WO2018051687A1 - Antibody test reagent - Google Patents

Abstract

The present invention makes it possible to build a highly sensitive and highly specific assay system, by incorporating a polyvinyl alcohol (PVA) in an amount equal to 6.67×10^-6%(w/v) to 6.25×10^-3%(w/v), inclusive, into a specimen-diluting liquid for diluting a specimen. Thus, accurate and reproducible detection is possible even when the antibody titer of an antibody is extremely low.

Description

Reagent for antibody test

The present invention relates to an immunoassay reagent. In particular, the present invention relates to a specimen diluent for anti-peptide antibody testing capable of measuring a low concentration detection target with high accuracy and reproducibility.

Currently, the main treatment methods for cancer are surgery, radiation therapy, and chemotherapy, and these are selected. Furthermore, in recent years, antibody drugs targeting receptor molecules expressed on the surface of cancer cells have been developed and are actively used in clinical practice. However, there are cases where these treatment methods are not effective, and there are cases where the progression of cancer cannot be stopped although it is effective in the early stages of treatment. Research on immunotherapy has been conducted as a treatment method with a new mechanism of action. Has been done.

Immunotherapy is a treatment expected as the “fourth treatment” after surgery, radiation therapy, and chemotherapy. Immunotherapy is a method in which the patient's own immune mechanism is activated to perform treatment, such as vaccine therapy, immune cell therapy, and cytokine therapy. However, each immunotherapy has advantages and disadvantages, and none of them are widely used.

Among the above immunotherapy, vaccine therapy has few side effects. In addition, the mechanism of action is significantly different from that of main therapies such as radiation therapy and chemotherapy. Therefore, it is a therapeutic method that has been attracting attention because even a patient who does not succeed in these therapeutic methods may have a therapeutic effect.

Cancer vaccines are broadly divided into those intended for cancer prevention and those intended for treatment. There is a preventive vaccine for cervical cancer to prevent cancer. As a vaccine for preventing cervical cancer, a vaccine developed by Merck and GSK has already been approved. Vaccines that prevent cervical cancer prevent infections of human papillomavirus type 16 and 18 that cause cervical cancer, do not act on cancer itself, stop its progression, and treat it Absent.

On the other hand, there is a vaccine therapy in which cancer progression is stopped by inoculating a patient with a so-called “cancer antigen” expressed in cancer cells. Antigens expressed by cancer cells are presented on antigen-presenting cells by human leukocyte antigen (HLA) molecules, and cytotoxic T lymphocytes (CTLs) that recognize them damage cancer cells. There is a mechanism that prevents the progression of cancer. A cancer peptide vaccine is a method of activating CTLs in the immune system by administering a peptide that is a cancer antigen, killing cancer cells with CTLs, and trying to treat them.

Development of cancer peptide vaccines at home and abroad only administered one peptide specified for each cancer type. However, patients with advanced cancer have reduced immunity, and it takes a considerable amount of time to induce activated CTLs. In some cases, the patient dies while the immune system is activated. I couldn't. In fact, peptide vaccines derived from MAGE-A3 and EGFRvIII, which are cancer vaccines currently undergoing clinical trials, administer peptide antigens derived from antigens expressed in cancer cells of patients, and have therapeutic effects Is what you expect. However, since protein expression in cancer cells in each patient varies from person to person (Non-Patent Document 1), depending on the patient, the administered antigen may not be expressed in cancer cells, and sufficient effects are obtained. There are cases where it cannot be expected. In fact, there is a report that the success rate of such immunotherapy is not sufficient (Non-patent Document 2).

The group of Ito et al. Of Kurume University, which has been conducting clinical research on peptide vaccine therapy since the early 1990s, is a heterogeneous group of cancer cells in each patient, and the antigens that cause an immune response to cancer differ for each patient Focusing on this, research and development of a so-called “tailor-made cancer peptide vaccine” in which a plurality of optimal peptides capable of obtaining an immune response was administered to each patient was promoted. As a result, an excellent clinical effect that has not been recognized in the past was reported (Non-patent Document 3).

