JP6604081B2 - Method for measuring pancreatic ribonuclease 1 to which an N-type sugar chain is added - Google Patents

Description

  The present invention relates to a technique in the measurement field. More specifically, the present invention relates to a method for measuring pancreatic ribonuclease 1 to which an N-type sugar chain is added in a sample.

  As a method for detecting pancreatic cancer, it is preferable to use a non-invasive sample derived from a living body, for example, a body fluid such as blood or urine that is relatively easy to collect. Serum markers used in current diagnosis of pancreatic cancer include CA19-9 and DUPAN-2. However, these markers have the disadvantage that they are not definitive definitive diagnoses because they have low organ specificity and do not respond to the markers for genetic reasons.

  Pancreatic ribonuclease 1 (hereinafter referred to as “RNase 1”), which is one of the ribonuclease family, is a glycoprotein that is specifically expressed in the pancreas and is secreted into extracellular body fluids. This protein is translated as a peptide consisting of 156 amino acids, and after secretory signal removal and sugar chain modification, it is secreted extracellularly as a mature glycoprotein having a 128 amino acid peptide sequence (SEQ ID NO: 1). The N-type sugar chain-modifiable sites, that is, amino acid residues that may be subjected to N-type sugar chain modification are the 34th, 76th, and 88th asparagine residues in SEQ ID NO: 1.

  According to Patent Document 1, in RNase 1 in the serum of healthy individuals, the ratio of sugar chains added to the 88th asparagine residue (hereinafter referred to as “Asn88”) is very low. In contrast, RNase 1 in serum obtained from pancreatic cancer patients has been reported to increase sugar chain addition in Asn88. Furthermore, it is described that a pancreatic cancer patient is detected by determining the ratio of RNase 1 in serum to the total RNase 1 added to Asn88 with an N-type sugar chain added.

International Publication No. 2013/187371 Pamphlet

  An object of the present invention is to provide a method for measuring RNase 1 in which an N-type sugar chain is added to Asn88 in a sample.

As a result of intensive studies to solve the above problems, the present inventors have completed the present invention. That is, the present invention is as follows.
(1) A treatment is performed so that RNase 1 in which no N-type sugar chain is added to Asn88 in the sample is not involved in the reaction with an antibody that binds to RNase 1 regardless of the presence or absence of the N-type sugar chain,
RNase 1 in which an N-type sugar chain is added to Asn88 in the sample is measured using an antibody that binds to RNase 1 regardless of the presence or absence of the N-type sugar chain. A method for measuring RNase 1 to which a sugar chain is added.
(2) The treatment is to remove RNase 1 with no N-type sugar chain added to Asn88 by reacting with an antibody that specifically binds to RNase 1 with no N-type sugar chain added to Asn88. The method according to (1).
(3) The method according to (2), wherein the antibody that specifically binds to RNase 1 in which an N-type sugar chain is not added to Asn88 is the RrhRN0723 antibody or the RrhRN117 antibody.
(4) The treatment coexists an antibody that specifically binds to RNase 1 in which no N-type sugar chain is added to Asn88 in the sample, and the N-type sugar chain is added to the antibody and Asn88. The method according to (1), wherein an antigen-antibody complex formed with no RNase 1 cannot bind to an antibody that binds to RNase 1 regardless of the presence or absence of an N-type sugar chain.
(5) The method according to (4), wherein the antibody that binds to RNase 1 regardless of the presence or absence of an N-type sugar chain is an RrhRN203 antibody.
(6) RNase 1 in which an N-type sugar chain is added to Asn88 in a sample is measured by the method according to any one of (1) to (5), and is compared with a measured value of a healthy person A method for detecting cancer.
(7) A method for detecting cancer, characterized in that a ratio of values obtained by the following A and B is obtained and compared with a ratio of values of healthy persons.
A: RNase 1 in which an N-type sugar chain is added to Asn88 in a sample is measured by the method according to any one of (1) to (5).
B: Total RNase 1 in the sample is measured using an antibody that binds to RNase 1 regardless of the presence or absence of N-type sugar chains.

