JPH0275955A - Method for measuring protein in human urine by reverse passive antibody hemagglutination reaction and kit for this method - Google Patents

Abstract

PURPOSE:To allow the exact and rapid measurement of the specific protein contained in the human urine by adjusting the pH of the urine to be inspected to 8.0 to 9.2 pH and using the supernatant liquid after the adjustment as the human urine to be inspected. CONSTITUTION:The pH of the urine to be inspected is previously adjusted to 8.0 to 9.2 and is subjected to RPHA (reversed passive hemaggutination assay) as it is, or if insoluble matter is formed, this matter is removed from the urine and the supernatant liquid is subjected to the above-mentioned assay. The RPHA consists in adding the suspension of the sensitized red cells contg. an effective quantity of antibodies to the liquids to be inspected of various concns. and observing, measuring and weighing the generated hemagglutination. The measuring and weighing are exactly executed by correlatively contrasting the results with the results of the contrast tests which are simultaneously carried out and regard a positive control and a negative control respectively as the liquids to be inspected. The non-specific hemagglutination is minimized in this way and the measurement accuracy is improved.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Technology] The present invention relates to a method for preventing non-specific reactions in an immunoassay for human-specific proteins using urine as a test liquid.

Due to physiological abnormalities or diseases, various specific proteins are expressed or changed in human urine, and this is often used for diagnosis. For example, the pregnancy bed contains pregnancy-specific protein (PR).
egnancy 5pecif'1cβ1-glyco
expression of protein (hereinafter referred to as SPl) was observed,
Its amount is considered an indicator of placental function and fetal growth. The amount of kallikrein in the urine of patients with essential or renal hypertension is also lower than that of normal humans, and higher in patients with Bertha syndrome.

Examples of other interrelated relationships between specific proteins and physiological abnormalities are as follows.

Disease or Abnormality - Specific Protein Myeloma - Pence-Jones protein Acute pancreatitis - amylase Chorionic disease-hCG.

Sanfilippo B syndrome - N-acetylglucosaminidase hyperfibrinolysis - FDP.

Therefore, there is a need for an accurate and rapid method for measuring correlated specific proteins for disease diagnosis.

[Conventional technology]

Immunoassay methods that label specific proteins with radioactive isotopes are accurate, but in addition to the instability, toxicity, and expense of the labeled proteins caused by the radioactive isotopes, the quantification method itself is complicated to clean up. The disadvantage is that it takes a long time. That is, in the conventional method in which the reaction is carried out in a liquid phase, the two-antibody method is used to separate the antigen (8 forms) that has formed a complex with the antibody and the free antigen (F form) (so-called B,/F separation). Alternatively, a polyethylene glycol precipitation method is employed. This requires incubation and centrifugation operations, which increases the time required for measurement and complicates the operations.

The applicant previously proposed a method of avoiding the complexity of the B/F separation by performing the antigen-antibody reaction in the same liquid heterogeneous phase. One of these is a radioimmunoassay based on the reaction between an anti-human specific protein antibody purified from the antiserum of an animal that has been sensitized and immobilized on a container such as polystyrene, and a labeled human-specific protein. . (JP-A-58-129364, European Patent No. 85402 regarding Kallikrein).

The other is a method based on the so-called reverse passive antibody hemagglutination reaction using the anti-specific protein antibody sensitized to immobilized red blood cells of animals or humans (Japanese Patent Application Laid-open No. 17476/1989 regarding S Pl; Unexamined Japanese Patent Publication No. 5 on Kallikrein
No. 9-026065 and European Patent No. 100395).

The latter method < reversed passivehe
maggluNation assay, hereinafter referred to as RPH
It is called A. ) compensates for the above-mentioned disadvantages of using radioisotopes and is clinically advantageous in that a large number of analytes can be measured easily and quickly.

[Problems to be solved by the invention and means thereof] When urine is used as the test solution in the latter method, non-specific agglutination may be observed in addition to specific red blood cell agglutination due to antigen-antibody reaction. Non-pregnant urine may give false positive results for SP1. This tendency cannot be prevented by the presence of stroma, animal serum, etc. in the reaction system to prevent the generation of non-specific reactions.

The present invention aims to improve the method for measuring specific proteins in human urine by RHPA, to minimize non-specific red blood cell agglutination, and to improve the accuracy of the method.

