JP4286256B2 - Bence Jones protein assay - Google Patents

Description

  The present invention relates to an efficient assay method for urinary Bence Jones protein using a combination of urine protein electrophoresis and immunoassay.

Monoclonal free immunoglobulin light chain (FLC) in a state dissociated from immunoglobulin heavy chain is a monoclonal protein (M (Br. J. Haematol., 1969, 16, 599-606).
In 1847, Henry Bence Jones discovered a unique thermal aggregation reaction in patient urine and named the protein responsible for it as Bence Jones protein (BJP) (Lancet, 1847, 2, 88-92). . It was later revealed that this was a monoclonal FLC in urine.
BJP is a useful clinical marker such as multiple myeloma, and its assay is performed as one item of daily clinical examination. BJP has a small molecular weight, easily passes through the glomerulus, and most of it moves into urine. Therefore, in actual clinical examination, BJP is detected by electrophoresis (DRG / PPS-compliant clinical laboratory guidelines second draft, 2000, p. 78-82).
The measurement of BJP by electrophoresis is generally referred to as “screening assay” or simply “screening”. Usually, since immunoglobulins in human specimens have a polyclonal composition, immunoglobulins in specimens show a broad band when electrophoresis is performed. On the other hand, since the M protein has a single charge and exhibits a single band (M band), the two can be distinguished by electrophoresis.
Although electrophoresis of urine proteins is widely adopted as a screening method for BJP, its detection sensitivity and specificity depend on the analytical system and the degree of concentration of urine before testing, and are not necessarily satisfactory. For example, cellulose acetate membranes that are widely used as electrophoresis supports have poor immunoglobulin resolution, and the detection sensitivity varies depending on the type of staining dye for protein detection after electrophoresis. Must be 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.
Therefore, the screening method for BJP using the electrophoresis method has a large difference in test accuracy between facilities, and has various problems as clinical tests. In fact, in a control survey between facilities conducted in recent years, it became clear that 60 mg / dl of BJP was overlooked in about 30% of facilities, which is a problem.
In addition, since the immunophenotype (subclass) of immunoglobulin cannot be identified by the above electrophoresis method, the subclass of M protein (BJP, IgG type) detected using the immunofixation method (or immunoelectrophoresis method) M protein or IgM type M protein) must be confirmed. In addition, the immunofixation method used in these confirmation tests is very complicated and the test unit price is expensive. Therefore, in clinical testing, the M protein is first screened by electrophoresis, and then the immunofixation method is used. Therefore, the current situation is that a method for determining BJP must be taken.

Whether or not it is possible to overcome the current situation, use two types of methods, electrophoresis and immunoassay, improve the reliability of the test, and reduce the number of times of the immunofixation method for the definitive test (Clin. Chim. Acta, 1997, 262, 121-130). That is, in the above report, the results of electrophoresis, immunoassay, and immunofixation were compared, and the κ-type light chain and λ-type light in urine, including the light chain bound to intact immunoglobulin, were included. If there is no abnormality in the total amount of the chain (total amount) and the amount ratio (total amount and κ / λ ratio) and no abnormal band is observed by electrophoresis, it can be judged as BJP negative. Suggests.
However, the above examination has various problems as described below, and it is not always a reliable conclusion when it is used in an actual clinical test.
(1) Sensitivity problem As a result of the above examination, it is concluded that the sensitivity is 100%, but it is not necessarily BJP-specific. That is, the total amount of κ-type light chain and λ-type light chain in urine, including the light chain bound to intact immunoglobulin, is quantified, and the amount and ratio are calculated. When an immunoglobulin M protein (such as IgG-κ-M protein) is present in urine, it is not FLC specific, and the amount and ratio are necessarily abnormal in an immunoassay that measures the total amount of light chain. This amount and ratio abnormality is an abnormality derived from the presence of immunoglobulin M protein other than BJP, and is not necessarily BJP-specific. Here, sensitivity refers to the percentage (%) of samples that showed a positive result among urine samples with BJP (see Example (4)).
Further, the measurement sensitivity of the immunoassay employed in the above examination is poor, and about half of the samples subjected to the test are below the measurement sensitivity, and no further examination has been made, so it cannot be said to be reliable data.
(2) Problem of reduction in the number of confirmation tests As a result of the above examination, it has been concluded that the number of confirmation tests by the immunofixation method can be reduced by 52%, but there is a merit of combining two methods of electrophoresis and immunoassay. Not shown at all. That is, in the above report, the test result by electrophoresis was simply confirmed by immunoassay, and specimens showing M protein by electrophoresis require confirmation test of immunofixation method regardless of the result of immunoassay. There is no difference from this method. There is no rationale for why confirmation testing can be reduced by 52%.
In addition, in the confirmation test by the immunofixation method, there are cases in which B protein is positive even if M protein is not detected by electrophoresis, and if M protein is not confirmed, all are BJP negative regardless of the immunoassay result ( The conclusion of NO BJP) is only brute force and the scientific reliability is low.
As a result of intensive studies on a more realistic and efficient BJP assay, the present inventors have (1) based on the ratio of κ chain to λ chain (κ / λ ratio) of FLC in immunoassay. , BJP positivity can be determined, (2) The combination of electrophoresis and immunoassay, constant based on the presence of M protein in electrophoresis and the ratio of κ chain to λ chain (κ / λ ratio) in the immunoassay It was found that it is possible to efficiently test BJP by judging by the processing procedure (algorithm) of the above, and the present invention was completed.
That is, the present invention includes the following contents.
[1] A method for assaying Bence Jones protein (BJP) in a urine sample by carrying out the following steps in combination with electrophoresis and immunoassay.
(1) Step 1
Confirming the presence of M protein in the urine sample by electrophoresis (2) Step 2
Step (3) Step 3 of measuring the content of free immunoglobulin κ chain and free immunoglobulin λ chain in the urine sample by immunoassay and determining the ratio (κ / λ ratio)
When M protein is confirmed and the κ / λ ratio is 0.1 ± 0.05 or less or 5.5 ± 2.75 or more, a step of judging BJP positive [2] Step 1, Step 2, Step 3 The method according to [1], which is performed in the order of:
[3] The method described in [1] above, which is performed in the order of step 2, step 1, and step 3.
[4] A method for assaying Bence Jones protein (BJP) in a urine sample by using electrophoresis and immunoassay together and performing the following steps.
(1) Step 1
Confirming the presence of M protein in the urine sample by electrophoresis (2) Step 2
Step (3) Step 3 of measuring the content of free immunoglopurin κ chain and free immunoglobulin λ chain in a urine sample by immunoassay and determining the ratio (κ / λ ratio)
When M protein is confirmed and the κ / λ ratio is 0.2 ± 0.1 or less or 1.67 ± 0.84 or more, a step of judging BJP is positive [5] Combined electrophoresis and immunoassay A method for assaying Bence Jones protein (BJP) in a urine sample by performing the following steps.
(1) Step 1
Confirming the presence of M protein in the urine sample by electrophoresis (2) Step 2
Step (3) Step 3 of measuring the content of free immunoglobulin κ chain and free immunoglobulin λ chain in the urine sample by immunoassay and determining the ratio (κ / λ ratio)
When M protein is confirmed and the κ / λ ratio is more than 0.2 ± 0.1 and less than 1.67 ± 0.84, it is judged that there is a suspicion of BJP positive [6] Electrophoresis and immunoassay And testing the Bence Jones protein (BJP) in the urine sample by performing the following steps.
(1) Step 1
Confirming the presence of M protein in the urine sample by electrophoresis (2) Step 2
Step (3) Step 3 of measuring the content of free immunoglobulin κ chain and free immunoglobulin λ chain in the urine sample by immunoassay and determining the ratio (κ / λ ratio)
If M protein is not confirmed and the κ / λ ratio is 0.2 ± 0.1 or less or 1.67 ± 0.84 or more, a step of judging that there is a suspicion of BJP positive [7] A method for assaying BJP in a urine sample by performing the following steps in combination with an immunoassay.
(1) Step 1
Confirming the presence of M protein in the urine sample by electrophoresis (2) Step 2
Step (3) Step 3 of measuring the content of free immunoglobulin κ chain and free immunoglobulin λ chain in the urine sample by immunoassay and determining the ratio (κ / λ ratio)
If the M protein is not confirmed and the κ / λ ratio is more than 0.2 ± 0.1 and less than 1.67 ± 0.84, it is determined that the BJP is negative [8] Step 1, Step 2, Step The method according to any one of [4] to [7], which is performed in the order of 3.
[9] The method according to any one of [4] to [7], which is performed in the order of step 2, step 1, and step 3.
[10] A method for assaying Bence Jones protein (BJP) in a urine sample by using electrophoresis and immunoassay together and performing the following steps.
(1) Step 1
Confirming the presence of M protein in the urine sample by electrophoresis (2) Step 2
Step (3) Step 3 of measuring the content of free immunoglobulin κ chain and free immunoglobulin λ chain in the urine sample by immunoassay and determining the ratio (κ / λ ratio)
Based on the presence or absence of M protein and the κ / λ ratio, in case A, it is determined that BJP is positive, in cases B and C, it is determined that there is a suspicion of BJP. Further confirmation, in the case of Case D, a step of judging BJP negative (Case A)
M protein is confirmed, and the κ / λ ratio is 0.2 ± 0.1 or less or 1.67 ± 0.84 or more.
(Case B)
M protein is identified and the κ / λ ratio is greater than 0.2 ± 0.1 and less than 1.67 ± 0.84.
(Case C)
M protein is not confirmed, and κ / λ ratio is 0.2 ± 0.1 or less or 1.67 ± 0.84 or more.
(Case D)
M protein is not confirmed, and the κ / λ ratio is more than 0.2 ± 0.1 and less than 1.67 ± 0.84.
[11] The method described in [10] above, which is performed in the order of step 1, step 2, and step 3.
[12] The method according to [10], which is performed in the order of step 2, step 1, and step 3.
[13] The content of free immunoglobulin κ chain and free immunoglobulin λ chain in a urine sample is measured by immunoassay, and the ratio (κ / λ ratio) is determined. The κ / λ ratio is 0.1 ± 0. Bence Jones protein (BJP) test method for judging BJP positive when .05 or less or 5.5 ± 2.75 or more.

FIG. 1 is a diagram showing an example of a standard curve of an FLC measurement method in an immunoassay.
FIG. 2 is a diagram showing an example of the measurement result of urinary FLC. In the figure, ◯: BJPκ positive specimen, Δ: BJPλ positive specimen, X: BJP negative specimen, respectively, and two lines in the figure show the cutoff value of κ / λ ratio determined by ROC analysis.
FIG. 3 is a diagram illustrating an example of a BJP test method (algorithm) when combining the FLC measurement method and the electrophoresis method.

The sample used in the present invention is not particularly limited as long as it is a urine sample, and specifically, a urine collection form (preservative-added urine, daily urine, early morning first urine, etc.) collected clinically is used as a sample. Can be used.
In the present invention, as described above, the method of assaying BJP positivity by immunoassay (Method 1), the method of assaying BJP in a urine sample by a specific step using both electrophoresis and immunoassay (Method 2) These two methods are included.
(Method 1)
As a method of testing for BJP positivity by immunoassay, the content of FLCκ chain and FLCλ chain in a urine sample is measured by immunoassay, and the quantitative ratio (κ / λ ratio) is determined. When it is ± 0.05 or less or 5.5 ± 2.75 or more, it is a BJP test method for determining BJP positive.
The immunoassay employed in the present invention is not particularly limited as long as it can measure urinary free immunoglobulin κ chain and free immunoglobulin λ chain. Specifically, it has sufficient measurement sensitivity for FLC present in trace amounts in urine, shows little cross-reactivity to intact immunoglobulin (for example, 1% or less), and contains components other than FLC in urine. A method that is not affected is suitable, and an enzyme immunoassay developed by the present inventors can be exemplified (J. Immunol. Methods, 2003, 275, 9-17).
After the measurement, the quantitative ratio (κ / λ ratio) is calculated based on the measured values of the κ chain and λ chain of FLC, and the κ / λ ratio is 0.1 ± 0.05 or 5.5 or 5.5. When it is ± 2.75 or more, it is judged as BJP positive.
Since the κ / λ ratio of BJP-positive specimens fluctuates greatly, the κ / λ ratio used as an index, as shown in the examples below, is determined based on the operating characteristics of the examinee (ROC) (Clin. Chem., 1993, 39, 561-577), so that the positive prediction rate of the BJP test becomes a numerical value of 80% or more even when compared with the result of the immunofixation method, 0.1 ± 0.05 or 5.5 ± 2. By making a determination using a criterion of 75 or more, preferably 0.1 ± 0.03 or less or 5.5 ± 1.65 or more, diagnosis can be performed with high accuracy. In addition, a positive prediction rate means the ratio (%) with which the sample which showed the BJP positive result is actually accompanied by BJP (refer Example (4)).
In addition, since 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.
(Method 2)
The method of assaying BJP in a urine sample using both electrophoresis and immunoassay is preferably performed in the following order of Step 1, Step 2, and Step 3, but Step 1 and Step 2 are in reverse order. It does not matter.
(1) Step 1
Step 1 is a step of confirming the presence of M protein in the urine sample by electrophoresis.
The electrophoresis method employed in the present invention is a known method. Specifically, the protein contained in the specimen is electrophoresed and electrophoresed using a cellulose acetate membrane or agarose gel as a support. Is a staining method as well as a physicochemical detection method (High-Resolution Electrophoresis and Immunofixation, 1994, Chapter 2, Butterworth-Heinemann). After electrophoresis, the presence or absence of M protein is determined by the presence or absence of M band.
(2) Step 2
Step 2 is a step of measuring the contents of free immunoglobulin κ chain and free immunoglobulin λ chain in the urine sample by the same method as the above-described immunoassay, and determining the quantitative ratio (κ / λ ratio). is there.
(3) Step 3
Step 3 is a step of determining that there is a suspicion of BJP negative, BJP positive, and / or BJP positive for each case, using the presence or absence of M protein and the κ / λ ratio as indices.
For the purpose of judging only BJP positive, the same as in the above immunoassay, 0.1 ± 0.05 or less or 5.5 ± 2.75 or more, preferably 0.1 ± 0.03 or less or 5.5 ± 1 A numerical value of .65 or more is adopted as the κ / λ ratio, the M protein is confirmed in Step 1, and the κ / λ ratio obtained in Step 2 is 0.1 ± 0.05 or 5.5 ± 2.75 or more. If it is, it can be determined that the BJP is positive.
In addition, although it is possible to determine only BJP positive by immunoassay alone (method 1) as described above, it is possible to add the confirmation of M protein by electrophoresis in step 1 and to make a determination by taking this into account. A reliable determination can be made.
Next, for the purpose of determining not only BJP positive but also suspicion of negative and / or BJP positive, immunofixation is performed by a patient operating characteristic (ROC) analysis (Clin. Chem., 1993, 39, 561-577). Even when compared with the results of the method, the diagnostic accuracy of the BJP test is a numerical value of 80% or more, preferably 90% or more, specifically, the measured value of a BJP negative sample as shown in the examples described later. Are numerical values within the range of 0.2 ± 0.1 and 1.67 ± 0.84, preferably 0.2 ± 0.03 and 1.67 ± 0.5. It can be employed as the λ ratio.
The determination of BJP in this case is made in the following four cases from the results of the presence or absence of the M protein in step 1 and the κ / λ ratio in step 2. That is, in case A, it is determined that BJP is positive, in cases B and C, it is determined that there is a suspicion of BJP positive, the presence or absence of BJP is further confirmed by immunofixation method, and in case D, BJP is negative Judge.
(Case A)
M protein is confirmed, and the κ / λ ratio is 0.2 ± 0.1 or less or 1.67 ± 0.84 or more.
(Case B)
M protein is identified and the κ / λ ratio is greater than 0.2 ± 0.1 and less than 1.67 ± 0.84.
(Case C)
M protein is not confirmed, and κ / λ ratio is 0.2 ± 0.1 or less or 1.67 ± 0.84 or more.
(Case D)
M protein is not confirmed, and the κ / λ ratio is more than 0.2 ± 0.1 and less than 1.67 ± 0.84.

この区分に基づき、感度、特異度、陽性予想値、正確度を以下のように定義した。
感度：式ａ／（ａ＋ｂ）×１００で算出され、ＢＪＰ陽性検体の中で、陽性の検査結果を示した検体の割合を意味する。
特異度：式ｄ／（ｃ＋ｄ）×１００で算出され、ＢＪＰ陰性検体の中で、陰性の検査結果を示した検体の割合を意味する。
陽性予測率：式ａ／（ａ＋ｃ）×１００で算出され、ＢＪＰ陽性の検査結果を示した検体の中で、実際にＢＪＰを伴う検体の割合を意味する。
正確度：式（ａ＋ｄ）／（ａ＋ｂ＋ｃ＋ｄ）×１００で算出され、検査された検体のうち、ＢＪＰの陽性と陰性が正しく診断された検体の割合を意味する。
（５）統計解析
受診者動作特性（ＲＯＣ）分析には統計解析ソフトウェアパッケージ（ＳｔａｔＦｌｅｘ）を用いた。
（６）結果
イムノアッセイの測定結果より、κ／λ比０．１又は５．５をカットオフとした場合、表２に示すように、該当する検体は２８７例中５６例存在した。その中で、κ／λ比５．５以上に該当する全１８例あり、そのうち１６例がＢＪＰκ陽性で、ＢＪＰ以外の２例のうち１例は、免疫固定法でも分析不可能であった。一方、κ／λ比０．１以下で該当する全３８例あり、そのうち２９例がＢＪＰλ陽性であった。残りの９例のうち、５例はインタクトイムノグロブリンＭ蛋白陽性、３例はＢＪＰ陰性、１例は免疫固定法で解析不能の検体であった。このように、κ／λ比０．１又は５．５を基準として評価することにより、免疫固定法で解析不能だった２検体を除いて計算すると、８３．３％の陽性予測率（５４例中４５例）でＢＪＰ陽性と判定できる。
［表２］

次に、イムノアッセイ、電気泳動法ならびに免疫固定法の結果を表３に示す。表３から明らかなように、２８７臨床尿検体のうち免疫固定法により５８例にＢＪＰが同定され、１４例にインタクトイムノグロブリンＭタンパク質が検出された。また、尿蛋白の電気泳動法ではＢＪＰ陽性の５８例中５０例にＭバンドが観察された。
［表３］

また、図２に示すように、ＢＪＰ陽性検体のκ／λ比の中央値はＢＪＰκ型で５５．５、ＢＪＰλ型で０．００１２であった。一方、ＢＪＰ陰性検体では０．６３であった。
これらのデータを基に、ＲＯＣ分析によりカットオフ値を１．６７（ＢＪＰκ用）と０．２（ＢＪＰλ用）とに定め、図３に示すアルゴリズムにより電気泳動とκ／λ比を組み合わせ、２８７尿検体について解析を試みた。その結果を表４に示す。
表４から明らかなように、尿蛋白の電気泳動法でＭバンドが検出された６２例のうち、５３例をケースＡと判断し、そのうち４６検体（８６．８％）がＢＪＰ陽性であった。
また、Ｍバンドが検出されなかった２２５例のうち、１８３例にκ／λ比の異常は見られなかった。このうち９８．９％（１８３例中１８１例）がＢＪＰ陰性であった。
さらに、２８７例のうち５０例はＭバンドまたはκ／λ比の異常のどちらかを示した。このうち１０例はＢＪＰ陽性であった。
このように、上記のようなカットオフ値を用いた本発明方法により、ＢＪＰを感度８９．７％（５８検体中５２例）、特異度８１．６％（２２９例中１８７例）、正確度８４．３％（２８７例中２４２例）で検定できることが明らかとなり、ＢＪＰの可能性の高い検体を容易に抽出する事が可能となった。
［表４］

EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited to a following example at all.
[Example]
(1) Urine sample 287 samples of urine collected from 287 patients suspected of having BJP were used. All samples were anonymized for non-connection according to the guidelines of the Japanese Society for Clinical Laboratory Medicine (Clinical Pathology, 2002, 50; 438-439).
(2) Electrophoresis method and immunofixation method Electrophoresis method and immunofixation method were carried out using a Helena kit. Antisera used for immunofixation was purchased from Dako. Urine samples were concentrated up to 100 times as needed.
(3) FLC measurement method About the FLC measurement method, it carried out according to literature (J.Immunol.Methods, 2003,275,9-17). That is, a sample or a standard concentration solution was added every 100 μl to a 96-well microplate (Nunc) coated with a monoclonal antibody and reacted at room temperature for 2 hours. After washing, 100 μl each of anti-κ light chain antibody and horseradish peroxidase labeled anti-λ light chain antibody as secondary antibodies were added and reacted at 37 ° C. for 30 minutes. After the reaction, the plate was washed with PBS containing 0.05% Tween 20, and a color developing solution (OPD, Sigma) was poured every 100 μl to cause color development. After the color development was stopped with 1N sulfuric acid, the absorbance at 492 nm was measured. As the assay buffer, 50 mM Tris-HCl (pH 7.5) containing 1% bovine serum albumin was used. As a standard, FLC isolated and purified from urine was used. An example of a standard curve is shown in FIG.
(4) Evaluation of diagnostic accuracy The diagnostic accuracy was evaluated using a calculation method generally used in the clinical laboratory field. That is, the presence or absence of BJP is used as a control, and is classified as shown in Table 1 by the immunofixation method as a reference method and the method of the present invention as an evaluation target.
[Table 1]

Based on this category, sensitivity, specificity, expected positive value, and accuracy were defined as follows.
Sensitivity: Calculated by the formula a / (a + b) × 100, and means the proportion of samples showing a positive test result among BJP positive samples.
Specificity: Calculated by the formula d / (c + d) × 100, and means the proportion of samples showing a negative test result among BJP negative samples.
Positive predictive rate: means the ratio of samples actually accompanied by BJP among the samples calculated by the formula a / (a + c) × 100 and showing the test result of BJP positive.
Accuracy: Calculated by the formula (a + d) / (a + b + c + d) × 100, and means the proportion of samples that are correctly diagnosed as positive or negative for BJP, among the tested samples.
(5) Statistical analysis A statistical analysis software package (StatFlex) was used for the operation characteristic of the examinee (ROC).
(6) Results From the immunoassay measurement results, when the κ / λ ratio of 0.1 or 5.5 was used as the cutoff, as shown in Table 2, 56 of the 287 samples were present. Among them, there were 18 cases corresponding to a κ / λ ratio of 5.5 or more, of which 16 cases were BJPκ positive, and 1 case out of 2 cases other than BJP could not be analyzed by the immunofixation method. On the other hand, there were a total of 38 cases with a κ / λ ratio of 0.1 or less, of which 29 were BJPλ positive. Of the remaining 9 cases, 5 cases were intact immunoglobulin M protein positive, 3 cases were BJP negative, and 1 case was a sample that could not be analyzed by immunofixation. As described above, when the evaluation was performed based on the κ / λ ratio of 0.1 or 5.5 and the two samples that could not be analyzed by the immunofixation method were excluded, the positive prediction rate of 83.3% (54 cases) 45 cases) can be determined as BJP positive.
[Table 2]

Next, Table 3 shows the results of immunoassay, electrophoresis and immunofixation. As is clear from Table 3, BJP was identified in 58 cases by immunofixation method among 287 clinical urine specimens, and intact immunoglobulin M protein was detected in 14 cases. In addition, by electrophoresis of urine protein, M band was observed in 50 out of 58 BJP positive cases.
[Table 3]

Further, as shown in FIG. 2, the median value of the κ / λ ratio of the BJP positive specimen was 55.5 for the BJPκ type and 0.0012 for the BJPλ type. On the other hand, it was 0.63 in the BJP negative specimen.
Based on these data, the cutoff value is set to 1.67 (for BJPκ) and 0.2 (for BJPλ) by ROC analysis, and the electrophoresis and κ / λ ratio are combined by the algorithm shown in FIG. We tried to analyze urine samples. The results are shown in Table 4.
As is clear from Table 4, 53 cases were judged to be case A out of 62 cases where M band was detected by electrophoresis of urine protein, and 46 specimens (86.8%) were BJP positive. .
Of the 225 cases in which no M band was detected, no abnormality in the κ / λ ratio was found in 183 cases. Of these, 98.9% (181/183 cases) were BJP-negative.
In addition, 50 of the 287 cases showed either M-band or abnormal κ / λ ratios. Of these, 10 were BJP positive.
Thus, according to the method of the present invention using the cutoff value as described above, the sensitivity of BJP was 89.7% (52 out of 58 samples), the specificity was 81.6% (187 out of 229), and the accuracy was It became clear that the test could be performed at 84.3% (242 out of 287 cases), and it became possible to easily extract a specimen having a high possibility of BJP.
[Table 4]

According to the method of the present invention, it is possible to provide a simple and reliable BJP assay method for the first time by judging by a certain processing procedure (algorithm) by combining immunoassay alone or immunoassay and electrophoresis. It becomes.
In the method of the present invention, the BJP subclass can be identified at the same time by measuring the content of urinary free immunoglobulin κ chain and urinary free immunoglobulin λ chain and determining the ratio of the contents. Thus, it is possible to reduce the number of inspections of the licensed fixation method, which is a confirmation inspection, to about 1/6.
In addition, this application is based on Japanese Patent Application No. 2003-388617 for which it applied in Japan, The content is altogether included by this specification.

Claims (13)

A method of assaying Bence Jones protein (BJP) in a urine sample by performing the following steps using electrophoresis and immunoassay together.
(1) Step 1
Confirming the presence of M protein in the urine sample by electrophoresis (2) Step 2
Step (3) Step 3 of measuring the content of free immunoglobulin κ chain and free immunoglobulin λ chain in the urine sample by immunoassay and determining the ratio (κ / λ ratio)
The step of judging BJP positive when the M protein is confirmed and the κ / λ ratio is 0.1 ± 0.05 or less or 5.5 ± 2.75 or more The method according to claim 1, wherein the method is performed in the order of step 1, step 2, and step 3. The method according to claim 1, wherein the method is performed in the order of step 2, step 1, and step 3. A method of assaying Bence Jones protein (BJP) in a urine sample by performing the following steps using electrophoresis and immunoassay together.
(1) Step 1
Confirming the presence of M protein in the urine sample by electrophoresis (2) Step 2
Step (3) Step 3 of measuring the content of free immunoglobulin κ chain and free immunoglobulin λ chain in the urine sample by immunoassay and determining the ratio (κ / λ ratio)
A step of judging BJP positive when the M protein is confirmed and the κ / λ ratio is 0.2 ± 0.1 or less or 1.67 ± 0.84 or more A method of assaying Bence Jones protein (BJP) in a urine sample by performing the following steps using electrophoresis and immunoassay together.
(1) Step 1
Confirming the presence of M protein in the urine sample by electrophoresis (2) Step 2
Step (3) Step 3 of measuring the content of free immunoglobulin κ chain and free immunoglobulin λ chain in the urine sample by immunoassay and determining the ratio (κ / λ ratio)
A step of judging that there is a suspicion of BJP positive when M protein is confirmed and the κ / λ ratio is more than 0.2 ± 0.1 and less than 1.67 ± 0.84 A method of assaying Bence Jones protein (BJP) in a urine sample by performing the following steps using electrophoresis and immunoassay together.
(1) Step 1
Confirming the presence of M protein in the urine sample by electrophoresis (2) Step 2
Step (3) Step 3 of measuring the content of free immunoglobulin κ chain and free immunoglobulin λ chain in the urine sample by immunoassay and determining the ratio (κ / λ ratio)
The step of judging that there is a suspicion of BJP positive when the M protein is not confirmed and the κ / λ ratio is 0.2 ± 0.1 or less or 1.67 ± 0.84 or more A method of assaying Bence Jones protein (BJP) in a urine sample by performing the following steps using electrophoresis and immunoassay together.
(1) Step 1
Confirming the presence of M protein in the urine sample by electrophoresis (2) Step 2
Step (3) Step 3 of measuring the content of free immunoglobulin κ chain and free immunoglobulin λ chain in the urine sample by immunoassay and determining the ratio (κ / λ ratio)
The step of judging BJP positive when the M protein is not confirmed and the κ / λ ratio is more than 0.2 ± 0.1 and less than 1.67 ± 0.84 The method as described in any one of Claims 4-7 performed in order of step 1, step 2, and step 3. The method as described in any one of Claims 4-7 performed in order of step 2, step 1, and step 3. A method of assaying Bence Jones protein (BJP) in a urine sample by performing the following steps using electrophoresis and immunoassay together.
(1) Step 1
Confirming the presence of M protein in the urine sample by electrophoresis (2) Step 2
Step (3) Step 3 of measuring the content of free immunoglobulin κ chain and free immunoglobulin λ chain in the urine sample by immunoassay and determining the ratio (κ / λ ratio)
Based on the presence or absence of M protein and the κ / λ ratio, in case A, it is determined that BJP is positive, in cases B and C, it is determined that there is suspicion of BJP, and the presence or absence of BJP is determined by immunofixation. Further confirmation, in the case of Case D, a step of judging BJP negative (Case A)
M protein is confirmed, and the κ / λ ratio is 0.2 ± 0.1 or less or 1.67 ± 0.84 or more.
(Case B)
M protein is identified and the κ / λ ratio is greater than 0.2 ± 0.1 and less than 1.67 ± 0.84.
(Case C)
M protein is not confirmed, and κ / λ ratio is 0.2 ± 0.1 or less or 1.67 ± 0.84 or more.
(Case D)
M protein is not confirmed, and the κ / λ ratio is more than 0.2 ± 0.1 and less than 1.67 ± 0.84. The method according to claim 10, wherein the method is performed in the order of step 1, step 2, and step 3. The method according to claim 10, wherein the method is performed in the order of step 2, step 1, and step 3. The content of free immunoglobulin κ chain and free immunoglobulin λ chain in a urine sample is measured by immunoassay, and the ratio (κ / λ ratio) is determined. The κ / λ ratio is 0.1 ± 0.05 or less. Alternatively, a Bence Jones protein (BJP) test method for determining BJP positive when 5.5 ± 2.75 or more.

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