  The present invention is described in further detail below. In the present invention, RNase 1 in which an N-type sugar chain is added to Asn88 is hereinafter referred to as “Asn88N-type sugar chain-added RNase 1”. Further, RNase 1 in which an N-type sugar chain is not added to Asn88 is hereinafter referred to as “Asn88N-type sugar chain non-added RNase 1”.

  In the present invention, the sample is not particularly limited, and examples thereof include body fluids obtained from humans, particularly serum, plasma, saliva, urine and the like. Moreover, as a sample, one or both of Asn88N type sugar chain addition RNase 1 and Asn88N type sugar chain non-addition RNase 1 may be included.

  In the present invention, the treatment is performed so that the Asn88N-type non-glycan added RNase 1 in the sample is not involved in the reaction with the antibody that binds to RNase 1 regardless of the presence or absence of the N-type sugar chain. This treatment is not particularly limited. For example, Asn88N-type non-glycan-added RNase 1 is removed by reacting with an antibody that specifically binds to Asn88N-type non-glycan-added RNase 1, that is, removed from the sample. There are methods. At this time, an antibody that specifically binds to Asn88N-type non-glycosylated RNase 1 is immobilized on an appropriate carrier and brought into contact with the sample to specifically remove Asn88N-type non-glycosylated RNase 1. it can.

  The antibody that specifically binds to Asn88N-type non-glycosylated RNase 1 is not particularly limited, but the RrhRN0723 antibody or RrhRN117 antibody described in Patent Document 1 is preferable. RrhRN117 antibody can be produced in the same manner as RrhRN0723 antibody by the method described in Example 1 of Patent Document 1.

  In the present invention, Asn88N-type glycosylated RNase 1 present in a sample is measured using an antibody that binds to RNase 1 regardless of the presence or absence of an N-type sugar chain. This measurement may be performed after the above-described processing or may be performed simultaneously with the processing. The antibody used at this time is not particularly limited, but RrhRN1111 antibody and MrhRN0614 antibody described in Patent Document 1 are preferable.

  The measurement method is not particularly limited, and examples thereof include a sandwich method, a sandwich ELISA method, a competitive method, a method using an automated immunoassay device using them, a Western blot method, etc., preferably a sandwich ELISA method. is there.

  In the present invention, as the above-described treatment, an antibody that specifically binds to RNase 1 in which no N-type sugar chain is added to Asn88 coexists in the sample. Examples of the antigen-antibody complex formed with RNase 1 to which RNase 1 is not added include those that cannot bind to an antibody that binds to RNase 1 regardless of the presence or absence of an N-type sugar chain. At this time, the antibody that cannot bind to the antigen-antibody complex formed by the Asn88N-type non-glycan added RNase 1 and the antibody that specifically binds to the Asn88N-type non-glycan added RNase 1 is not particularly limited. The RrhRN1111 antibody described in Patent Document 1 and the RrhRN203 antibody described in this specification are preferable.

The present invention also relates to a method for detecting cancer by determining a ratio of values obtained from the following A and B and comparing the ratio with the value of a healthy person.
A: Asn88N-type glycosylated RNase 1 in the sample is measured by the above-described method (1) or (2).
B: Total RNase 1 in the sample is measured using an antibody that binds to RNase 1 regardless of the presence or absence of N-type sugar chains.

  In this method, the sample is measured by the method A. The sample is measured by the method B. Then, the ratio of these values is obtained. This method is shown in FIGS. The sample at this time is exemplified as described above. The same antibodies as those described above are also exemplified.

  Cancer can be detected by comparing the ratio of the values obtained by Asn88N-type glycosylated RNase 1 and methods A and B measured by the present invention with those of healthy individuals. Specifically, the measured value of Asn88N-type glycosylated RNase 1 is higher in cancer patients than in healthy individuals, and the ratio A / B of values obtained by methods A and B is large (even if B / A is small). I can say). As cancer, for example, pancreatic cancer, gastric cancer, bile duct cancer and the like can be detected, and in particular, pancreatic cancer can be detected.

