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WO2005062044A1 - Procede de dosage de la proteine bence-jones - Google Patents

Procede de dosage de la proteine bence-jones Download PDF

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Publication number
WO2005062044A1
WO2005062044A1 PCT/JP2004/017645 JP2004017645W WO2005062044A1 WO 2005062044 A1 WO2005062044 A1 WO 2005062044A1 JP 2004017645 W JP2004017645 W JP 2004017645W WO 2005062044 A1 WO2005062044 A1 WO 2005062044A1
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WIPO (PCT)
Prior art keywords
protein
ratio
bjp
electrophoresis
chain
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2004/017645
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English (en)
Japanese (ja)
Inventor
Takanari Nakano
Atsuo Nagata
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Yamasa Corp
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Yamasa Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yamasa Corp filed Critical Yamasa Corp
Priority to JP2005516435A priority Critical patent/JP4286256B2/ja
Publication of WO2005062044A1 publication Critical patent/WO2005062044A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/6857—Antibody fragments

Definitions

  • the present invention relates to an efficient method for assaying urinary vents joint protein using urine protein electrophoresis and Immnothase together.
  • Monoclonal free light chain which is dissociated from the immunoglobulin heavy chain, is a monoclonal protein found in blood diseases such as plasma cell abnormalities, particularly in multiple myeloma and macroglobulinemia. (M protein) (Br. J. Haematol., 1969, 16, 599-606).
  • BJP is a useful clinical marker such as multiple myeloma, and its test is performed as an item of routine clinical examination.
  • BJP has a small molecular weight, easily passes through glomeruli, and most of it is transferred into urine. Therefore, in actual clinical tests, BJP in urine is detected by electrophoresis ( DRG / PPS Compatible Clinical Laboratory Guidelines Second Draft, 2000, pp. 78-82).
  • Urine protein electrophoresis is widely used as a screening method for BJP, but its detection sensitivity and specificity depend on the analysis system and the degree of urine preconcentration before testing. However, it is not always a satisfactory method. For example, cellulose acetate membrane, which is widely used as a support for electrophoresis, has poor resolution of imnoglobulin, and its detection sensitivity also changes depending on the type of staining dye for protein detection after electrophoresis. The electrophoresis method must have low sensitivity. Furthermore, since urine is used as a sample, it is necessary to concentrate urine in advance, but the degree of concentration in that case is not clearly defined.
  • the BJP screening method using electrophoresis has a large difference in test accuracy between facilities, and has various problems inherent in clinical tests. In fact, a recent control survey between facilities revealed that 60 mg Bd of Zd1 was overlooked in about 30% of the facilities, which is a problem.
  • the immunophenotype (subclass) of immunoglobulin cannot be identified by the above-mentioned electrophoresis, the subclass (BJP, IgGG) of the M protein detected using the immunofixation (or immunoelectrophoresis) method Type M protein or IgM type M protein).
  • the results of the electrophoresis method, the immunoassay, and the immunofixation method were compared, and the urinary / c-type light chain, including the light chain bound to intact immunoglobulin, was BJP-negative can be determined if there is no abnormality in the total (total) amount with the type I light chain and its ratio (to the total amount) and no abnormal band is observed by electrophoresis. Suggest that.
  • the sensitivity was 100%, but it is not necessarily BJP-specific. That is, the total (total) of the / c-type light chain and the ⁇ -type light chain in urine, including the light chain bound to the intact immunoglobulin, is quantified, and the amount and ratio are calculated.
  • an intact immunoglobulin M protein for example, IgG- ⁇ M protein
  • the amount and ratio of the immunoglobulin which is not FLC-specific and measures the total amount of light chain, is inevitable. It becomes abnormal. This abnormality in the amount and ratio is due to the presence of an immunoglobulin M protein other than BJP and is not necessarily BJP-specific.
  • the sensitivity refers to a percentage (%) of a urine sample with BJP that showed a positive result (see Example (4)).
  • the present inventors have conducted intensive studies on a more realistic and efficient BJP test method. (1) Based on the ratio (K ⁇ ⁇ ratio) of the KC and ⁇ chains of FLC in Imnoassay. (2) The use of both electrophoresis and Imnoassay to determine the presence or absence of the M protein in electrophoresis and the / c chain of FLC in Imnoassay The present inventors have found that BJ ⁇ ⁇ ⁇ can be tested efficiently by making a judgment based on a constant processing procedure (algorithm) based on the ratio of ⁇ chains ( ⁇ / ratio), and completed the present invention.
  • algorithm constant processing procedure
  • the present invention includes the following contents.
  • step 1 The method described in [1] above, in which step 1, step 2, and step 3 are performed in this order.
  • step 2 step 1, and step 3 are performed in this order.
  • the step to judge BJP positive If the M protein is confirmed, and the ratio is less than 0.2 ⁇ 0.1 or 1.67 ⁇ 0.84 or more, the step to judge BJP positive
  • step 2 step 1, and step 3 are performed in this order.
  • the M protein was confirmed, and the ratio was 0.2 ⁇ 0.1 or less or 1.67 / 0.84 or more.
  • the M protein was confirmed, and the ⁇ L ratio was more than 0.2 ⁇ 0.1 and less than 1.67 ⁇ 0.84.
  • the 1: 1 ratio was 0.2 or less, 0.1 or less, or 1.67 ⁇ 0.84 or more.
  • FIG. 1 is a diagram showing an example of a standard curve of an FLC measurement method in Imnoassy.
  • FIG. 2 is a view showing an example of a measurement result of urine FLC.
  • Two lines in the figure indicate the cut-off value of the ⁇ / ⁇ ratio determined by ROC analysis.
  • FIG. 3 is a diagram showing an example of a BJP test method (algorithm) when the FLC measurement method and the electrophoresis method are combined.
  • the sample used in the present invention is not particularly limited as long as it is a urine sample.
  • a urine collection form (urine with preservatives, daily urine, early morning urine, etc.) collected clinically is used as a sample. Can be used.
  • the present invention provides a method for assaying BJP positivity by Imnoassay (Method 1), and a method for assaying BJP in a urine sample by specific steps using electrophoresis and Imnoassay (Method 2). Encompasses the two methods of
  • the content of the FLC / c chain and the FLCA chain in the urine sample is measured by the Imnoassay, and the amount ratio ( ⁇ ratio) is obtained, and the ⁇ specific power SO. 1 ⁇ If it is 0.05 or less or 5.5 ⁇ 2.75 or more, it is a BJP test method that determines that BJP is positive.
  • the immunoassay used in the present invention is not particularly limited as long as it can measure the free imnoglobulin K chain and the free immunoglobulin I chain in urine.
  • the quantitative ratio ( ⁇ ratio) was calculated based on the measured values of the K chain and the I chain of FLC, and the ⁇ / 1 ratio was 0.1 ⁇ 0.05 or less or 5.5 soil. 2. If it is 75 or more, it is judged as BJP positive.
  • the ratio used as an indicator is determined by the operating characteristics of the patient (ROC) analysis (Clin. Chem., 1993, 39, 561-577). Therefore, even when compared with the results of the immunofixation method, the positive predictive value of the BJP test is 80% or more, so that it is 0.1 ⁇ 0.05 or less or 5.5 ⁇ 2.75 or more, preferably By using the criteria of 0.1 ⁇ 0.03 or less or 5.5 ⁇ 1.65 or more, diagnosis can be performed with high accuracy.
  • the positive prediction rate refers to the percentage (%) of the sample that showed a BJP positive result actually associated with BJP (see Example (4)).
  • this ratio may vary depending on the target patient group, it is necessary to set a clinically appropriate ratio each time within the above range.
  • the method for testing BJP in a urine sample by using both electrophoresis and Immonoassay is preferably performed in the following order of step 1, step 2, and step 3, but steps 1 and 2 are reversed. It does not matter.
  • Step 1 is a step for confirming the presence of M protein in the urine sample by electrophoresis.
  • the electrophoresis method used in the present invention is a known method.
  • Step 2 is a step of measuring the respective contents of the free immunoglobulin ⁇ chain and the free immunoglobulin; chain in the urine sample in the same manner as in the above-described Imuno-Assy, and obtaining the amount ratio ( ⁇ ratio). .
  • Step 3 is a step for judging that there is a suspicion of BJP negative, BJP positive, and / or BJP positive for each case, using the presence or absence of ⁇ protein and the ⁇ ratio as indices.
  • 0.1 ⁇ 0.05 or less or 5.5 ⁇ 2.75 or more, preferably 0.1 ⁇ 0.03 or less A value of 5 ⁇ 1.65 or more was adopted as the ⁇ / ⁇ ratio, the M protein was confirmed in step 1, and the ⁇ ratio obtained in step 2 was 0.1 ⁇ 0.05 or less or 5.5. If it is ⁇ 2.75 or more, it can be determined that BJJ is positive.
  • the diagnostic accuracy of the BJ ⁇ test is at least 80%, preferably at least 90%, specifically, as shown in the Examples below. , 0.2 ⁇ 0.1 and 1.67 ⁇ 0.84, preferably 0.2 ⁇ 0.03 and 1.67 A numerical value within the soil 0.5 range can be adopted as the ⁇ ratio.
  • the determination of BJP is based on the presence or absence of the M protein Judgment is made in the following four cases from the result of the ⁇ ratio. In other words, in case ⁇ , it is determined that BJP is positive, and in case: B and C, it is determined that BJP is suspected, and the presence or absence of BJP is further confirmed by the immunofixation method. Judge as negative.
  • the M protein was confirmed, and the / 1/1 ratio was 0.2 ⁇ 0.1 or less or 1.67 ⁇ 0.84 or more.
  • the M protein was confirmed, and the ⁇ I ratio was more than 0.2 ⁇ 0.1 and less than 1.67 ⁇ 0.84.
  • the ⁇ ratio was 0.2 ⁇ 0.1 or less or 1.67 ⁇ 0.84 or more.
  • ⁇ No protein was confirmed, and the ⁇ ratio was more than 0.2 ⁇ 0.1 and less than 1.67 ⁇ 0.84.
  • the immunofixation method was performed using a Helena kit.
  • the antiserum used for the immunoassay was purchased from Dako. Urine samples were concentrated up to 100-fold as needed.
  • the FLC assay was performed according to the literature (J. Immunol. Methods, 2003, 275, 9-17). That is, a sample or a standard concentration solution was added every 100 1 to a 96-well microplate (Nunc) coated with a monoclonal antibody, and reacted at room temperature for 2 hours. After washing, 100 ⁇ of each of the anti- ⁇ light chain antibodies and 100 ⁇ of horseradish peroxidase-labeled anti-light chain antibodies were added as secondary antibodies and reacted at 37 ° C. for 30 minutes. After the reaction, wash with PBS containing 0.05% Tween 20,
  • the diagnostic accuracy was evaluated using a calculation method generally used in the field of clinical testing. That is, the presence or absence of BJP is used as a control, and is classified as shown in Table 1 by the immunoassay method as a reference method and the method of the present invention as an evaluation object.
  • Sensitivity Calculated by the formula a / (a + b) x 100, and means the proportion of samples that showed a positive test result among BJP positive samples.
  • Positive prediction rate calculated by the formula a / (a + C ) x 100, means the proportion of samples that actually have BJP among the samples that show BJP-positive test results.
  • Accuracy Calculated by the formula (a + d) (a + b + c + d) x 100, and means the proportion of samples tested positive or negative for BJP correctly.
  • the statistical analysis software package (StatF1ex) was used for the analysis of the patient's operating characteristics (ROC).
  • Table 3 shows the results of the immunofixation method for the immunoassay and electrophoresis methods.
  • BJP was identified in 58 cases by immunofixation, and intact immunoglobulin M protein was detected in 14 cases.
  • M band was observed in 50 of 58 BJP-positive cases.
  • d Number of specimens in which abnormalities in ⁇ / ⁇ ratio were observed (c: ratio is 0.2 or less or 1.67 or more).
  • BJP Benth Jones protein.
  • the method of the present invention using the cutoff value as described above allows It is clear that the assay can be performed with a sensitivity of 89.7% (52 of 58 samples), a specificity of 81.6% (187 of 229), and an accuracy of 84.3% (242 of 287). It has become possible to easily extract highly-reactive samples.
  • the method of the present invention it is possible to provide, for the first time, a simple and highly reliable BJP test method by performing determination by a fixed processing procedure (algorithm) by combining ImnoAssy alone or by combining ImnoAssy and electrophoresis. It becomes.
  • the BJP subclass can be identified at the same time by measuring the contents of urinary free immunoglobulin K chain and urinary free immunoglobulin; I chain, and determining the amount ratio. It is possible, and it is possible to reduce the number of inspections under the fixed duty method, which is a confirmation inspection, to about 1-6.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
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  • Urology & Nephrology (AREA)
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  • Hematology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

L'invention concerne une méthode de dosage de la protéine de Bence-Jones (BJP) dans un échantillon d'urine au moyen d'une combinaison d'électrophorèses et d'un immunoessai. Ledit procédé consiste en la mise en oeuvre des étapes suivantes: (1) étape 1: confirmation de la présence de la protéine M dans un échantillon d'urine par électrophorèse; (2) étape 2: quantification de la chaîne d'immunoglobuline λ libre et de la chaîne d'immunoglobuline μ libre dans l'échantillon d'urine par immunoessai et détermination du rapport de contenus (rapport λ/μ) de ce dernier; et (3) étape 3: détermination de BJP-positif au cas que la présence de M protéine soit confirmée et que le rapport λ/μ soit d'au plus 0,1?0,05 et d'au moins 5,5?2,75.
PCT/JP2004/017645 2003-11-19 2004-11-19 Procede de dosage de la proteine bence-jones Ceased WO2005062044A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2005516435A JP4286256B2 (ja) 2003-11-19 2004-11-19 ベンスジョーンズ蛋白質の検定法

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JP2003-388617 2003-11-19
JP2003388617 2003-11-19

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008528995A (ja) * 2005-01-27 2008-07-31 ザ バインディング サイト リミテッド 悪性プラズマ細胞疾患の検出又はモニタリング方法
CN115684606A (zh) * 2022-10-21 2023-02-03 南方医科大学珠江医院 一种m蛋白检测的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03199966A (ja) * 1989-12-27 1991-08-30 Kazutomo Wakasugi 遊離型ｌ鎖の測定方法
JP2002156377A (ja) * 2000-09-08 2002-05-31 Tohoku Techno Arch Co Ltd 吸光ラベルを用いる尿中疾患マーカータンパク質の迅速・簡易検出方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03199966A (ja) * 1989-12-27 1991-08-30 Kazutomo Wakasugi 遊離型ｌ鎖の測定方法
JP2002156377A (ja) * 2000-09-08 2002-05-31 Tohoku Techno Arch Co Ltd 吸光ラベルを用いる尿中疾患マーカータンパク質の迅速・簡易検出方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LEVINSON S.S. ET AL: "An algorithmic approach using kappa/lambda ratios to improve the diagnostic accuracy of urine protein electrophoresis and to reduce the volume required for immunoelectrophoresis.", CLINICA CHIMICA ACTA, vol. 262, 1997, pages 121 - 130, XP002987743 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008528995A (ja) * 2005-01-27 2008-07-31 ザ バインディング サイト リミテッド 悪性プラズマ細胞疾患の検出又はモニタリング方法
CN115684606A (zh) * 2022-10-21 2023-02-03 南方医科大学珠江医院 一种m蛋白检测的方法
CN115684606B (zh) * 2022-10-21 2023-11-28 南方医科大学珠江医院 一种m蛋白检测的方法

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JP4286256B2 (ja) 2009-06-24

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