Since tailor-made cancer peptide vaccines have different HLA class I types for each patient, they confirm the immunity against many tumor antigen peptides and select and administer the optimal peptide recognized by each patient. is there.

Explain the outline of treatment. First, blood is collected from a cancer patient and the HLA type and anti-peptide antibody titer are measured. The top four peptides that are compatible with the HLA type and have the highest antibody titer are selected and administered. Further, the antibody titer of the patient against the regularly administered peptide is examined to confirm the degree of immunostimulation, and the peptide to be administered is reselected. (Non-patent document 4, Patent documents 1 to 3).

From the results of clinical studies, there are different patterns of peptides that can induce an immune response depending on the individual patient even in the same HLA type, that is, there are unique patterns of peptides that function as vaccines for each patient. It has become clear that there are individual differences as to whether it works effectively. Therefore, a step of diagnosing and selecting a peptide capable of inducing an immune response from a plurality of cancer peptides capable of inducing a specific immune response for each patient is very important. By performing a tailor-made treatment for selecting an optimal peptide, the efficiency of inducing a specific immune response for each patient is increased, and the possibility of obtaining a clinical effect is increased. At present, clinical research is further advanced using 31 kinds of peptides.

The inventors are exploring a method for performing a more effective treatment in applying to the clinic in response to the research results of the Kurume University group. That is, in order to select a peptide set effective for a patient, an immunoassay with a small non-specific reaction and a high sensitivity is being intensively studied.

The anti-peptide antibody titer present in the blood collected in order to select peptides at the stage of examination before vaccine therapy is performed on patients is very low for all peptides. However, peptides that can be expected to induce an immune response that can function as a vaccine have a slightly higher antibody titer than the background even before vaccine therapy. For effective vaccine therapy, it is important to assess antibody titers prior to vaccine administration and select peptides that can induce a specific immune response in the patient. Therefore, antibody values slightly higher than the background must be compared with each other and evaluated. Furthermore, the peptide to be administered in the next course is again measured for anti-peptide antibody titer, and the effect of the administered peptide is examined and reviewed. As a result, a peptide having a high possibility of obtaining a clinical effect is selected again. Therefore, in order to select an optimal peptide, it is necessary to obtain not only high sensitivity and specificity but also a highly reproducible measurement result after a certain period of time.

Generally, when measuring antibody titer by immunoassay, it is often evaluated how much the antibody titer against a specific antigen has increased by comparing before and after immunization. Generally, when an immunogen is inoculated, the antibody titer rises at least 10 times or more. However, here, as described above, antibody values with a low background level must be compared with each other and evaluated. Therefore, compared to ordinary immunoassay, both sensitivity and specificity are higher. Desired. In addition, since it is necessary to continuously observe the patient after vaccine administration, high reproducibility is also required.

The present inventors have conducted intensive studies and found that the composition of the specimen diluent among various reagents is important in terms of sensitivity and specificity. In particular, the present inventors have found that the type and concentration of the water-soluble polymer strongly influences the sensitivity and specificity among the compositions of the sample diluent, and the present invention has been completed.

Various studies have been made on the sample diluent used for immunoassay. Conventionally, water-soluble polymers contained in an immunoassay system include polybilyl alcohol (hereinafter sometimes referred to as PVA), polyvinylpyrrolidone (hereinafter sometimes referred to as PVP), polyethylene glycol, and the like. Have been used.

Rodda et al. Show that 0.5% (w / v) PVA as a blocking agent in immunoassay remarkably suppresses non-specific binding as compared to a commonly used blocking agent ( Non-patent document 5). In addition, Studentsov et al. Report that the sensitivity and specificity of ELISA increase when PVA-50 (PVA with a molecular weight of 50,000) and PVP-360 (PVP with a molecular weight of 360,000) are added. In particular, it has been reported that adding non-specific binding by adding PVA-50 to the blocking solution. Further, it is described that PVA-50 has the maximum blocking effect when used at 0.5%, and that PVP-360 has an optimum concentration of 0.8% (Non-patent Document 6).