  According to the present invention, Asn88N-type glycosylated RNase 1 in a sample can be measured using an antibody that binds to RNase 1 regardless of the presence or absence of an N-type sugar chain, and the total RNase 1 in a sample can be measured. You can also Since these can be measured using the same measurement reagent, it is possible to minimize the performance difference / variation caused by the measurement reagent, compared to the case where measurement is performed using separate measurement reagents for each.

It is the figure which showed the method in the case of removing Asn88N type sugar chain non-addition RNase 1 from a sample among the methods of this invention. It is the figure which made Table 1 of Example 1 into a graph. In Table 2 of Example 2, total amount of RNase 1 (untreated—using R1111 measuring reagent), Asn88N type sugar chain added RNase 1 amount (using RrhRN0723 fixed magnetic fine particle treatment—using R1111 measuring reagent), Asn88N type sugar chain not added It is the figure which made the amount of RNase 1 (Untreated-R0723 measurement reagent use) graphed. FIG. 3 is a graph showing the ratio of Asn88N-type glycosylated RNase 1 shown in Table 2 of Example 2. FIG. 4 is a graph showing the reactivity of anti-RNase 1 antibodies RrhRN117 and RrhRN203 with respect to Asn88N-type non-glycosylated RNase 1 and Asn88N-type glycosylated RNase 1; It is the figure which each measured the measured value when it measured by adding RrhRN0723 or RrhRN117 to a sample as an inhibitory antibody using the measurement reagent using the alkaline phosphatase-labeled RrhRN1111 antibody or RrhRN203 antibody as a detection antibody. It is the figure which made the relative value of the measurement result in the measurement reagent which changed the density | concentration of the RrhRN117 antibody added to the sample, and used the measurement reagent using the alkaline phosphatase-labeled RrhRN203 as a detection antibody. It is the figure which showed the method in the case of inhibiting Asn88N type sugar chain non-addition RNase 1 among the methods of this invention. G3 / t ratio, total RNase 1 amount (inhibitory antibody non-addition measurement), Asn88N-type glycosylated RNase 1 amount (inhibitory antibody addition measurement) when human serum was measured by the method described in Example 4 in a graph FIG. Among the measurement results using human serum described in Example 4, for the cancer specimen, the G3 / t ratio obtained by the method described in the present invention and the G3 / t determined by conversion according to the method described in Patent Document 1 were used. It is the figure which compared ratio.

Example 1 Measurement of Asn88N-type glycosylated RNase 1 amount using RNase 1 standard product In a phosphate buffer containing 150 mM sodium chloride, magnetic fine particles (MX200 / Carboxyl, manufactured by JSR) having a dry weight of 100 μg were used. The suspension was suspended at a concentration of 10%, and 50 μg of RrhRN0723 antibody was added to 1 ml of the resulting magnetic fine particle suspension, and mixed for 16 hours to adsorb and immobilize the antibody on the magnetic fine particles. After washing with PBS, the magnetic microparticles to which the antibody has been immobilized are further suspended in a Tris buffer containing 3% bovine serum albumin and 150 mM sodium chloride, and kept at 37 ° C. for 3 hours to suspend the RrHRN0723 fixed magnetic microparticles. Liquid. Using a solution containing about 50 ng of recombinant RNase 1 expressed in CHO-K1 cells as a sample and using 10, 20, 40, and 80 μl of RrHRN0723-fixed magnetic microparticle suspension, respectively, King Fisher Flex (Thermo Fisher Scientific) In the sample, Asn88N type sugar chain non-added RNase 1 was selectively adsorbed on the magnetic fine particles and removed from the sample. The sample from which the magnetic fine particles were removed was measured for RNase 1 in the sample in the same manner as in Example 5 [0057] of Patent Document 1. A reagent for measuring Asn88N-type non-glycosylated RNase 1 amount using antibody RrhRN0723 labeled with alkaline phosphatase is hereinafter referred to as “R0723 measuring reagent”, and N-type sugar using antibody RrhRN1111 labeled with alkaline phosphatase. A reagent capable of measuring the amount of RNase 1 regardless of the presence or absence of a chain is hereinafter referred to as “R1111 measuring reagent”. In both measurement reagents, MrhRN0614 antibody is used as the solid phase side antibody. As a control experiment, the same study was performed using magnetic fine particles to which no antibody was immobilized. The measurement results are shown in Table 1 and FIG.

