JPS5920838A - Turbidity measuring device - Google Patents

Abstract

PURPOSE:To make it possible to perform accurate automatic analysis when the components of a living body are determined by turbidity measurement, by making a specific dispersing agent to coexist in a reactive system, preventing sedimentation and cohesion of turbid particles, dispersing the particles, and avoiding the blocking of tubes and coils. CONSTITUTION:A dispersing agent is selected from the groups comprising carrageenin, xanthane gum, locust been gum, and tragacanth gum and mixture of these materials. The dispersing agent is added in a reacting system when the components in serum, urine, and the like are determined by turbidity measurement. In this way, formed turbid particles are evenly dispersed, and cohesion and sedimentation do not occur. Therefore, the tubes and coils of an automatic measuring device are not blocked. Since the dispersing agent does not give adverse effects on the components of a living body and a reagent, highly accurate analysis can be performed.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a turbidity measurement method, and more particularly to a method for quantifying biological components by turbidity measurement, which is particularly useful in the field of clinical testing.

When quantifying biological components, it is often necessary to react a sample with a quantitative reagent and quantify fine particles suspended in the reaction system. Such methods for quantifying fine particles can be roughly divided into turbidimetry, which measures transmitted light of a reaction suspension, and turbidimetry, which measures scattered light, both of which are widely employed in the field of clinical testing.

Because turbidimetry uses fluorescent light or laser light for measurement, its detection sensitivity is higher than that of turbidimetry, and it can detect insoluble particles that are invisible to the naked eye. It has been applied to the quantification of minute amounts of precipitates, and its automatic analysis has also become commonplace.

On the other hand, although the detection sensitivity of the turbidimetric method is lower than that of the nephelometric method, it is widely used for quantifying urine protein, colloid reaction, β-lipoprotein, etc. due to the convenience of being able to carry out ordinary colorimeter-C measurements. has been done.

However, the turbidity method requires a level of turbidity that is visible to the naked eye, and therefore the size and density of the insoluble particles are relatively high.
Turbid particles within the reaction system tend to settle. Therefore, in order to obtain an appropriate quantitative value, it is necessary to homogeneously disperse these particles in the reaction system, and the reaction system must be shaken and mixed during measurement, which is a problem in the turbidity method. It imposes constraints on the application of automated analysis.In particular,
In recent years, automatic analyzers using a so-called continuous flow system, such as the autoanalyzers SMA and SMAC manufactured by Technicon Corporation in the United States, have been developed. In addition to their tendency to settle, particles also tend to aggregate and become large due to interactions between particles and contact with the inner walls of the tubes and coils while flowing through the tubes and coils of the C1 analyzer, and the dispersion of particles is reduced. This results in non-uniformity and eventually occlusion of the tube or coil, making it virtually impossible to automate with a continuous flow system.

In view of these circumstances, the present inventor conducted extensive research to apply the turbidity method to automatic analysis, particularly to a continuous °70-system, and unexpectedly found that carrageenan, xanthan gum, and locust were used as dispersants. We have discovered that when bean gum, tragacanth gum, or a mixture of these coexists in the reaction system, the homogeneous dispersion of turbid particles can be removed without interfering with measurements, and particle sedimentation and agglomeration can be prevented, thereby achieving the purpose. Ta.

That is, the present inventor aims to homogeneously disperse turbid particles in the turbidity method, prevent sedimentation and aggregation of particles, and coexist in the reaction system as a dispersant with a substance that does not interfere with measurement. One way to do this is to consider the following and search for various substances that have the expected effect.

β-lipoprotein quantitative method described below (sample: 900 μ/dl,
According to the reaction final concentration of the specimen: 26 mg/l), various dispersants were added to the reaction system at a final reaction concentration of 0.7 g/l, and the appearance of turbid particles, sedimentation state when left still, continuous ゛70 - We observed the state of giant particle formation after passing through the mixing coil in the system.

The results are shown in Table 1. The evaluation of sedimentation and giant particle formation due to standing in Table 1 is carried out according to the following criteria.

Sedimentation due to standing still - It does not settle even after being left for several hours and remains homogeneous.

±... Precipitation occurs in 1 to 2 hours after the start of antigen-antibody reaction.

+...-C precipitates around 30 minutes after the start of the antigen-antibody reaction.

廿...After the start of antigen-antibody reaction, C-precipitate for 10 to 15 minutes.

n, Precipitates simultaneously with the start of the antigen-antibody reaction.

Formation of giant particles (when passed through a mixing coil (20 turns) in a 70-system) - remains completely homogeneous.

±...Fine particles are slightly observed with the naked eye.

+... Some large particles are observed.

廿・・・Many giant particles are formed.

Hexagram: Formation of significant giant particles.

Table J Table 2 also shows the results of measuring the Δ optical density at 66 Qnm of the reaction left standing for 1 hour as it was and after shaking.

Table 2 As shown in Tables 1 and 2, among the various substances searched, carrageenan, xanthan gum, locust bean gum, tragacanth gum, and mixtures thereof specifically disperse turbid particles homogeneously and It was found to prevent sedimentation and aggregation. Furthermore, as is clear from the optical density after shaking in Table 2, even when these substances are added, the optical density is almost the same as when no additives are added, indicating that these substances interfere with measurement. It was also found that this is not the case.

Similar searches were also conducted for various anionic and nonionic surfactants, but these inhibited antigen-antibody reactions and were not suitable as dispersants.

The present invention was completed based on this knowledge, and when quantifying biological components by turbidity measurement, a dispersion selected from the group consisting of carrageenan, xanthan gum, locust bean gum, tragacanth gum, and mixtures thereof is added to the reaction system. The present invention provides a turbidity measurement method characterized by the coexistence of an agent. Conventionally, for example, gum arabic was included in the reagent when measuring urine protein (ExLon
, J.

Lab, &CIiCl1n0. ．． 10: 722,
1925) and the use of gum arabic as a specific gravity adjuster in a turbidity standard solution (Japanese Unexamined Patent Publication No. 16845/1983). However, according to the method of the present invention, by using these dispersants, it is possible to perform not only manual analysis but also automatic analysis, especially continuous analysis.
The turbidity method can also be suitably applied to analysis using a zero-meter stem.

Thus, the turbidity measurement method of the present invention can be applied to the determination of various biological components such as urine protein, β-lipoprotein, γ-globulin, and immunoglobulin by the turbidity method in clinical tests. For example, quantification is carried out according to conventional methods such as serum colloid reaction or turbidity immunoassay, such as thymol turbidity test ('r'r'r), zinc diasulfate turbidity test (z'r ding), which are used as liver function testing methods. When performing this, the dispersant may be added to the reaction system.

The dispersing agent may be carrageenan, xanthan gum, locust bean gum, tragacanth gum alone or a mixture of two or more of these.
~1:2 mixtures are preferred.

The amount of dispersant added can be selected depending on the type and concentration of the sample, the test method used, etc., but the final reaction concentration is usually 0.
．． 01-1.0g/3 Preferably, for example, in the case of β-lipoprotein determination, 0.1-0.8g/l, and in the case of urine protein determination, about 0.3-1.0g// gives good results. is obtained. In addition, the addition method and addition time can be selected as appropriate. For example, it is sufficient to prepare an aqueous dispersant solution with an appropriate concentration and add it before, during, or after the reaction between the specimen and the quantitative reagent. Even when employing the 70-system, the tube and the mixing coil may be appropriately combined and a dispersant may be added to the reaction system as appropriate.

Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 (quantification of urine protein) Preparation of reagent (1) Reagent I: Sulfosalicylic acid/sodium sulfate 70 g of liquid crystalline sodium sulfate was dissolved under heating in approximately 800 ml of water, and after cooling, 50 g of sulfosalicylic acid was added and dissolved.
The total volume was made up to 100ml with water.

(2) Reagent (2): Dispersant liquid 2 g of carrageenan was added to 200-500 ml of water and dissolved by boiling. After cooling, add water to bring the total volume to 10001
nl, filtered through a glass filter, and used the P solution.

(3) Albumin standard solution 600 ml of bovine serum albumin was dissolved in water to make the total volume 100-. Dilute this with water and give 600 ml each.
．． Standard solutions of 300.150 and 75~/dl were prepared.

Measurement operation Urine sample 0. After adding 3.0 rnl of reagent I to I mZ and mixing, 11.5 rnl of reagent I was further added, mixed well, and left for 5 minutes. A similar operation was carried out using 0.1 mm of water instead of the urine specimen, and using this as a control, the optical density at 6601 m of the reaction suspension containing the specimen was measured. A calibration curve was created in the same manner using an albumin standard solution instead of the urine sample. The optical density of the sample-containing reaction suspension was 0.10, and the urine sample was found to contain 150-/d/d/d of protein.

Both reaction suspensions showed a homogeneous dispersion of turbid particles.

Example 2 (quantification of β-riboprotein) Preparation of reagents (1) Reagent engineering: β-riboprotein quantitative reagent BL-A immunoturbidimetric antiserum was mixed with BL-A buffer (both Nippon Shoji Co., Ltd.) (Released) and diluted 50 times.

(2) Reagent ■: Dispersant liquid Same as reagent ■ in Example].

(3) Serum standard solution BL-A standard serum (lyophilized product) (Nippon Shoji@) released)
Add 3rnl of water to dissolve, further dilute with water =C
1 each, 900.600.400.200 and 100
A standard solution of ``5'/dJ was prepared.

Measurement procedure: 0.5 μl of water was added to 50 μl of the serum specimen and mixed, followed by 0.6 μl of reagent IO and 0.6 μl of reagent 1, mixed, and allowed to stand for 5 minutes. Water 0.0% instead of serum sample. I
A similar operation was performed using 1 nl, and using this as a control, the optical density at 660 nm of the reaction suspension containing the specimen was measured. A calibration curve was created in the same manner using a serum standard solution instead of a serum sample. The optical density of the reaction suspension containing the specimen is 0.20, and the optical density of the serum specimen is 600.
It was found that it contained ~/di β-riboprotein. Both reaction suspensions showed a homogeneous dispersion of turbid particles.

Example 3 (Zinc sulfate turbidity test) Preparation of reagent (1) Reagent ■: Barbital mild buffer solution route Rubital 02 ■ and Parbital sodium 1
Add 90"f to about 900ml of water and dissolve while heating.
From now on, it was made up to 1000 rnl with water.

(2) Reagent (2): Zinc sulfate aqueous solution Zinc sulfate (Z n504 .7H20) 480"i was dissolved in water to give a solution of 1000".

(3) Reagent ■: Dispersant liquid Same as reagent ① in Example 1.

(4) Kunkel reagent I500m
115 ml of reagent and 200 d of reagent (1) were added to Z, and the total volume was made up to 11 with water. If necessary, 0°IN sodium hydroxide or 0. The pH was adjusted to 7.60 with IN hydrochloric acid.

Measurement procedure Kunkel's reagent 3.0- was added to 50 μl of serum, mixed gently, and left at 25° C. for 30 minutes, after which the optical density was measured at 660 nm.

At the same time, the state of dispersion of the turbid particles was observed; homogeneous dispersion was observed, and no agglomeration or precipitate was observed. On the other hand, when ordinary Kunkel's reagent (reagent technique + reagent ■) was used with high unit serum (12 units or more), precipitation of a occurred due to standing.

Example 4 (Thymol turbidity test) Preparation of reagent (1) Thymol reagent 100 (6° Og of thymol crystals was placed in a 7-volume Erlenmeyer flask and mixed and dissolved by pouring 300 rn1. of boiling distilled water. Add 3.09g and 1.69g of parbital sodium, add water 700+,
I/ was added and dissolved. After leaving it at room temperature, it was transferred to a 7-volume volumetric flask and diluted with water.
Furthermore, 1 g of thymol crystals was added and left overnight. The pH was adjusted to 7.55±0.03, and the supernatant was used for individual use.

(2) Dispersant liquid Same as reagent ① of Example 1.

Measurement procedure 2.71 nl of thymol reagent and 0.3 volume of dispersant solution were added to 50 μl of serum, mixed gently, and after standing at 25° C. for 30 minutes, 6600 m −C optical density was measured.

At the same time, the state of dispersion of the turbid particles was observed, and they were found to be homogeneous and no agglomeration or precipitate was observed. On the other hand, when only the thymol reagent was used with high-unit serum (6 units or more), aggregation and precipitation occurred upon standing.

Example 5 Urine protein was determined in the same manner as in Example 1 using the continuous 70-degree system shown in the diagram shown in the attached Figure 1.

First, flow the reagent ■ into the tube] at a flow rate of 1.5 rn 1.7 minutes, and then pour the urine sample from tube 2 into the tube at a flow rate of 0,1 m7.
! The mixture was added at a flow rate of /min and passed through the mixing coil 3. Next, reagent ■ was added from tube 4 at a flow rate of 0.5 m17 min, passed through mixing coil 5, and 66
The optical density at Q nm was measured.

The residence time in the mixing coils 3 and 5 is 1
I understand.

The particles in the suspension after passing through the mixing coil 5 were homogeneously dispersed, no formation of giant particles was observed, and no blockage of the tube or coil was observed.

Example 6 β-riboprotein was quantified in the same manner as in Example 2 using the continuous flow system shown in the diagram shown in the attached FIG. 2.