The group of the present inventors is measuring the antibody titer of patients before vaccine administration by multiplex flow cytometry assay. Multiplex flow cytometry assay not only can measure with high sensitivity, but also can measure antibody titers against multiple peptides simultaneously in a single well, enabling measurement with a small amount of patient specimens and the time required for testing. Can also be shortened.

Also in a system for measuring by a multiplex flow cytometry assay, addition of PVA and PVP has been studied and disclosed (Non-patent Documents 7 and 8). Non-Patent Document 7 describes that non-specific binding is increased by binding an antibody contained in human plasma directly to beads. Furthermore, it is described that PVA, PVP and Super ChemiBlock (registered trademark) are effective for suppressing non-specific binding. According to the method disclosed in Non-Patent Document 7, after diluting plasma with a sample diluent containing 0.5% PVA and 0.8% PVP, the diluted sample is preincubated for use. ing. As a result of examining the effects of PVA and PVP, it is described that the effects of PVA and PVP are additive, and both may be used as PVX by mixing them. Non-Patent Document 8 describes that non-specific binding is suppressed by adding 0.5% PVA and 0.8% PVP to the specimen diluent. In specimens not exposed to antigen, it has been shown that non-specific binding is significantly suppressed when 0.5% PVA and 0.8% PVP are added to the specimen diluent.

As described above, the effects of suppressing non-specific binding by adding PVA and PVP to the sample diluent in the case of ELISA, multiplex flow cytometry assay, and any immunoassay have been shown so far. Yes. Moreover, the concentration was 0.5% for PVA and 0.8% for PVP, or a combination of both as PVX, that is, 1.3%.

The present inventors have also studied the composition of the specimen diluent, but when PVA and PVP are used at the concentrations disclosed in the prior art, good results are obtained with proteins, but when peptides are used. Was unable to obtain satisfactory sensitivity and inhibition of non-specific binding. As described above, in the case of tailor-made vaccine therapy, it is necessary to measure a very low antibody titer against a peptide presented on antigen-presenting cells in a vaccine-naïve state. Therefore, not only high sensitivity is required as compared with the prior art, but measurement is repeated throughout the vaccination period, so it is necessary to be able to measure with good reproducibility. Therefore, it is considered that there is a demand for higher sensitivity and suppression of nonspecific binding than the conditions disclosed in the prior art.

As a result of intensive studies, the present inventors have found that the concentration of PVA, which is a water-soluble polymer, among compounds added as a blocking agent is very important for reducing nonspecific binding and increasing sensitivity. I found out. Also, the concentration was much lower than that disclosed in the prior art.

International Publication No. 2005/041982 International Publication No. 2005/123122 International Publication No. 03/025569

An object of the present invention is to detect a sample having a very low antibody titer with high accuracy and with high reproducibility. In particular, it is an object to provide a specimen diluent used in a test for selecting a peptide that can be efficiently presented on antigen-presenting cells in a vaccine-naïve state prior to administration of a peptide vaccine, and an immunoassay method And

The present invention is a specimen diluent shown below and an immunoassay method using the specimen diluent.
(1) A sample in an immunoassay characterized by containing polyvinyl alcohol (PVA) in a range of 6.67 × 10 ⁻⁶ % (w / v) to 6.25 × 10 ⁻³ % (w / v). Sample diluent used for dilution.
(2) The specimen diluent according to (1), which contains 1.67 × 10 ⁻⁵ % (w / v) or more and 6.25 × 10 ⁻³ % (w / v) or less of PVA.
(3) The specimen diluent according to (1) or (2), which is used for measurement of an anti-peptide antibody.
(4) The specimen diluent according to any one of (1) to (3), wherein the sample is plasma, serum, or blood.
(5) An immunoassay characterized by diluting a sample with a specimen diluent containing 6.67 × 10 ⁻⁶ % (w / v) or more and 6.25 × 10 ⁻³ % (w / v) or less of PVA Law.
(6) The specimen diluent contains 1.67 × 10 ⁻⁵ % (w / v) or more and 6.25 × 10 ⁻³ % (w / v) or less of PVA ( 5) The immunoassay method described.
(7) An immunoassay method, wherein the immunoassay method according to (5) or (6) is a multiplex flow cytometry assay using flow cytometry.
(8) The immunoassay method according to any one of (5) to (7), which measures an anti-peptide antibody.
(9) The immunoassay method according to any one of (5) to (8), wherein the sample is plasma, serum, or blood.