表１から、当初の（磁性微粒子による処理前の）試料中のＲＮａｓｅ １量は、磁性微粒子を添加しない試料（０μｌ添加）の結果より、Ａｓｎ８８Ｎ型糖鎖未付加ＲＮａｓｅ １が４４．６５μｌ／ｍｌ、総ＲＮａｓｅ １が５０．８９ｎｇ／ｍｌであり、これらの値から換算するとＡｓｎ８８Ｎ型糖鎖付加ＲＮａｓｅ １の比率は、［（５０．８９−４４．６５）／５０．８９］×１００＝１２％であることがわかる。

From Table 1, the amount of RNase 1 in the initial sample (before treatment with magnetic fine particles) is 44.65 μl / ml of Asn88N-type non-glycosylated RNase 1 from the result of the sample without addition of magnetic fine particles (0 μl addition). The total RNase 1 is 50.89 ng / ml, and when converted from these values, the ratio of Asn88N-type glycosylated RNase 1 is [(50.89-44.65) /50.89] × 100 = 12% It can be seen that it is.

  It can also be seen that the measured value decreases depending on the amount of RrHRN0723 antibody-immobilized magnetic fine particles added during the treatment. On the other hand, when the treatment is performed using magnetic fine particles to which the antibody is not immobilized, the measured value does not fluctuate greatly even if the amount of magnetic fine particles added is increased. From this, the decrease in the measured value depending on the amount of RrHRN0723 antibody-immobilized magnetic microparticles is not due to non-specific binding to the magnetic microparticles, but the decrease dependent on the amount of RrHRN0723 antibody-immobilized magnetic microparticles, ie, the amount of RrhRN0723 antibody It was confirmed that it was dependent. When 80 μl of RrhRN0723 antibody-immobilized magnetic fine particle suspension is used, the amount of RNase 1 detected by the R0723 measurement reagent is not more than the detection limit of measurement (2 to 100 ng / ml), and the Asn88N type sugar chain is not detected from the sample. It was confirmed that the added RNase 1 was sufficiently removed.

  “Asn88N-type non-glycosylated RNase 1” in the sample was removed by the RrhRN0723 antibody, and then RNase 1 (6.27 ng / ml) measured using the R1111 measuring reagent is Asn88N-type glycosylated RNase 1 . Further, from the result of the sample to which no magnetic fine particles were added (0 μl addition), the total RNase 1 was 50.89 ng / ml, so the Asn88N-type glycosylated RNase 1 was (6.27 / 50.89) × 100. It can be seen that = 12%. This ratio agrees with the value obtained by conversion from the aforementioned Asn88N-type non-glycan added RNase 1 and total RNase 1. As described above, it was shown that the amount of Asn88N-type glycosylated RNase 1 can be measured.