First, reagent (2) was flowed from tube 6 at a flow rate of 0.6 d/min. On the other hand, water was flowed from tube 7 at a flow rate of 0.5 m/min, and a serum sample was added from tube 8 at a rate of 0.1 rn.
Flow rate of l/min -C plus mixing coil 9teU fD
After that, it was introduced into tube 6. These mixed liquids are passed through the mixing coil 10, and the flow rate is 0 from the tube
．． Reagent (2) fed at 6 ml/min was added and passed through the mixing coil 12, and the optical density at 660 nm was measured. The residence times in mixing coils 10 and 12 were 5 minutes and 1 minute, respectively.

Mixing Coil] The particles in the suspension after passing through 2 were homogeneously dispersed, no formation of giant particles was observed, and no clogging of the tube or coil was observed.

Example 7 (quantification of β-riboprotein) Preparation of reagents (1) Reagent I: β-riboprotein quantitative reagent Same as the reagent technique in Example 2.

(2) Reagent ■: Dispersant liquid xanthan gum 1g and locust bean gum 0.5
g was added to about 500 ml of water and boiled to dissolve. From now on, the solution was made up to 100 Oynl with water, passed through a glass filter, and used as P solution.

(3) Serum standard solution Same as the standard solution in Example 2.

Measurement procedure Same as Example 6, except that the above reagent ■ is used as the reagent ■.
β-lipoprotein was quantified using a continuous flow system.

The particles in the suspension after passing through the mixing filter 12 were homogeneously dispersed, and no formation of giant particles was observed, nor was there any clogging of tubes or coils.

Example 8 (Zinc sulfate turbidity test) A Kunkel reagent was prepared in the same manner as in Example 3, except that reagent ① in Example 7 was used instead of reagent ②, and a zinc sulfate turbidity test was conducted.

As a result, no formation of insoluble particles was observed over a wide range of C in Kunkel units, and there was no sedimentation after standing, allowing for good measurement. On the other hand, when using ordinary Kunkel's reagent (without dispersant) with high-unit serum (12 units or more),
Coagulation and precipitation occurred upon standing.

Example 9 (Thymol turbidity test) Preparation of dispersant liquid 2 g of xanthan gum was added to about 500 ml of water and dissolved by boiling. From now on, water was added to bring the temperature to 10,100 O, filtered through a glass filter, and the p solution was used.

Measurement procedure The measurement was carried out in the same manner as in Example 4, except that the above-mentioned dispersant liquid was used.

As a result, no insoluble particles were observed over a wide range of Maclagan units, and there was no precipitation after standing, allowing for good measurements.

On the other hand, when only the thymol reagent was used with high-unit serum (6 units or more), aggregation and precipitation occurred upon standing.

Example 10 (Quantification of Urine Protein) Urine protein was quantified in the same manner as in Example 5, except that the dispersant solution in Example 9 was used in a continuous flow system.

The particles in the suspension after passing through the mixing coil 5 were homogeneously dispersed, no formation of giant particles was observed, and no blockage of the tube or coil was observed.

[Brief explanation of drawings]

Figures 1 and 2 are both representative diagrams of the method of the present invention performed in a continuous flow system. 1.2.4.6.7.8 and 1]...Tube, 3
．． 5.9, 1O and I2...Mixing coil patent applicant Nippon Shoji Co., Ltd. Agent Aoyama Hajime and 2 others Figure 1

Claims (10)

[Claims] (1) Turbidity measurement characterized by coexisting a dispersant selected from the group consisting of carrageenan, xanthan gum, locust bean gum, tragacanth gum, and mixtures thereof in the reaction system when quantifying biological components by turbidity measurement. Law. (2) The measurement method of item (1) above, in which the measurement is performed using a manual method. (3) The measurement method of item (1) above, in which the measurement is performed by automatic analysis. (4) The above-described (
3) Measurement method. (5) The final reaction concentration of the dispersant is “rO,01~i,og
/l any one of the above clauses (1) to (4) to coexist
Two measurement methods. (6) The measurement method according to any one of items (1) to (5) above, which uses a dispersant or carrageenan. (7) Item (1) above, wherein the dispersant is xanthan gum.
Any one of the measurement methods in Clause (5). (8) The dispersant is locust bean gum.
) to (5). (9) The measuring method according to any one of items (1) to (5) above, wherein the dispersant is gum tragacanth. (10) The dispersant is a mixture of xanthan gum and locust bean gum in a weight ratio of 1:0.1 to 1:2.
) to (5).