Even a sample with a low antibody titer can be measured with high sensitivity and specificity with good reproducibility.

The figure which shows the outline | summary of the measuring method of an antibody titer. Examination of PVA concentration for non-specific binding using peptide non-solid phase beads. Examination of PVA concentration for non-specific binding using peptide solid phase beads. Examination of PVA concentration related to sensitivity using various peptide beads and specimens. Examination of PVA concentration related to sensitivity and specificity. The figure which shows the examination result of the dilution rate of a sample.

In addition to PVA, a compound that is generally added as a blocking agent that suppresses nonspecific binding can be added to the specimen diluent of the present invention. In that case, it may be added at a concentration usually added in an immunoassay such as multiplex flow cytometry assay or ELISA. Examples of blocking agents that can be added include water-soluble polymers such as PVP and polyethylene glycol, polypeptides such as skim milk, bovine serum albumin (BSA), ovalbumin, and gelatin, and surfactants such as Tween 20 and Triton X-100. It is possible to add. Moreover, you may add Block Ace (trademark), ChemiBlock (trademark), etc. which are commercially available blocking agents.

Furthermore, it is preferable to use a buffer solution in order to maintain the pH and ionic environment appropriately. As the buffer to be used, TBS, PBS or the like usually used in immunoassays can be used. In order to improve the storage stability of the solution, about 0.05% (w / v) sodium azide may be added.

In the tailor-made vaccine treatment, plasma and serum samples are used as samples for measuring antibody titers. In this treatment method, it is necessary to measure the antibody titer against the peptide before and during treatment. When storing a sample, it may be stored frozen. Further, the present invention can be applied to general immunoassays. In this case, it is needless to say that any sample may be used in addition to the blood-derived sample.

The present invention was developed as a specimen diluent used in a test prior to inoculation with a peptide vaccine, but it is sensitive and can suppress nonspecific binding. Therefore, the present invention is not limited to this, and it can be used in any immunoassay method such as antibody titer test after peptide vaccination and normal test.

In the present invention, the antibody titer is measured by a multiplex flow cytometry assay using flow cytometry, but any immunoassay may be used as long as the antibody titer can be measured. Such immunoassays include flow cytometry, ELISA, RIA and the like.

In the present invention, a PVA having a molecular weight of 30,000 to 70,000 is used, but a PVA having a lower molecular weight can also be used. Moreover, when adding PVP, PVP360 (having a molecular weight of 360,000) is used, but a low molecular weight having a molecular weight of about 40,000 can be used. Regardless of the molecular weight, high sensitivity and specific binding can be obtained by using equivalent concentrations.

[Method for measuring antibody titer]
In the present invention, it is necessary to measure antibody titers against a large number of peptides, and thus antibody titers are measured by a multiplex flow cytometry assay based on the flow cytometry method. First, the measurement method will be outlined (FIG. 1). Here, the Luminex (registered trademark) system, which is a fluorescence flow cytometry system, is used, but analysis may be performed using any method.

First, different types of peptides are immobilized on a plurality of identifiable beads. By detecting a combination of two kinds of fluorescent dyes colored on the beads, the beads can be identified, and the peptide immobilized on the surface can be identified. A sample obtained from a patient is diluted with the peptide-immobilized beads and reacted with the sample diluent of the present invention (FIG. 1, incubation). If the patient specimen contains an antibody that recognizes the peptide, it specifically binds to the peptide. Usually, as a diluted sample, patient plasma is used after being diluted 100-fold with a sample diluent, but can be measured by appropriately adjusting according to MFI (Media Fluorescence Intensity, median fluorescence intensity).

Next, the secondary antibody is bound to the peptide recognition antibody contained in the patient sample (FIG. 1, secondary antibody, labeled incubation). Specifically, an anti-human IgG antibody is used as a secondary antibody and is bound to a peptide recognition antibody derived from a patient. Here, the secondary antibody is biotinylated, and detection is performed by binding fluorescently labeled streptavidin. Peptides bound to the carrier can be identified from the fluorescence derived from the beads, and the reactivity of the patient specimen to each peptide can be analyzed from the fluorescence labeled with streptavidin.

Here, the secondary antibody uses biotin as a label and is detected by a biotin-streptavidin system, but may be detected using any assay system used in immunoassays. For example, a secondary antibody that is directly fluorescently labeled may be used, or an antibody that recognizes the secondary antibody may be fluorescently labeled. Any fluorescent label may be used as long as it is different from the detection wavelength of the solid phase as a carrier.

Although not shown in FIG. 1, after each step, such as incubation of the diluted specimen and the solid-phase support, washing is performed with a washing solution to remove unbound specimen and the like. As the washing solution, those commonly used in immunoassays can be used. For example, a buffer solution containing a salt close to physiological salt concentration such as PBS or TBS containing 0.01 to 0.1% (w / v) of a surfactant such as Tween 20 or Triton X-100. Can be preferably used.

[Examination of PVA and PVP concentrations]
In the process of incubating the diluted specimen and the peptide-immobilized carrier, the present inventors have found that the non-specific binding of the specimen to the solid phase increases the background during measurement and decreases the sensitivity. Revealed. Therefore, the composition of the specimen diluent was examined. The specimen dilution solution contained a water-soluble polymer, a surfactant, a blocking agent containing a protein, and a buffer as a composition. As a result of investigation, non-specific binding and sensitivity did not change greatly depending on the type and concentration of the surfactant and the type of blocking agent and buffer. However, it was revealed that the concentration of PVA, which is a water-soluble polymer, affects nonspecific binding and sensitivity, and the concentration of PVP affects the enhancement of reactivity. In particular, it was revealed that the PVA concentration is very important in measuring antibody titer at a low concentration. Hereinafter, it demonstrates in detail, showing a result.

Conventionally, as described above, the concentration of PVA as a water-soluble polymer is 0.5% (w / v), the concentration of PVP is 0.8% (w / v), or the concentration of PVX is 1.3% (w / v). It has been reported that non-specific binding is suppressed and sensitivity is enhanced by performing an immunoassay (Non-Patent Documents 6 to 8). In the inventors' examination, the measurement of the antibody against the protein showed the usefulness at this PVA concentration. Therefore, 2.0% (w / v) Block Ace (manufactured by DS Pharma Biomedical), 0.05% (w / v) Tween20 (manufactured by MP Biomedicals), 0.5% (w / v) PVA, Analysis of a patient sample before immunization was performed using a sample diluent containing 0.8% PVP (w / v), but the sensitivity was low and the measured value was not stable. Therefore, the types and concentrations of the surfactant, blocking agent, and water-soluble polymer included in the composition of the sample diluent were examined. As a result, it became clear that the water-soluble polymer had the most influence on the sensitivity and specificity of the measurement system. Therefore, various compositions of the water-soluble polymer in the sample diluent were examined. Unless otherwise specified, the following PVA and PVP concentrations are shown in% (w / v).

It was found that PVP has the effect of increasing the reactivity as described in Non-Patent Document 6. Although the results are not shown here, PVP was added to PBS, 0.05% Tween 20, or 2.0% Block Ace, 0.05% Tween 20 at concentrations of 0, 0.008, 0.08, and 0.8%. When multiplex flow cytometry assay was performed, the reaction was enhanced in a concentration-dependent manner in both systems. On the other hand, since an increase in non-specific reaction is also observed, when adding PVP, it is preferable to add it at a concentration lower than 0.8%. In addition, PVP may not be added as long as the sample can obtain sufficient reactivity and does not need to enhance the reaction.

It became clear that the influence of PVA is greater than that of PVP on the sensitivity and nonspecific binding of the measurement system. Therefore, the optimal condition for the concentration of the water-soluble polymer was determined using a sample dilution solution obtained by sequentially diluting the concentration of PVA contained in the sample dilution solution.

First, the concentration of PVA that suppresses non-specific reaction was examined using beads (non-solid phase beads, Ctl (control) beads in FIG. 1) in which peptides used as controls were not immobilized. From 0.5% PVA, a specimen dilution solution was prepared by sequentially diluting PVA. Commercially available human purified IgG (MP Biomedicals) and healthy human plasma (sample A, sample B) were diluted 100-fold with sample dilutions that differed only in PVA concentration. The beads were incubated only with commercially available human purified IgG, healthy human serum, and specimen diluent (no specimen), reacted with the secondary antibody and label, and fluorescence measurement was performed.

As shown in FIG. 2, although the amount of the antibody that nonspecifically binds to the non-solid phase beads varies depending on the specimen, the nonspecific binding rapidly increases at the boundary of the PVA concentration of 6.67 × 10 ⁻⁶ %. It became clear. Also, for example, there is a specimen that causes nonspecific binding from a concentration of 1.67 × 10 ⁻⁵ % depending on the specimen, such as specimen A. Therefore, in order to avoid non-specific binding to the beads, the PVA concentration is preferably 6.67 × 10 ⁻⁶ % or more, more preferably 1.67 × 10 ⁻⁵ % or more. It was judged.

Next, the PVA concentration at which non-specific binding occurs was examined using beads on which peptides were immobilized. In the same manner as described above, human purified IgG was diluted with specimen diluents having different PVA concentrations, and measurement was performed. The results in FIG. 3 are the results of analysis using peptide-11 (AYDFLYNYL; SEQ ID NO: 1), but the results were similar when measured with other peptides.

As shown in FIG. 3, nonspecific binding increased rapidly at PVA concentrations below 6.67 × 10 ⁻⁶ %. Therefore, it was concluded that the PVA concentration should be used at a concentration of 6.67 × 10 ⁻⁶ % or higher. The concentration of 6.67 × 10 ⁻⁶ % PVA is 1 / 100,000 or less, much lower than the concentration of 0.5% PVA that has been recommended in the prior art. It is.

Next, a region with a high PVA concentration was examined using beads on which peptides were immobilized and human plasma specimens. When using peptide-immobilized beads, the reactivity may decrease when the concentration of PVA is high, although the degree varies depending on the specimen. Peptide-10 (DYSARWNEI; SEQ ID NO: 2) immobilized beads and healthy human plasma (sample B), peptide-1 (DYVREHKDNI; SEQ ID NO: 3) immobilized beads and human purified IgG, peptide-9 (RYLTQETNKV; SEQ ID NO: 4) The analysis result of a solid-phased bead and a healthy subject's plasma (specimen A) is shown (FIG. 4).

The concentration of PVA in which a decrease in reactivity was observed was different depending on the combination of the specimen and the beads used. For example, in the combination of Peptide-10 immobilized beads and Specimen B (top of FIG. 4), although the reactivity with the Specimen was decreased in the region of PVA concentration higher than 3.13 × 10 ⁻² % On the other hand, the combination of peptide-1 immobilized beads and human purified IgG (center of FIG. 4) is 2.50 × 10 ⁻² %, and in the case of peptide-9 immobilized beads and specimen A (bottom of FIG. 4). A decrease in reactivity was observed when diluted with a sample diluent containing PVA at a concentration higher than 6.25 × 10 ⁻³ %. Moreover, although the PVA concentration at which the decrease in reactivity occurs depends on the combination of the specimen and the bead, in any case, at a high concentration of PVA of 0.13% or more, errors and variations for each measurement are large and reproducibility is poor.

Although the reactivity differs depending on the patient specimen and peptide, the above results revealed that PVA added to the specimen diluent has various problems in terms of sensitivity in a high concentration region. That is, if the concentration of PVA is higher than 0.13%, it is not preferable because the measurement error increases. Further, depending on the combination of the specimen and the bead, there was a case where the sensitivity was lowered at a concentration higher than 6.25 × 10 ⁻³ %. Accordingly, the PVA concentration is preferably 0.13% or less, and more preferably 6.25 × 10 ⁻³ % or less.

Table 1 summarizes the results of specificity and sensitivity when using specimen dilutions containing various PVA concentrations. In the table, ○ indicates good, × indicates inappropriate, Δ indicates slightly inappropriate, and NT indicates that no inspection was performed. Instability indicates that the measured values varied and the reproducibility was low. From the viewpoint of specificity, the concentration of PVA contained in the specimen diluent is preferably 0.5% or less, and preferably 6.67 × 10 ⁻⁶ % or more. Further, the concentration is more preferably 1.67 × 10 ⁻⁵ % or more. From the viewpoint of sensitivity, a PVA concentration of 0.13% or less is preferable, and 6.25 × 10 ⁻³ or less is more preferable.

Next, the effect of PVA on specificity as well as sensitivity was analyzed. Using the serum before peptide vaccination, a competitive inhibition test was performed, and the specificity and sensitivity according to the PVA concentration were analyzed (FIG. 5).

Using two different specimens (C) and (D) before vaccination using peptide-9 immobilized beads, with no competitive inhibition (-) and with competitive inhibition by peptide-9 (+) Analysis was performed by fluorescence intensity. Experimental conditions without competitive inhibition indicate sensitivity and experimental conditions with competitive inhibition indicate specificity. In addition, since the fluorescence intensity shows tens of thousands of FI after peptide vaccination, both samples are measured and judged at very low values. Further, when FI is smaller than 5, it is judged as negative, and 5 or more samples are judged as positive.

First, when the sensitivity was examined, as shown in FIG. 5, an increase in sensitivity was observed when the PVA concentration was lower than the PVA concentration of 0.5%. In particular, in the sample shown in specimen D (bottom of FIG. 5), FI was below the detection limit at 0.5% PVA, but the fluorescence intensity was 6FI at 6.25 × 10 ⁻³ and 3.33 × 10 ⁻⁵ %, respectively. 10FI was shown and it could be judged as positive.

When the specificity is examined, the inhibition rate (%) at 0.5, 6.25 × 10 ⁻³ , 3.33 × 10 ⁻⁵ % PVA concentration is shown in the sample shown in specimen C (upper part of FIG. 5). 70/78/95% and N / A (uncalculated), 100/80% in the sample shown in specimen D (lower part of FIG. 5), respectively. It is clear that the inhibition rate (%) at 6.25 × 10 ⁻³ , 3.33 × 10 ⁻⁵ % PVA concentration is 80% or more in all samples, and the specificity is sufficiently high. The results of the competitive inhibition test using peptides also revealed that favorable results were obtained in both specificity and sensitivity when the concentration of PVA was 6.25 × 10 ⁻³ % or less.

From the above examination results, the preferred PVA concentration from both the specificity and sensitivity is 6.67 × 10 ⁻⁶ % or more and 0.13% or less, and further considering the reproducibility, 0.13 In% PVA, since the measured value variation was poor and reproducibility was poor, the concentration is preferably 6.25 × 10 ⁻² % or less. Further, a concentration of 1.67 × 10 ⁻⁵ % or more is more preferable because the background does not increase and good results can be obtained in both specificity and sensitivity regardless of the combination of peptide and specimen. In addition, if the concentration is 6.25 × 10 ⁻³ % or less, there is no decrease in sensitivity or instability for each measurement, and good results in both specificity and sensitivity can be obtained regardless of the combination of peptide and specimen. More preferred.

In addition to PVA alone, PVP alone and PVX (combination of PVA and PVP) were similarly examined for nonspecific binding and sensitivity. No results are shown here, but unlike the results reported so far, PVP enhances reactivity but has little effect on inhibiting non-specific binding, PVA lowers background and enhances reactivity It became clear that there was an effect of making it. Therefore, it is more preferable to add PVP as appropriate when enhancement of sensitivity is required after adding PVA within the above concentration range.

Next, the specimen dilution rate was examined (FIG. 6). In the same manner as described above, the sample was reacted with the peptide-immobilized beads, and the fluorescence intensity was measured. Peptide-3 (DYLRSVLEDF; SEQ ID NO: 5) solid-phase beads and specimen B which is healthy human plasma, or peptide-5 (NYSVRYRPGL; SEQ ID NO: 6) solid-phase beads and specimen E which is normal human plasma Study was carried out. The specimen dilution solution used contains 3.33 × 10 ⁻⁵ % of PVA. Each sample was diluted 25, 50, 100, and 200 times with the sample diluent, and the fluorescence intensity was measured.

In any combination, linearity was observed at a specimen dilution rate of 50 to 200 times. In addition, the fluorescence intensity tends to decrease at a dilution factor of 25, and the reactivity is considered to be reduced. Therefore, the specimen is preferably diluted in a range where linearity is recognized, that is, 50 to 200 times, and more preferably diluted to 100 to 150 times near the center where linearity is recognized. Since the fluorescence intensity as a measurement value differs depending on the sample, the sample may be appropriately diluted with the sample diluent so that the measurement error is small within the above range.

In addition, since the method of the present invention uses PVA or PVP at a lower concentration than conventional ones, many samples can be stably measured even when measured by a multiplex flow cytometry assay. Since PVA and PVP are high molecular weight polymers, they give viscosity to the solution at high concentrations. Therefore, when PVA or PVP is used at a conventional concentration, the multiplex flow cytometry assay that measures the fluorescence intensity on the solid phase beads through a fine tube causes clogging and a decrease in flow rate when measuring multiple samples. Occurred and affected the measurement results. By using the sample diluent adjusted in the concentration range of the present invention, the measurement could be performed stably without causing a decrease in flow rate.

By using the sample diluent of the present invention, even a sample having a low antibody titer can be measured with high specificity, high sensitivity, and good reproducibility.

Claims (9)

1. To dilute a sample in an immunoassay characterized by containing polyvinyl alcohol (PVA) in a range of 6.67 × 10 ⁻⁶ % (w / v) to 6.25 × 10 ⁻³ % (w / v) Sample diluent used for
2. 2. The specimen diluent according to claim 1, wherein PVA is contained in an amount of 1.67 × 10 ⁻⁵ % (w / v) to 6.25 × 10 ⁻³ % (w / v).
3. 3. The specimen diluent according to claim 1 or 2, which is used for measurement of an anti-peptide antibody.
4. The specimen diluent according to any one of claims 1 to 3, wherein the sample is plasma, serum, or blood.
5. An immunoassay method comprising diluting a sample with a specimen diluent containing 6.67 × 10 ⁻⁶ % (w / v) or more and 6.25 × 10 ⁻³ % (w / v) or less of PVA.
6. The specimen diluent is
   6. The immunoassay method according to claim 5, wherein the immunoassay contains 1.67 × 10 ⁻⁵ % (w / v) or more and 6.25 × 10 ⁻³ % (w / v) or less of PVA.
7. The immunoassay method according to claim 5 or 6,
   An immunoassay characterized by being a multiplex flow cytometry assay using flow cytometry.
8. The immunoassay method according to any one of claims 5 to 7, which measures an anti-peptide antibody.
9. The immunoassay method according to any one of claims 5 to 8, wherein the sample is plasma, serum, or blood.
   
   
   

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