Example 2 Measurement of Asn88N-type glycosylated RNase 1 in serum obtained from healthy subjects and pancreatic cancer patients Serum obtained from healthy subjects and pancreatic cancer patients using the method described in Example 1 (each 10 samples) Asn88N type glycosylated RNase 1 amount was measured. As the pancreatic cancer specimen, a specimen with diagnosis obtained from Bio Theme was used. Serum was diluted 11-fold with Tris buffer containing 1% bovine serum Albun, 150 mM sodium chloride and 0.05% Tween-20, and one of the two samples was treated with RrhRN0723-fixed magnetic microparticles. One was an untreated sample. For each sample, the amount of RNase 1 was measured using R0723 measurement reagent or R1111 measurement reagent. The measured value is the amount of RNase 1 contained in the sample diluted 11 times. Table 2 shows the ratio between the respective measured values and the Asn88N-type glycosylated RNase 1 determined from the measured values and those determined by the method of the present invention.

また、表２の値をプロットしたグラフを図３と図４に示す。図３には、健常者と膵臓癌患者血清の希釈試料中の、総ＲＮａｓｅ １量、Ａｓｎ８８Ｎ型糖鎖付加ＲＮａｓｅ １量、Ａｓｎ８８Ｎ型糖鎖未付加ＲＮａｓｅ １量をそれぞれグラフとして示した。

Moreover, the graph which plotted the value of Table 2 is shown in FIG. 3 and FIG. FIG. 3 is a graph showing the total amount of RNase 1, the amount of Asn88N-type glycosylated RNase 1, and the amount of Asn88N-type non-glycosylated RNase 1 in diluted samples of serum from healthy subjects and pancreatic cancer patients.

  4 shows the relationship between the ratio (1-[(A) / (B)]) obtained by conversion and the ratio ((D / (B)) according to the method of the present invention. Although both values were obtained, it was possible to clearly distinguish the group of healthy subjects (× in the figure) from the group of patients with pancreatic cancer (black circles in the figure) by either method. It is judged that the difference is caused by the material used for the measurement, in particular, a calibration curve is created for each of the two measurement reagents (R0723 measurement reagent and R1111 measurement reagent) in the ratio obtained by conversion, Since the measurement value is obtained, an error due to the quality of the RNase 1 serving as a standard used for each calibration curve is likely to occur, whereas in the method of the present invention, the total RNase 1 is obtained with one measurement reagent (R1111 measurement reagent). And Asn88N-type glycosylated RNas Because you are measuring the 1, it is considered to be less affected by the quality of RNase 1 as a standard.

  From the above results, it was shown that Asn88N-type glycosylated RNase 1 can be measured by the method of the present invention. Furthermore, it was shown that healthy individuals and pancreatic cancer patients can be distinguished.

Example 3 Measurement of Asn88N-type glycosylated RNase 1 amount by adding an inhibitory antibody to a measurement sample Purification of “Asn88N-type glycosylated RNase 1” and “Asn88N-type glycosylated RNase 1” were expressed in CHO cells. The recombinant RNase 1 has little N-type glycosylation in Asn88, and about 10% of the recombinant RNase 1 secreted from cells has N-type glycosylation. In order to obtain purified “Asn88N-type glycosylated RNase 1” and “Asn88N-type non-glycosylated RNase 1”, HiRrap is used as an antibody that specifically binds to Asn88N-type non-glycosylated RNase 1. An affinity purification column bound to a column (GE) was used. Affinity purification was carried out according to a conventional method to obtain Asn88N-type non-glycosylated RNase 1 free from contamination with Asn88N-type glycosylated RNase 1. Further, the fraction that did not bind to the affinity purification column was repeatedly applied to the same affinity purification column to obtain “Asn88N-type glycosylated RNase 1” from which Asn88N-type non-glycosylated RNase 1 was removed. The purity of each sample was confirmed by measuring the amount of “Asn88N-type non-glycan added RNase 1” and the total amount of RNase 1 in the same manner as in Example 5 [0057] of Patent Document 1.

Examination of antigen binding specificity of RrhRN117 antibody and RrhRN203 antibody Next, using the purified “Asn88N-type glycosylated RNase 1” and “Asn88N-type non-glycosylated RNase 1”, the RrhRN117 antibody was transformed into “N-type sugar It was confirmed that the antibody specifically binds to pancreatic ribonuclease 1 having the 88th asparagine residue to which no chain is added.