The above purpose is to adjust the pH of the raw wave test solution in advance to 8 or higher, preferably 8.8 to 9.2, and then perform RHPA on the clarified solution either directly or by removing insoluble matter if it is generated. This is achieved by

That is, the present invention provides immobilized sensitized red blood cells (hereinafter simply referred to as sensitized red blood cells), which are obtained by sensitizing an animal with a human specific protein and sensitizing the red blood cells with an anti-human specific protein antibody obtained from the generated antiserum; In a method for detecting or quantifying specific proteins in urine by a reverse passive antibody hemagglutination reaction in which this is reacted with a specific protein antigen in a human urine test fluid, the pH of the test urine is set to 8.0.
The present invention relates to an improved method for detecting or quantifying a specific protein in human urine, which comprises using as an antigen solution a clarified solution adjusted to a concentration of 9.2 to 9.2, either as it is, or after removing any insoluble matter that may be present as an antigen solution.

RHPA is well known (Japanese Unexamined Patent Publication No. 59-174)
No. 76, etc.), a suspension of sensitized red blood cells containing an effective amount of antibodies is added to a test solution of various concentrations (dilution factor), and the resulting red blood cell agglutination is observed, measured, and weighed. Measurement and weighing are performed accurately by correlating and comparing with the results of a control test in which the positive control and negative control are treated as test liquids. A neutral buffer solution is usually used in the reaction system.

By the way, when urine is used as the test liquid as in the case of the present invention,
Using urine as it is often causes inaccurate measurements. For example, when SP1 is measured using non-pregnant urine as a test liquid, positive cases may be observed even though the results should be negative, and this naturally affects the original determination of SP1DI. Insoluble substances in urine and insoluble substances generated during standing are thought to be the cause, but even in urine that does not produce insoluble substances during standing, this phenomenon due to a nonspecific reaction may be observed. The above phenomenon is unique to the method test liquid and is not observed in negative controls such as physiological saline.

When urine is made alkaline (pH 8 or higher), insoluble matter is produced in many cases. When using a clear liquid from which insoluble matter has been removed by filtration or centrifugation as the test solution, the non-specific reaction described in 2. is prevented. On the other hand, if urine is alkaline and does not produce insoluble matter, non-specific reactions may occur if it is measured as is. Even in this case, it has been found that non-specific reactions can be prevented by measuring as alkaline.

That is, in the present invention, urine has a pH of 8 or higher, preferably 8.8 to 9.
．． 2, and if insoluble matter is produced, it is removed, and if no insoluble matter is produced, it is left as is, and the clear solution after pH adjustment is used as the test solution.

For pH adjustment, it is convenient to use, for example, a Tris-hydrochloric acid buffer or a boric acid buffer.

Although SP1 has been explained above as an example, the method is also common to the measurement and quantification of other specific proteins such as kallikrein, hCG, FDP, amylase, Bence Jones protein, and N-acetylglycosaminase as specific proteins in urine.

The present invention also provides: (a) anti-human specific protein antibody-sensitized red blood cells (b) standard positive control (c) standard negative control (d) measurement buffer (neutral) (e) urine pH adjustment buffer ( The present invention provides a human urine-specific protein ΔP1 determination kit for reverse passive antibody hemagglutination reaction consisting of a combination of pH 8 to 9.2).

Regarding kits (a) to (d), the above-mentioned Japanese Patent Application Laid-open No. 1983-
This can be fully understood from the detailed explanation in No. 26065. (e) is described in this specification.

Example 1 100 human non-pregnant urine samples were collected for S P1 detection.
Applied to RHPA. One series is urine as it is, and the other series is 10ml of urine and 3M Tris-HCl buffer (pH 9, 0).
1 ml was added, and if any insoluble material was generated, this was removed.

The results are shown in Table 1.

Table 1 [Operations and Effects of the Invention] As described above, the present invention provides a method that makes it possible to accurately and quickly measure specific proteins contained in human urine by RHPA, and a kit used therein. This method prevents the inherent non-specific reactions based on the forensic sample liquid, and has high value as a clinical diagnostic method.

Patent applicant: Midori Juji Co., Ltd.

Claims (2)

[Claims] (1) In the method of measuring protein in urine by reverse passive antibody hemagglutination reaction, the pH of the test urine is adjusted to 8.0 to 9.2, and the adjusted clear liquid is used as the human urine test solution. A method for measuring specific proteins in human urine. (2) (a) Anti-human specific protein antibody sensitized red blood cells (b) Standard positive control (c) Standard negative control (d) Measurement buffer (neutral) and (e) Urine pH adjustment buffer (pH 8~ 9.2) A human urine-specific protein measurement kit for reverse passive antibody hemagglutination reaction comprising the combination of: