relationship between the intensity of scattered light and the amount of antigen, as shown in Figure 1. Two different amounts of antigen correspond to one scattered light intensity.

In the conventional nephelometry method for immunological measurements, the first
Measurements are made so that measurements are carried out within the range 1 in the figure. However, since the sample may contain an unexpectedly high concentration of antigen, there is a possibility that the measurement is actually performed within the antigen excess area 2 in FIG. Therefore, in order to achieve more accurate measurements, for example, prepare two samples with different dilution ratios from one specimen, perform the same measurement on each sample, and examine the proportional relationship between the scattered light intensity and the dilution ratio. This requires complicated procedures such as determining whether the antigen is in the antigen-excessive region or not. Furthermore, it takes a long time to measure one object. The present invention provides a method for accurately and easily quantifying the amount of antigen by measuring only one sample per specimen, even if the concentration of the test component in the sample falls within the antigen-excessive range. purpose.

More specifically, nephelometry for immunoassay is characterized by irradiating the same subject with light of two different wavelengths simultaneously or sequentially, and detecting the intensity of scattered light from the subject for each wavelength. J on the method and the nephelometer that performs it. Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 The present invention was carried out by using human immunoglobulin G (hereinafter referred to as 1gG) as a measurement target and measuring the scattered light intensity at two wavelengths using a Hitachi spectrofluorophotometer (MPFC (1)).

That is, first, 20μ' of standard antigen (IgG) solution of each concentration was added to 2ml of a 16-fold diluted anti-1gG serum (derived from rabbit), and after incubation for 3 hours at room temperature, the primary light wavelength was changed to 400nm and 800nm. The scattered light intensity was measured for each case, and the primary light wavelength was 400 r.
Calibration curves were determined for each of 1m and 800I1m.

This calibration curve is shown in Figure 2, and in this figure, the scattered light intensity of a sample prepared by adding 20 μl of physiological saline to 2 ml of 16-fold diluted anti-1gG serum (hereinafter referred to as The value obtained by subtracting the antiserum blank) was further divided by the antiserum blank and displayed as the value (hereinafter referred to as relative difference scattered light intensity). Next, 1@ with physiological saline
Add 20Pe of human serum with unknown GG concentration diluted to 2ml of diluted anti-1gG antiserum, calculate the relative difference scattered light intensity at primary light wavelengths of 400r1m and 800r1m in the same way, and from Figure 2 Determination of IgG concentration was performed. The concentration was determined as follows.
From the calibration curve at the primary light wavelength of 400r1m in Figure 2,
Read the antigen amount at two points relative to the relative difference scattered light intensity at the primary light wavelength of 400 nm of the sample with unknown concentration determined above (the smaller value is set as CtOO, and the larger value is set as C3O). Similarly, for the primary light wavelength of 800 nm, the antigen amount at two points is read (same as C arm 0 and C?0). Here, if Ct(X) and C. ? 00 is equal, Yang He takes this value as the IgG concentration of human serum diluted 10 times, and C
If 00 and C00 are equal, this value is determined to be the IgG concentration of human serum diluted to 1. Furthermore, as can be seen from Fig. 2, for any sample, Ct'' and C 0
0 are equal, and C-AOO and CHOO are never equal, and this fact supports the principle of the present invention. As shown in this example, even samples containing a large amount of antigen, which could not be quantified using the conventional nephelometric method without photometry by changing the dilution ratio of the sample, can be easily quantified. The present invention has.

Example 2 The present invention was carried out by dual wavelength simultaneous photometry using the apparatus shown in FIG.

A tungsten lamp was used as the light source 12, and the light from it was passed through a lens system 13 and turned into primary light 14, which was applied to a sample 16 to be measured in a sample cell 15. 1 cm for sample cell 15
A four-sided transparent fluorescent cell with corners was used. 90 left and right for primary light
Scattered lights 17 and 18 in the 0 direction pass through monochromatic light devices 19 and 20 that can selectively transmit only the scattered lights with wavelengths of 450r1m and 650nm by combining an interference filter and an absorption filter, respectively, and are sent to a light receiving detector 21 and 22 to be detected. ) Photomultiplier tubes were used as the photodetectors 21 and 22, and their respective outputs were guided to preamplifiers 23 and 24, amplified by amplifiers 25 and 26, and simultaneously recorded by a recorder 27. . Incidentally, the primary scattered lights 17 and 18 were guided to a monochromatic light device through a slit. Using the above device, immunoassay using dual wavelength simultaneous photometry for IgGl human immunoglobulin A (hereinafter referred to as 1gA), human immunoglobulin M (hereinafter referred to as 1gM), and human complement third component (hereinafter referred to as C3) I went there.

In addition, all antisera are diluted with 1 volume, and 2 ml of this is diluted with standard antigen solution or human serum with unknown antigen concentration diluted with 1 volume, and for IgG, 20 μe 1 of other IgA, I
For gM and C3, 50 μe was added and measurements were performed after incubation for 1 hour at room temperature. Further, the antigen concentration of human serum with unknown antigen concentration was determined in the same manner as in Example 1. As a result, it was revealed that all the substances measured in this example can be easily quantified by dual wavelength photometry even if the sample contains an excessive amount of antigen. Embodiment 3 FIG. 4 is a system diagram of an apparatus in which the present invention is implemented using one light source and one light receiving detector.

In this embodiment, the light source 28 is a tungsten lamp, and the sample cell 31 is
A glass cylindrical cell was used for the unit, a combination of an interference filter and an absorption filter was used for the monochromatic light device 34, and a photomultiplier tube was used for the photodetector 37. Also, as a monochromatic light device, 4
A device that transmits light of 50 nm or 65 Qr1 m is prepared, and these are arranged concentrically on a disk 35, and this disk is rotated by a motor 36 that receives a control signal 41 from a computer 40 to generate scattered light. Each monochromatic light device was placed sequentially on the road. The measurement target is I
gG, and using the above-mentioned apparatus, immunoassays of samples with unknown antigen concentrations were performed by dual-wavelength photometry. The method for preparing the sample to be measured was the same as in Example 1. First, the computer 40 stores the scattered light intensity at each wavelength of a standard sample to which a standard antigen solution of each concentration has been added, and from this and the scattered light intensity at each wavelength of a test sample to which an unknown antigen concentration sample has been added, the antigen The antigen concentration of the sample with unknown concentration was determined by calculation using the computer 40.

This calculation method was based on the method of Example 1. In this example as well, immunoassays could be easily and accurately performed regardless of whether the sample contained an excessive amount of antigen or not. In this example, the monochromatic light device was placed on the scattered light path, but similar results could be obtained even if it was placed on the primary light path. In addition, a combination of an interference filter and an absorption filter was used as the monochromatic light device, but similar results were obtained even if this was replaced with a diffraction grating and the diffraction surface was rotated by a motor receiving control signals from a computer. Embodiment 4 FIG. 5 is a system diagram of an apparatus in which the present invention is implemented using two light sources and one light receiving detector.

In this embodiment, two light emitting diodes with different peak wavelengths are used as the light sources 45 and 46 (each peak wavelength is 73 cm).
A two-wavelength photometry method was used using 0nm and 830I1m). These two light emitting diodes are made to alternately emit light by passing a direct current 43 from a power source 42 to a switching circuit 44 which is operated manually or by a signal 58 from a computer 57. Further, a glass cylindrical cell was used as the sample cell 51, and a photomultiplier tube was used as the light receiving detector 54. Immunological measurements were carried out using the same arithmetic processing method as in Example 4 using the computer 57, using Isei as the measurement target. In this example as well, it was possible to easily determine the amount of antigen in a sample with unknown antigen concentration, regardless of whether the sample contained excess antigen or not.
In this example, two light emitting diodes with different peak wavelengths were used as light sources, but two tungsten lamps were used instead, and interference filters with different transmitted light wavelengths (450r1m and 650r1m) were installed on each primary optical path. The same results as in this example were obtained even when a monochromatic light device combining a light source and an absorption filter was installed, and shutters further provided on each primary light path were alternately opened and closed. Although the present invention is not limited to the above-mentioned embodiments, as explained above, according to the present invention, it is impossible to quantify with the conventional nephelometry method for immunoassay without changing the dilution ratio of the sample. Even samples containing excess antigen can be accurately and easily quantified.

Furthermore, the present invention, which covers the antigen-excessive region as a measurement range, allows the dilution ratio of antiserum used as a reagent to be increased accordingly, and has the effect of reducing the cost of reagents required for quantifying one sample. Regarding the two-wavelength photometry method of the present invention, it has been experimentally confirmed that the present invention can be fully implemented as long as the difference between the two wavelengths is 50 r1 m or more.

[Brief explanation of drawings]

FIG. 1 is a diagram showing a calibration curve for immunoassay by nephelometry. In this figure, 1 represents the measurement range used in conventional immunoassays, and 2 represents the antigen excess area. FIG. 2 is a diagram showing a calibration curve of IgG. In this figure, 3 and 4 each have a primary light wavelength of 400 nm.
There is also a calibration curve at 800r1m. FIG. 3 is a system diagram of the apparatus described in Example 2. In this figure, 12 is a light source, 13 is a lens system, 14 is a primary light, 15 is a sample cell, 16 is a sample to be measured, 17 and 18 are scattered lights, 19 and 20 are monochromatic light devices, 21 and 22
is a photodetector, 23 and 24 are preamplifiers, 25 and 2 are
6 represents an amplifier, and 27 represents a recorder. Figure 4 shows Example 3
FIG. 2 is a system diagram of the device described in FIG.

Claims (1)

[Claims] 1. Irradiate a measurement sample containing an antigen-antibody conjugate with primary light of two wavelengths, measure the scattered light intensity of the primary light, and calculate multiple antigen concentrations including a virtual antigen concentration obtained from the scattered light intensity. A nephelometry method for immunological measurement is characterized in that the true antigen concentration is determined by numerically comparing these multiple antigen concentrations.