  RrhRN117 antibody and RrhRN203 antibody were prepared in the same manner as RrhRN0723 antibody by the method described in Example 1 of Patent Document 1.

  Below, the preparation method and evaluation method of the measurement reagent of the immunoassay system constructed in order to evaluate said antibody are shown. MrhRN0614 was physically adsorbed to 90 ng / carrier on a water-insoluble carrier (manufactured by EVA having a particle diameter of about 1.5 mm kneaded with ferrite inside), and after the adsorption, blocking treatment was performed using BSA. As for the water-insoluble carrier, about 100 ng of protein can be physically adsorbed per one. After 12 carriers are placed in a magnetically permeable container (capacity: 1.2 ml), antibody RrHRN117 labeled with 0.50 μg / mL alkaline phosphatase or antibody RrhRN203 labeled with 0.25 μg / mL alkaline phosphatase (1% BSA, 2.5% dextran, 150 mM NaCl, 0.05% Tween-20, 20 mM Tris buffer, pH 7.4) was added and lyophilized. Using this commercially available fully automated immunoassay apparatus (trade name AIA-600II, manufactured by Tosoh Corporation), the purified “Asn88N-type glycosylated RNase 1” and “Asn88N-type glycosylated Measurement of “addition RNase 1” was performed. The measurement principle is as follows. That is, 100 μL of a measurement sample was added, the water-insoluble carrier was moved using a magnet at 37 ° C. for 10 minutes, and an immunoreaction was performed with the mixture stirred. After the reaction, B / F separation operation is performed to separate and remove the free labeled antibody, and 4-methylumbelliferyl phosphate which is a substrate of alkaline phosphatase is added, and the enzyme reaction from 20 seconds to 295 seconds after the addition of the substrate The production rate (nM / sec) per unit time of the degradation product (4-methylumbelliferone) was measured.

  As shown in FIG. 5, the RrhRN117 antibody and the RrhRN203 antibody showed an increase in measured value depending on the amount of antigen relative to the Asn88N-type non-glycosylated RNase 1, and both antibodies showed an Asn88N-type non-glycosylated RNase. It was confirmed that the antibody was bound to 1. On the other hand, with respect to Asn88N-type glycosylated RNase 1, it was confirmed that the RrhRN203 antibody increased in the measured value depending on the antigen amount, and was confirmed to be an antibody that binds to Asn88N-type glycosylated RNase 1. The RrhRN117 antibody was confirmed to be an antibody that does not bind to Asn88N-type glycosylated RNase 1 at all. That is, the RrhRN203 antibody is “an antibody that binds to pancreatic ribonuclease 1 regardless of the N-glycan addition state to the 88th asparagine residue”, whereas the RrhRN117 antibody is “Asn88N-glycan unadded RNase”. It was revealed that the antibody specifically binds to “1”.

Examination of the inhibitory activity of the RrhRN0723 antibody and the RrhRN117 antibody on the binding ability of the RrhRN203 antibody and the RrhRN1111 antibody In order to examine the inhibitory ability of the RrhRN0723 antibody and the RrhRN117 antibody against the binding ability of the RrhRN203 antibody, the RrHRN203 antibody labeled with the alkaline phosphatase was detected. An immunoassay reagent was prepared. A dilution series of “Asn88N-type glycosylated RNase 1” and “Asn88N-type glycosylated RNase 1” was prepared, and an antibody-free diluent (1% bovine serum albumin, 150 mM sodium chloride, and 0.05 Tris buffer containing% Tween-20), diluent containing 25 μg / ml RrhRN0723 antibody, or diluent containing 25 μg / ml RrhRN117 antibody. The diluted antigen was measured with the aforementioned immunoassay reagent. Moreover, it measured similarly using the RrhRN1111 antibody labeled with alkaline phosphatase.

  As shown in FIG. 6, the addition of the RrhRN0723 antibody or the RrhRN117 antibody to the Asn88N-type non-glycan-added RNase 1 inhibits the measurement of the Asn88N-type non-glycan-added RNase 1 by the reagent using the RrhRN203 antibody. Became clear. On the other hand, it has been clarified that the addition of the RrhRN0723 antibody or the RrhRN117 antibody to the Asn88N-type glycosylated RNase 1 does not significantly affect the measurement of the Asn88N-type glycosylated RNase 1 by the reagent using the RrhRN203 antibody. It was. That is, considering that the RrhRN0723 antibody and the RrhRN117 antibody are antibodies that specifically bind to Asn88N-type non-glycosylated RNase 1, these antibodies formed an antigen-antibody complex with Asn88N-type non-glycosylated RNase 1. In some cases, the antibody was confirmed to be an antibody that inhibits the binding of the RrhRN203 antibody to RNase 1.

  FIG. 6 reveals that when the antibody concentration is the same, the RrhRN117 antibody inhibits the binding of the RrhRN203 antibody and the RrhRN1111 antibody to Asn88N-type non-glycosylated RNase 1 slightly stronger than the RrhRN0723 antibody. It was revealed that the inhibition efficiency was highest when the RrhRN117 antibody was used and the RrhRN203 antibody was used as the detection antibody.

  When the inhibitory ability of the RrhRN203 antibody to inhibit Asn88N-type non-glycosylated RNase 1 is inhibited by changing the concentration of the inhibitory antibody, it inhibits depending on the concentration of the RrhRN117 antibody in the reaction solution. This was confirmed (FIG. 7). In the measurement using 0.25 μg / ml RrhRN203 antibody as the detection antibody, 98.2% of “Asn88N type non-glycan added RNase 1” was inhibited when the RrhRN117 antibody concentration was 50 μg / ml. That is, in the RNase 1 measurement to which a sufficient amount of the RrhRN117 antibody concentration was added, it was revealed that the amount of “Asn88N type non-glycan added RNase 1” detected can be approximately zero.

  From the above results, the RrhRN117 antibody was added to a sample in which “Asn88N-type glycosylated RNase 1” and “Asn88N-type glycosylated RNase 1” were mixed, and the RrhRN203 antibody was used as the detection antibody, and the amount of RNase 1 When “Asn88N type non-glycan added RNase 1” and RrhRN117 antibody form an antigen-antibody complex, which does not bind to the RrhRN203 antibody, the amount of “Asn88N type non-glycan added RNase 1” It is possible to say that the amount of RNase 1 measured is “Asn88N-type glycosylated RNase 1” without being reflected.

Example 4 Measurement of Asn88N-type glycosylated RNase 1 amount in human serum The method described in Example 3 except that 0.25 μg / mL alkaline phosphatase-labeled antibody RrhRN203 was used as an antibody for detection. An RNase 1 measurement reagent that can be measured with a measuring device (trade name AIA-600II, manufactured by Tosoh Corporation) was prepared. Using this reagent, the total amount of RNase 1 and the amount of Asn88N-type glycosylated RNase 1 in human serum were measured as follows. When measuring the total amount of RNase, the sample is diluted 10 times with a diluent containing no inhibitory antibody (Tris buffer containing 1% bovine serum albumin, 150 mM sodium chloride, and 0.05% Tween-20). Dilute and measure. When the amount of Asn88N-type glycosylated RNase 1 was measured, a diluent (1% bovine serum albumin, 150 mM sodium chloride, and 0.05% Tween-20) containing RrRNRN117 antibody at a concentration of 30 μg / ml as an inhibitory antibody was measured. The sample was diluted 10 times with a Tris buffer solution) and measured. From the measured values obtained by the respective measurement methods, the total amount of RNase 1 contained in each sample and the amount of Asn88N-type glycosylated RNase 1 were converted using a calibration curve determined using an RNase 1 standard. Furthermore, the ratio (G3 / t ratio) of Asn88N-type glycosylated RNase 1 to the total RNase 1 in the sample is obtained from the converted Asn88N-type glycosylated RNase 1 amount (D) and total RNase 1 amount (E). It was.

  In addition, Asn88N type sugar chain non-added RNase 1 amount (A) and total RNase 1 amount (B) were measured by the method described in Patent Document 1 as a control experiment, and Asn88N type sugar chain addition to the total RNase 1 in the sample was performed. The ratio of RNase 1 (G3 / t ratio) was determined.

  FIG. 9 shows the results of measuring 39 healthy human subjects, 34 pancreatic cancer patients, 10 gastric cancer patients, and 10 bile duct cancer samples by the above method. The G3 / t ratio of serum samples obtained from pancreatic cancer, gastric cancer, and bile duct cancer was clearly higher than that of healthy subjects.

  FIG. 10 shows the G3 / t ratio (1-[(A) / (B)]) obtained by conversion according to the method described in Patent Document 1 and the method of the present invention for the above-mentioned 54 cancer patient samples. The relationship of G3 / t ratio ((D) / (E)) was also shown. Although different numerical values were obtained by the two methods, the results showed a strong correlation. The G3 / t ratio obtained by the method of the present invention was the same as that obtained by the method described in Patent Document 1. It shows that they are essentially equivalent. It is judged that the numerical difference between the two methods is due to the material used for the measurement. In particular, in the ratio obtained by conversion, a calibration curve is created for each of the two measurement reagents (R0723 measurement reagent and R1111 measurement reagent), and a measurement value is obtained. Error due to quality is likely to occur. In contrast, in the method of the present invention, the total RNase 1 and Asn88N-type glycosylated RNase 1 are measured with one measurement reagent (measurement reagent using RrhRN203 antibody as a labeled antibody), so the quality of the standard RNase 1 is measured. It is thought that there is little influence caused by.

Claims (5)

Treatment in which pancreatic ribonuclease 1 in which an N-type sugar chain is not added to the 88th asparagine residue in a sample is not involved in the reaction with an antibody that binds to pancreatic ribonuclease 1 regardless of the presence or absence of the N-type sugar chain And
Pancreatic ribonuclease 1 in which an N-type sugar chain is added to the 88th asparagine residue in the sample is measured using an antibody that binds to pancreatic ribonuclease 1 regardless of the presence or absence of the N-type sugar chain. The method for measuring pancreatic ribonuclease 1 in which an N-type sugar chain is added to the 88th asparagine residue.   An antibody that specifically binds pancreatic ribonuclease 1 with no N-type sugar chain added to the 88th asparagine residue and pancreatic ribonuclease 1 with no N-type sugar chain added to the 88th asparagine residue The method of Claim 1 which is made to react and remove.   The treatment coexists an antibody that specifically binds to pancreatic ribonuclease 1 in which an N-type sugar chain is not added to the 88th asparagine residue in the sample, and the antibody and the 88th asparagine residue 2. The antigen-antibody complex formed with pancreatic ribonuclease 1 to which no N-type sugar chain is added is one in which an antibody that binds to pancreatic ribonuclease 1 cannot bind regardless of the presence or absence of an N-type sugar chain. the method of. The pancreatic ribonuclease 1 in which an N-type sugar chain is added to the 88th asparagine residue in the sample is measured by the method according to any one of claims 1 to 3 , and is compared with the measured value of a healthy person A method for detecting cancer, which is characterized. A method for detecting cancer, characterized in that a ratio of values obtained by the following A and B is obtained and compared with a ratio of values of healthy persons.
A: Pancreatic ribonuclease 1 in which an N-type sugar chain is added to the 88th asparagine residue in the sample is measured by the method according to any one of claims 1 to 3 .
B: Total pancreatic ribonuclease 1 in the sample is measured using an antibody that binds to pancreatic ribonuclease 1 regardless of the presence or absence of N-type sugar chains.

Images