A method for detecting abnormal results in immunoassays caused by insufficient delivery of polyhapten reagents.

Abstract

To provide a method for solving problems related to the delivery of R2 polyhapten reagents, such as short delivery times or bubble collapse. [Solution] A novel and improved method for separating absorbance caused solely by the addition of assay reagents, and a novel and improved method for detecting and flagging problems, particularly those caused by the delivery of R2 polyhapten reagents, as well as apparatus and compositions for use herein, are provided.

Description

Technical Field

[0001] Cross - reference to related applications / Incorporation by reference statement Not applicable

[0002] Statement regarding federally - sponsored research or development Not applicable

Background Art

[0003] By accurately controlling blood glucose, many of the morbidity and mortality rates associated with type 2 diabetes can be improved. Therefore, many different assays for hemoglobin have been developed based on the physical and chemical properties of hemoglobin or on its specific antibody - recognition epitopes. In clinical studies, HbA1c results have been shown to improve decision - making, patient compliance, and outcomes (Non - Patent Document 1); and Non - Patent Document 2).

[0004] [[ID=2 (24)]]Immunoassays are the most common type of hemoglobin assay currently used in clinical laboratory settings. These immunoassay methods utilize antibodies that recognize epitopes of hemoglobin and, in certain cases, epitopes of glycated hemoglobin (HbA1c), such as (but not limited to) at least a portion of its N - terminal glycated amino acids. For example, the turbidimetric inhibition immunoassay (TINIA) for the analyte HbA1c utilizes an R1 reagent (i.e., an anti - HbA1c antibody) and an R2 polyhapten reagent (i.e., a synthetic molecule containing multiple HbA1c epitopes that cause aggregation with the free antibody). When no HbA1c analyte is present, the polyhapten reacts with the free anti - HbA1c antibody to form an insoluble antibody - polyhapten complex, which results in turbidity and light scattering when the sample is illuminated with a light source. When the target analyte, HbA1c, is present in a biological sample (e.g., but not limited to, a whole - blood sample), the HbA1c analyte reacts with the anti - HbA1c antibody to form a soluble analyte - antibody complex, which results in a decrease in the amount of light scattering observed. The reaction rate can be measured by turbidimetry and is inversely proportional to the amount of HbA1c analyte present in the biological sample.

[0005] The main problems with this immunoassay include (but are not limited to) delivery issues of the R2 polyhapten reagent, such as short delivery times or bubble collapse, which lead to abnormal results that should be reported. R2 is a polypeptide solution with absorption at a wavelength of 293 nm, available on several clinical chemistry analyzers, such as (but not limited to) DIMENSION VISTA® Systems (Siemens Healthcare Diagnostics, Tarrytown, NY), but there is no read time available immediately after the addition of the polyhapten reagent on these types of systems. Furthermore, even when a read time is available, using absorbance measured at the time of delivery does not accurately measure the delivery of the R2 polyhapten reagent because it contains interference signals due to agglutination reactions caused by the presence of samples and antibodies already present in the mixing and reaction mixture.

[0006] Furthermore, there are currently no solutions available to detect problems with reagent delivery that also initiate assay signal generation. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Thaler et al. (Diabetes Care (1999) 22:1415~1421) [Non-Patent Document 2] Miller et al. (Diabetes Care (2003) 26:1158~1163) [Overview of the project] [Means for solving the problem]

[0008] Therefore, there is a need in the art for novel and improved methods for separating absorbances solely due to the addition of assay reagents, and in particular (but not limited to) novel and improved methods for detecting and flagging problems caused by the delivery of R2 polyhapten reagents. This disclosure covers such novel and improved methods, as well as apparatus and compositions used herein.

[0009] This patent or application file contains at least one drawing made in color. A copy of this patent or patent application publication containing the color drawing will be provided by the authorities upon request and payment of the required fees. [Brief explanation of the drawing]

[0010] [Figure 1] This graph shows the dynamic data of A1C assays using quality control (QC) and medical decision pool (MDP) samples. [Figure 2] This graph shows the dynamic data of a delivery check assay for R2 polyhapten constructed in accordance with this disclosure. [Figure 3] This graph shows R2 polyhapten delivery check data from assay parameters designed to simulate problems related to the delivery of polyhapten reagents. [Figure 4] This graph shows the delivery check data for the R2 polyhapten sample compared to the result monitoring limit. [Modes for carrying out the invention]

[0011] Before describing in detail at least one embodiment of the concept of the present invention by illustrative language and results, it should be understood that the concept of the present invention is not limited in its application to the configuration details and component arrangements described below. The concept of the present invention can be implemented or performed in other embodiments or in various ways. Therefore, the language used herein is intended to give the broadest possible scope and meaning, and the embodiments are illustrative and not exclusive. It should also be understood that the expressions and terminology used herein are for illustrative purposes only and should not be considered limiting.

[0012] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural terms and plural terms shall include singular terms. The techniques and procedures described herein are generally well known in the art and are performed in accordance with conventional methods as described in the various general and more specific references cited and discussed throughout this specification. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, as well as their experimental procedures and techniques, are well known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0013] All patents, published patent applications, and non-patent literature referenced herein represent the level of expertise of those skilled in the art to which this disclosure belongs. All patents, published patent applications, and non-patent literature referenced in any part of this application are expressly incorporated herein by reference in whole to the same extent as if each individual patent or document were specifically and individually indicated to be incorporated by reference.

[0014] All articles, compositions, kits, and / or methods disclosed herein can be manufactured and performed without excessive experimentation in relation to the present disclosure. While articles, compositions, kits, and / or methods are described in relation to specific embodiments, it will be apparent to those skilled in the art that modifications can be made to the articles, compositions, kits, and / or methods, as well as to the steps or sets of steps of the methods described herein, without departing from the concept, spirit, and scope of the present disclosure. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

[0015] When used in accordance with this disclosure, the following terms should be understood to have the following meanings unless otherwise indicated.

[0016] When used in conjunction with the term "comprising" in the claims and / or specification, the use of the terms "a and an" may mean "one," but also coincides with the meanings of "one or more," "at least one," and "one or more than one." Therefore, the terms "a and an" and "the" include multiple referents unless otherwise clearly indicated by the context. Thus, for example, a reference to "compounds" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or more than one compound. The term "plural" refers to "two or more."

[0017] The use of the term "at least one" is understood to include one and any quantity more than one including, but not limited to, two, three, four, five, ten, fifteen, twenty, thirty, forty, fifty, one hundred, etc. The term "at least one" can extend up to 100 or more than 1000 depending on the term to which it is attached, and further, the 100 / 1000 quantity should not be considered limiting as higher limit values may also result in satisfactory outcomes. Further, the use of the term "at least one of X, Y and Z" is understood to include X alone, Y alone and Z alone, as well as any combination of X, Y and Z. The use of ordinal terms (i.e., "first", "second", "third", "fourth", etc.) is for the sole purpose of differentiating between two or more items and does not mean any particular arrangement, order or importance of one item with respect to another, or any additional order.

[0018] The use of the term "or" in the claims is used to mean an inclusive "and / or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, the condition "A or B" is satisfied by any of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0019] As used herein, any reference to "one embodiment", "an embodiment", "some embodiments", "one example", "for example" or "an example" means that a particular element, feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. For example, the appearances of the phrases "in some embodiments" or "one example" in various places in this specification are not necessarily all referring to the same embodiment. Further, all references to one or more embodiments or examples should be construed as not limiting the claims.

[0020] Throughout this application, the term "about" is used to indicate that a value includes the inherent variability of errors of a composition / apparatus / device, the method used to determine the value, or the variability that exists between test subjects. For example, without limitation, when the term "about" is utilized, such variability is such that, as appropriate to perform the disclosed method and as understood by one of ordinary skill in the art, the specified value can vary plus or minus 20 percent, 15 percent, 12 percent, 11 percent, 10 percent, 9 percent, 8 percent, 7 percent, 6 percent, 5 percent, 4 percent, 3 percent, 2 percent, or 1 percent from the specified value.

[0021] As used in this specification and the claims, the terms "comprising" (and any form of "comprising" such as "comprise" and "comprises"), "having" (and any form of "having" such as "have" and "has"), "including" (and any form of "including" such as "includes" and "include"), or "containing" (and any form of "containing" such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0022] As used herein, the terms “or any combination thereof” refer to all permutations and combinations of the items listed prior to the term. For example, “A, B, C or any combination thereof” is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and, where the order is important in a particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing this example, combinations containing repetitions of one or more items or terms are explicitly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. A person skilled in the art will understand that, unless otherwise evident from the context, there is typically no limit to the number of items or terms in any combination.

[0023] As used herein, the term “substantially” means that the event or situation described below will occur entirely, or will occur to a considerable extent or degree. For example, in relation to a particular event or situation, the term “substantially” means that the event or situation described below will occur with a probability of at least 80%, at least 85%, at least 90%, or at least 95%. The term “substantially adjacent” may mean that two items are 100% adjacent to each other, or that two items are close to each other but not 100% adjacent, or that a portion of one of the two items is close to the other but not 100% adjacent to the other.

[0024] As used herein, the terms “associate with” and “bond” include both direct association / bonding of two parts with each other and indirect association / bonding of two parts with each other. Non-limiting examples of association / bonding include, for example, covalent bonding of one part to another by direct bonding or via spacer groups, noncovalent bonding of one part to another by direct or specific bond pair members bonded to the part, incorporating one part into another by, for example, dissolving one part into another, and coating one part with another.

[0025] As used herein, the term “sample” is understood to include any type of biological sample available in accordance with this disclosure. Examples of available fluid biological samples include, but are not limited to, whole blood or any part thereof (including, but not limited to, plasma or serum), whole blood cells or lysed blood cells (including, but not limited to, whole red blood cells or lysed red blood cells), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder lavage fluid, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof.

[0026] As used herein, the term “target analyte-specific binding partner” is understood to refer to any molecule that can specifically associate with a target analyte. For example, but not limited to, a binding partner may be an antibody, receptor, ligand, aptamer, molecularly imprinted polymer (i.e., inorganic matrix), a combination or derivative thereof, and any other molecule that can specifically bind to a target analyte.

[0027] The term “antibody” is used herein in a broad sense and refers to, for example, intact monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments and conjugates thereof exhibiting desired bioactivity to analytes (e.g., Fab, Fab', F(ab')2, Fv, scFv, Fd, bispecific antibodies, short-chain antibodies, and other antibody fragments and conjugates thereof that retain at least a portion of the variable region of an intact antibody), antibody substitute proteins or peptides (i.e., modified binding proteins / peptides), and combinations or derivatives thereof. Antibodies may be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0028] As used herein, the term "hapten" refers to a small proteinaceous or nonproteinaceous antigenic determinant (or "epitope") that can be recognized by a target analyte-specific binding partner, such as (but not limited to) an antibody. As used herein, the term "polyhapten" is understood to refer to a synthetic molecule containing multiple epitopes / antigenic determinants that bind to it.

[0029] The "analyte" is a polymer that can be recognized by a target analyte-specific binding partner, such as (but not limited to) an antibody. Both the analyte and the hapten contain at least one antigenic determinant or "epitope," which is a region of the antigen or hapten that binds to the target analyte-specific binding partner (i.e., the antibody). Typically, an epitope on a hapten is an entire molecule.

[0030] As used herein, the term “reaction cuvette” includes any device capable of performing at least one of the diagnostic assays described herein. While a reaction cuvette may perform a diagnostic assay manually, in most cases, it is inserted into a system that automates the performance of the diagnostic assay. In one non-limiting embodiment, a reaction cuvette includes, for example, a reaction cuvette for use in an automated diagnostic assay performed in one of the DIMENSION VISTA® Systems, commercially available from Siemens Healthcare Diagnostics (Newark, DE). However, it is understood that a reaction cuvette may be any commercially available product or cuvette described herein or otherwise intended to perform one or more diagnostic assays in accordance with this disclosure.

[0031] As used herein, the term "turbidimetric method" is understood to refer to a method for measuring the intensity loss of transmitted light due to the scattering effect of particles suspended in a solution. Light passing through a filter generates light of known wavelengths, which then passes through a cuvette containing the test solution. A photometer collects the light passing through the cuvette, and a measurement of the amount of absorbed light is obtained. Thus, the turbidimetric method is a method for determining the concentration of a substance in a solution by the degree of cloudiness or turbidity caused by the substance, or by the degree of clarification induced by the substance in a turbid solution.

[0032] Referring next to the concept of the present invention, certain non-limiting embodiments of this disclosure relate to kits, apparatus and methods for improving the performance and reliability of immunoassays in general. One embodiment relates to a kit, apparatus, and method for detecting problems with the delivery of polyhapten reagents.

[0033] As described herein, the main problems with HbA1c immunoassays include (but are not limited to) delivery problems of the R2 polyhapten reagent, such as short delivery times or bubble collapse, which lead to abnormal results that should be reported. R2 is a polypeptide solution with absorption at a wavelength of 293 nm, available on several clinical chemistry analyzers, such as (but not limited to) DIMENSION VISTA® Systems (Siemens Healthcare Diagnostics, Tarrytown, NY), but there is no read time available immediately after the addition of the polyhapten reagent on these types of systems. Furthermore, even when a read time is available, using absorbance measured at the time of delivery does not accurately measure the delivery of the R2 polyhapten reagent because it contains interference signals due to agglutination reactions caused by the presence of samples and antibodies already present in the mixing and reaction mixture.

[0034] For the reasons stated above, the method of this disclosure was developed to estimate the absorbance change due to R2 delivery (293 nm–700 nm) using the slope of a regression line with respect to time from two subsequent readings. This method can predict the mAU absorbance of R2 delivery immediately before the start of the aggregation reaction. The method works because the absorbance change with respect to time is linear with respect to the time point used, and the predicted R2 delivery mAU signal converges with respect to the total HbA1c concentration.

[0035] This technique, which detects problems in reagent delivery that also trigger the generation of assay signals, can be applied to any chemical or immunoassay. Therefore, the description of this technique in the context of polyhapten reagents in HbA1c immunoassays is for illustrative purposes only and should not be interpreted as limiting.

[0036] In the method disclosed herein, calculations are applied to assay parameters for estimating the instrument signal upon delivery of the polyhapten reagent. This method uses regression of two subsequent readings over time to predict the signal at 0 time and subtracts the signal contributions from the sample and antibody reagents in the reaction mixture to provide accurate monitoring of the signal upon addition of the polyhapten reagent to the reaction. By removing the signal contributions from the sample and antibody reagents, the instrument software can compare the signals from the delivery of the polyhapten reagent for each test and flag tests affected by reagent delivery problems. This method is effective because the change in signal over time is linear with respect to the time of use.

[0037] In general, the novelty of this method lies in using zero-order readings to separate absorbances obtained solely from reagent delivery in the presence of other reaction signals. The calculations for monitoring polyhapten reagent delivery provide a criterion for monitoring the outcomes of this reagent delivery. If significant errors occur during reagent delivery, these errors can have a clinically significant impact on patient outcomes. Adding this outcome monitoring allows instrument software to flag outcomes that may be affected by problems with polyhapten reagent delivery using the reagent 2 probe. The calculations applied to the assay parameters allow for monitoring the consistency of polyhapten reagent delivery and detecting delivery problems. Because the change in the signal over time is linear, estimation is possible. Therefore, with the method of this disclosure, users do not need to report outcomes that may be affected by problems with polyhapten delivery.

[0038] The method disclosed herein offers several advantages (referred to as “reagent delivery checks” in certain parts of this specification). If a patient’s results are affected by a problem with the delivery of a reagent (e.g., but not limited to R2 polyhapten reagents) and subsequently reported to the physician, the physician may 1) question the results, or 2) adjust the patient’s treatment based on the results. Furthermore, This results monitoring not only flags results that may have been affected by polyhapten reagent delivery problems, but also provides operators with advice for more efficient troubleshooting in resolving potential instrument issues. When this results monitoring is triggered, operators can focus on specific reagent wells in the instrument's reagent cartridge and reagent 2 system instead of performing a work procedure on all components of the instrument. This can reduce the time and cost of resolving instrument issues (e.g., the cost of replacing parts in other components of the sample and reagent servers).

[0039] Some non-limiting embodiments of this disclosure relate to methods for detecting the presence and / or concentration of a target analyte in a biological sample. In certain (but not limited to) embodiments, the method can be further defined as a method for minimizing abnormal or inadequate results in an immunoassay caused by insufficient delivery of an immunoassay reagent (e.g., a polyhapten reagent, but not limited to such reagents).

[0040] The method involves simultaneously, in whole or in part, sequentially combining (1) a biological sample suspected to contain a target analyte, (2) at least one target analyte-specific binding partner (e.g., an antibody, but not limited to this), and (3) at least one immunoassay reagent (e.g., a polyhapten reagent or other type of particle agglutination assay reagent) that can specifically bind to the target analyte-specific binding partner. The at least one target analyte-specific binding partner is then bound to the target analyte or at least one immunoassay reagent.

[0041] In certain non-limiting embodiments, the signal generated by an immunoassay reagent can be detected by a turbidimetric (i.e., agglutination) assay. These types of assays are well known in the art and therefore require no further explanation.

[0042] Any target peptide or protein analyte detectable by immunoassay can be detected by the method of this disclosure. Examples of target analytes, but not limited to, include glycated hemoglobin (HbA1c), albumin, human chorionic gonadotropin (hCG), ferritin, growth hormone, prolactin, thyroglobulin (Tg), C-reactive protein (CRP), and rheumatoid factor (RF).

[0043] Alternatively, the immunoassay may be a therapeutic drug monitoring (TDM) immunoassay that measures the serum level of a drug to ensure that its concentration is within its therapeutic range. Examples of target drug analytes detectable by TDM immunoassays include, but are not limited to, gentamicin, tobramycin, CRP, digoxin, amikacin, caffeine, carbamazepine, digitoxin, disopyramide, ethosuxamide, lidocaine, lithium methotrexate, NAPA, phenobarbital, phenytoin, primidone, procainamide, quinidine, theophylline, tobramycin, valproic acid, and vancomycin.

[0044] Any biological sample known in the art for use in the immunoassays described herein can be used in accordance with this disclosure. Examples of usable biological samples include, but are not limited to, urine, whole blood or any part thereof (including, but not limited to, plasma or serum), whole blood (i.e., substantially unlysed) cells or lysed blood cells (including, but not limited to, whole red blood cells or lysed red blood cells), saliva, sputum, cerebrospinal fluid (CSF), intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, tears, mucus, bladder lavage fluid, semen, and combinations thereof.

[0045] In a non-limiting embodiment, this disclosure relates to poly(P) used in immunoassays. The present invention relates to a method for detecting abnormal results caused by insufficient delivery of hapten reagents. The method comprises the following steps: (A) reacting a biological sample suspected to contain a target analyte with a target analyte-specific binding partner in a reaction cuvette to form a soluble analyte / specific binding partner complex; (B) adding a polyhapten reagent to the reaction cuvette, wherein the polyhapten reagent reacts with excess target analyte-specific binding partner to form an insoluble polyhapten / target analyte-specific binding partner complex; (C) irradiating the reaction cuvette with light; and (D) measuring absorbance at at least three wavelengths at multiple time points after the addition of the polyhapten reagent. A step of measuring a value, wherein a first wavelength detects an insoluble polyhapten / target analyte-specific binding partner complex by turbidimetry, a second wavelength detects a protein, and a third wavelength functions as a blank; (E) a step of estimating the absorbance value at the time of delivery of the polyhapten reagent using regression of absorbance values ​​measured at the second and third wavelengths at two time points after the addition of the polyhapten reagent; and (F) a step of flagging the concentration value of the target analyte obtained by another algorithm if the estimated absorbance value at the time of delivery of the polyhapten reagent differs from the predicted value by an established flag constant, as this is unacceptable.

[0046] Any target analytes described or otherwise intended herein can be detected by the methods described herein. In certain (but not limited to) specific embodiments of any of the above methods, the analyte is HbA1c, the antibody is an anti-HbA1c antibody, and the polyhapten comprises a plurality of HbA1c epitopes.

[0047] In a non-limiting embodiment, the present disclosure relates to a method for detecting abnormal results resulting from insufficient delivery of a polyhapten reagent used in a glycated hemoglobin (HbA1c) immunoassay. The method comprises the steps of: (A) reacting a biological sample suspected to contain a target analyte containing HbA1c with an anti-HbA1c antibody against the target analyte in a reaction cuvette to form a soluble HbA1c-antibody complex; (B) adding a polyhapten reagent to the reaction cuvette, wherein the polyhapten reagent reacts with excess anti-HbA1c antibody to form an insoluble polyhapten / target analyte-specific binding partner complex; (C) irradiating the reaction cuvette with light; and (D) measuring absorbance values ​​at least three wavelengths at multiple time points after the addition of the polyhapten reagent. A step of measuring a target analyte, wherein a first wavelength detects an insoluble polyhapten / target analyte-specific binding partner complex by turbidimetry, a second wavelength detects a protein, and a third wavelength functions as a blank; (E) a step of estimating the absorbance value at the time of delivery of the polyhapten reagent using regression of absorbance values ​​measured at the second and third wavelengths at two time points after the addition of the polyhapten reagent; and (F) a step of flagging the concentration value of the target analyte obtained by another algorithm if the estimated absorbance value at the time of delivery of the polyhapten reagent differs from the predicted value by an established flag constant, as this is unacceptable.

[0048] Any method described herein or otherwise intended may further include the step of dissolving a biological sample in a first container / cubet, and then transferring the dissolved biological sample to the reaction cuvette used in step (A).

[0049] Any wavelength can be used as the first, second, and third wavelengths according to any of the methods of this disclosure, as long as the values ​​described herein can be determined at any wavelength. For example, any wavelength can be used as the first wavelength as long as the presence of a protein / peptide can be detected, and therefore the delivery of a reagent can be detected, thereby providing an indicator of the aggregation state of polyhapten (or any other type of protein / polypeptide). Similarly, any wavelength can be used as the second wavelength as long as the influence due to hapten observed at any wavelength is at least minimal. Furthermore, any wavelength can be used as the third wavelength as long as the detection of protein / peptide at any wavelength is minimal, thereby the third wavelength is the A "blanking wavelength" or "reference wavelength" (i.e., a wavelength at which absorbance does not change as much as at the first and second wavelengths) can be used to ensure that the measurements obtained at the second wavelength are reliable and reproducible.

[0050] In one non-limiting embodiment, the first wavelength is in the range of approximately 300 nm to approximately 650 nm, the second wavelength is in the range of approximately 190 nm to approximately 300 nm, and the third wavelength is in the range of approximately 650 nm to approximately 850 nm. In a particular embodiment (but not limiting), the first wavelength is approximately 340 nm, the second wavelength is approximately 293 nm, and the third wavelength is approximately 700 nm.

[0051] In certain (but not limited to) specific embodiments, the absorbance at the first wavelength is (mAU 第1の波長 -mAU 第3の波長 The absorbance at the second wavelength is calculated as the first change in absorbance defined as (mAU), where the absorbance at the second wavelength is (mAU). 第2の波長 -mAU 第3の波長This is a two-wavelength value calculated as a second change in absorbance defined as ). Any wavelength that functions as a "blanking wavelength" and enables the calculation of two-wavelength values ​​described herein or otherwise intended can be used as a third wavelength in accordance with this disclosure. Non-limiting examples of wavelengths that can be used as a third wavelength include (but are not limited to) wavelengths in the range of about 650 nm to about 850 nm, including about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm and about 850 nm.

[0052] Any suitable regression analysis can be used as the established regression in step (E) of the methods disclosed herein or otherwise intended. Non-exclusive examples of available regression analyses include linear and non-linear regressions, and, for example (but not limited to), logarithmic curves, exponential curves, hyperbolas, parabolas, S-shaped curves, Michaelis-Menten curves, polynomial curves, logistic regression (or logit) curves, etc.

[0053] In certain embodiments (but not limited to), the established flag constant used in process (F) is determined as follows: A moving average is calculated for the A1C tests. The average is based on values ​​from a minimum (but not limited to) 50 tests and a maximum (but not limited to) 500 tests. Once the average is established using the minimum 50 values, results monitoring is turned "on" and new values ​​from the A1C tests are actively compared to the results monitoring range centered on the average. The moving average includes the minimum 50 values ​​and the maximum 500 values. When values ​​exceeding 500 are collected for results monitoring, the values ​​are replaced on a "first-in, first-out" basis. As a result, the old values ​​are removed from the average and the new values ​​are added to the average, so that a total of 500 values ​​are used. Each lot has its own moving average and range based on a predetermined criterion. In Example 2, described later in this specification, the predetermined criterion for the range (i.e., the "established flag constant") was 12% above the average and 15% below the average.

[0054] As described above, for the A1C assay, the acceptable range for result monitoring values ​​is based on the acceptable percentage (%) above and below the mean. The measured value is compared to the acceptable range centered on the mean. If the value is within this range, it is included in the moving average calculation. If the value is outside the range, it is not included in the moving average calculation, and the A1C assay result is flagged as an "abnormal assay" warning the user that the result is invalid and should not be reported.

[0055] As used herein, the term "established flag constant" refers to the cutoff value beyond which a significant difference between the measured value and the predicted value is observed. The established flag constant represents a value that exceeds the acceptable margin of variation / range of absorbance obtained for the sample, based on the measured value of the sample compared to the predicted value of the sample by regression analysis. The established flag constant is an acceptable The upper limit of the variation margin / range, for example, these linear values ​​are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 6 It may be any number that represents any non-integer value between 5, 66, 67, 6, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, etc., any negative value of any of these (i.e., -5, -65, etc.), or any slight variation of any of the values ​​listed above (i.e., "about 11", "about -15, etc.).Alternatively, established flag constants are the upper end of the allowable variation margin / range, for example, but not limited to these, 5000%, 4000%, 3000%, 2000%, 1000%, 900%, 800%, 700%, 600%, 500%, 450%, 400%, 350%, 300%, 250%, 200%, 150%, 100%, 99%, 98%, 97%, 96%, 95%. ,94%,93%,92%,91%,90%,89%,88%,87%,86%,85%,84%,83%,82%,81%,80%,79%,78%,77%,76%,75%,74%,73%,72%,71%,70%,69%,68%,67%,66%,65%,64%,63%,62%,61%,60%,59%,58%,57%,56%,55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 1 The percentage may be any integer or non-integer percentage value between 4%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, etc., any negative value thereof (i.e., -15%, -12%, etc.), or any slight variation of any of the percentage values ​​listed above (i.e., "approximately 85%", "approximately -15%, etc.).

[0056] In a non-limiting embodiment, measurements obtained by methods for detecting anomalous results as described herein or otherwise intended are measured and calculated independently of the actual assay for the presence and / or concentration of the target analyte in the biological sample. Alternatively, one or more measurements obtained by methods described herein and / or intended may be used in the actual assay for the presence and / or concentration of the target analyte in the biological sample.

[0057] In certain non-limiting embodiments, if one or more values ​​are flagged in step (F), the method may further include a step instructing the user to repeat assay steps (A) to (F).

[0058] While the methods described herein are for use with polyhapten reagents, it should be understood that the methods for detecting abnormal results in this disclosure are also applicable to use with other types of poorly delivered particle agglutination assay reagents. Accordingly, the scope of this disclosure further includes any and all variations of the methods described herein, where the term “polyhapten reagent” is replaced by “particle agglutination assay reagent.”

[0059] Any of the methods and processes described herein are performed by a user, for example, but not limited to such methods. However, the term “user” as used herein is not limited to human use, and may include (for example, but not limited to) computers, servers, websites, processors, network interfaces, people, user terminals, virtual computers, and combinations thereof.

[0060] Various embodiments of this disclosure are available in any reflection spectroscopy diagnostic instrument capable of (or modified to function in) the methods described herein. In some non-limiting embodiments, the instrument may be a point-of-care instrument. The reflection spectroscopy diagnostic instrument may be a system capable of embodying and / or executing the logic of the methods / processes described herein. The logic, embodied in the form of software instructions and / or firmware, can be executed on any suitable hardware. For example, the logic, embodied in the form of software instructions and / or firmware, can be executed on one or more components and / or similar on a dedicated system, a personal computer system, a distributed processing computer system, etc. In some embodiments, the entire logic may be implemented in an isolated environment operating on an instrument (e.g., a point-of-care instrument, but not limited to this). In other embodiments, the logic may be implemented in a network environment, such as a distributed system in which, for example, multiple instruments collect data that is analyzed and sent to a central computer system to supply the instruments with the analysis results. Each element of the instrument may be partially or entirely network-based or cloud-based, and may or may not be located in a single physical location.

[0061] Circuit configurations used herein include (but are not limited to) analog and / or digital components, or one or more suitable programmed processors (e.g., microprocessors) and associated hardware and software, or hardwired logic. A “component” can perform one or more functions. The term “component” can refer to hardware, such as, but not limited to, processors (e.g., microprocessors), application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), combinations of hardware and software, and / or similar.

[0062] The software used herein may, when executed by one or more components, include one or more computer-readable media (i.e., computer-readable instructions) that cause the components to perform specific functions. The algorithms described herein should be understood to be able to be stored on one or more non-transient memories. Non-limiting and exemplary non-transient memories include random-access memory, read-only memory, flash memory, and / or similar. Such non-transient memories may be electrical-based, optical-based, and / or similar.

[0063] Some non-limiting embodiments of this disclosure relate to reagent kits useful for conveniently performing the immunoassay methods described herein. Reagent kits include at least one target analyte-specific binding partner (e.g., an antibody against the target analyte, but not limited to these) and at least one polyhapten reagent, each of which is described in detail herein.

[0064] Other non-limiting embodiments of this disclosure relate to an immunoassay instrument (e.g., an immunoassay cartridge, but not limited to one) containing the reagent kits described herein and intended for use in the immunoassay methods described herein. For example, the immunoassay instrument may include at least one compartment capable of containing a sample suspected to contain a target peptide or protein analyte, the at least one compartment containing at least one target analyte-specific binding partner (e.g., an antibody against the target analyte, but not limited to one) and at least one polyhapten reagent, but not limited to one, as described in detail herein.

[0065] Furthermore, the reagent kits and / or immunoassay devices of this disclosure are described herein. The reagent kit and / or immunoassay apparatus of this disclosure may further contain other components and / or reagents for performing any of the specific immunoassays intended. The types of these additional components / reagents depend on the specific immunoassay format, and their identification is well within the scope of the art of the art. Examples of additional reagents / components that may be present in the reagent kit and / or immunoassay apparatus of this disclosure include, but are not limited to, diluents, solvents (e.g., for lysing red blood cells), washing solutions (e.g., but are not limited to isotonic solutions), positive controls, negative controls, quality control and / or actuators, and any combination thereof.

[0066] The relative amounts of various components / reagents in a kit and / or immunoassay instrument vary widely, providing component / reagent concentrations that substantially optimize the reactions that need to occur during the assay and further substantially optimize the sensitivity of the assay.

[0067] The reagent kits of this disclosure may further include a set of written instructions describing how to use the kit. Such kits can be used in any of the immunoassay instruments and / or methods described herein or otherwise intended.

[0068] The immunoassay apparatus may have one or more associated manual functions (i.e., pipetting is required for the addition of one or more reagents and / or the transfer of mixtures between two compartments), or alternatively, the immunoassay apparatus may be a fully automated, sealed system in which the required reagents / components are placed in various compartments during the construction of the immunoassay apparatus (various compartments having continuous fluid communication (or being able to have continuous fluid communication)), and therefore no manual handling of the sample and / or reagents is required to perform the assay after the sample has been added to the immunoassay apparatus.

[0069] An immunoassay apparatus comprises one or more compartments containing the components / reagents described herein, and an immunoassay apparatus may have several compartments, any arrangement of compartments, and any distribution of components / reagents between them, insofar as the immunoassay apparatus can function in accordance with this disclosure. If there are multiple compartments, the compartments may be completely separated from each other, or one or more compartments may be in fluid communication with each other. Various structures of immunoassay apparatus that can be used in accordance with this disclosure are well known in the art and therefore do not require further explanation.

[0070] In one embodiment, the immunoassay apparatus includes at least first and second compartments. The first compartment can accommodate a biological sample and may include, if desired, a mechanism for separating proteins / peptides from the majority of the sample, lysing red blood cells, etc. Such separation mechanisms are well known in the art of immunoassay apparatus and therefore no further explanation is deemed necessary. The second compartment is in fluid communication with the first compartment and contains at least one target analyte-specific binding partner (e.g., an antibody against the target analyte, but not limited to this) and / or at least one immunoassay reagent for carrying out the immunoassay method described in detail herein. Alternatively, the immunoassay apparatus may include a third compartment for the storage of at least one immunoassay reagent, and at least one immunoassay reagent may be moved from the third compartment to the second compartment.

[0071] The immunoassay apparatus may also include an optical reading chamber that can be optically examined by a spectrometer. The optical reading chamber may be connected to any of the compartments described herein, or it may be connected to a compartment other than those described herein.

[0072] Inlet channels and compartments, as well as two compartments, may be described as "fluidly communicating" with each other, and this expression means that the compartments may still be sealed, but there or between them This indicates that when the seal is broken, fluid can flow between the two compartments.

[0073] The kits / immunoassay devices of this disclosure may have any other desired features that are known in the art or otherwise contemplated herein. For example, but not limited to, the kits / immunoassay devices of this disclosure may further include one or more additional compartments containing other solutions, such as but not limited to lysants (for lysing red blood cells), diluents, washing solutions, labeling agents, interference solutions, positive controls, negative controls, quality control and / or actuators, and any combination thereof. [Examples]

[0074] Examples are shown below. However, it should be understood that this disclosure is not limited to its application to the specific experiments, results, and experimental procedures disclosed herein. Rather, these examples are provided as merely one of various embodiments, and are illustrative and not exclusive. [Examples]

[0075] R2 Polyhapten Delivery Check Result Monitoring Procedure The purpose of monitoring the delivery check results of R2 polyhapten is to detect polyhapten delivery problems, which may include short-term delivery or bubble collapse. Aggregation begins when the polyhapten is delivered to the reaction mixture, and since there are no photometric readings available immediately after delivery, the delivery cannot be directly measured using mAU (293nm–700nm). Therefore, a method was developed to estimate the mAU (293nm–700nm) at the time of delivery (i.e., the mAU at 0 hours immediately after delivery and just before the start of the agglutination reaction). This method predicts the mAU at 0 hours using regression of two subsequent readings against time (see Example 2). This method works because the change in the signal against time is linear with respect to the time used.

[0076] To monitor the delivery of the polyhapten reagent by the reagent 2 (R2) probe, result monitoring was added to the A1C sample assay parameters. The calculation for this result monitoring was also added to the A1CC calibration assay parameters, but the result monitoring code was not added.

[0077] Calculation method for monitoring delivery check results of R2 polyhapten: • In the assay parameters, the R2 probe is treated with polyhapten reagent in cycle 67. • The R2 polyhapten reagent is not observable during the reaction until the time between cycles 67 and 68 (referred to as "cycle 67+"). The duration of "Cycle 67+" was estimated to be [Cycle 69 - 5.7 seconds]. Each cycle is 3.6 seconds long. • The R2 polyhapten reagent can be detected at wavelengths of 293 nm and 700 nm (blanking wavelength). • Good readings at 293nm and 700nm prior to cycle 67 were obtained in cycles 52, 57, and 64. • Since the sample is transferred from the Loci vessel to the cuvette in cycle 6, the sample is present during the reaction in cycles 52, 57, and 64. • Good readings at 293nm and 700nm after cycle 67 are observed in cycles 69 and 71. The polyhapten absorbance after delivery (mAU at 0 hours) was calculated as follows: Gradient (cycle 69-71) * (time in cycle 67+) + (intercept at Y-0 time) The delivery check for R2 polyhapten was calculated as follows: (Polyhapten absorbance in cycle 67+) - (Average absorbance in cycles 52, 57, and 64). [Examples]

[0078] These calculations were performed offline using photometric data recovered during validation testing of the A1C assay. The data recovered during validation testing showed no evidence of problems with R2 delivery of the polyhapten reagent. Therefore, assay parameters were modified to simulate various instrument problems that could occur during the delivery process.

[0079] To simulate various types of delivery problems, the A1C assay parameters were modified in the manner shown below (and in Table 1). • Rg21: An additional trace dose for short-term delivery of polyhapten reagents and to maintain a constant reaction volume; • Rg22: Short-term delivery of polyhapten reagent and additional tracking dose (to maintain a constant reaction volume), and removal of air bubbles; • Rg23: Short-term delivery of polyhapten reagent - no additional tracking (true short-term delivery); • Standard delivery and bubble removal using Rg24:A1C parameters (28 μL); • Standard delivery using Rg25:A1C parameters (28 μL), removal of air bubbles, and increased tracking from 15 μL to 25 μL of water; and • Standard delivery using Rg26:A1C parameters (28 μL), bubble removal, and increased tracking from 15 μL to 20 μL of water.

[0080] In this way, Rg21 to Rg26 each mimic the problem of delivering one or more R2 reagents.

[0081] [Table 1]

[0082] For each series of assay parameters, the following samples were tested: diabetes management levels 1 and 2 from LYPHOCHEK® Diabetes Control (Bio-Rad Laboratories, Hercules, CA), as well as medical decision pools (MDPs) 1-4. All MDPs were tested with n=5, and quality control (QC) samples were tested with n=2 or n=5 depending on the test. For each assay parameter test, the impact on HbA1c% results and the change in R2 polyhapten delivery check values ​​were calculated.

[0083] Figure 1 illustrates the dynamic data of DV A1C for QC and MDP samples. As shown, a small absorbance is observed at the start of the assay, corresponding to the addition of the antibody reagent. An increase in absorbance is observed when the sample is added in cycle 6. After the addition of the polyhapten reagent in cycle 67, the agglutination reaction between the free antibody and polyhapten occurs. Accordingly, the absorbance immediately begins to increase.

[0084] The HbA1c measurement during the reaction is based on the aggregation of free antibodies with polyhapten: • When more HbA1c is present during the reaction, more antibody binds to the HbA1c, and therefore less antibody is available for aggregation with the polyhapten. Less aggregation between the antibody and the polyhapten results in a lower HbA1c signal and a higher HbA1c analyte result. • When less HbA1c is present during the reaction, less antibody binds to the HbA1c, and therefore more antibody is available for aggregation with the polyhapten. More aggregation between the antibody and the polyhapten results in a higher HbA1c signal and a lower HbA1c analyte result.

[0085] Figure 2 illustrates how polyhapten delivery check monitoring is performed. After adding the polyhapten reagent in cycle 67, readings are obtained immediately in cycles 69 and 71, and these two readings are then used to linearly estimate the 0-hour mAU value of the polyhapten reagent (see the circled linear estimate).

[0086] The first trial compared the control parameter (A1C1 was a clone of the A1C parameter) with the Rg21 and Rg22 parameters, in which the amount of polyhapten reagent delivered was reduced and the follow-up amount was increased. The data from this trial are shown in Table 2.

[0087] [Table 2]

[0088] Reducing the delivery of the polyhapten reagent and replacing it with tracking water resulted in a decrease in the HbA1c signal and a higher HbA1c analyte result. Since the polyhapten was added after hemoglobin measurement, the hemoglobin concentration was unaffected, and the increase in HbA1c results led to an increase in the HbA1c% result. The values ​​for the polyhapten delivery check in these parameters were lower than those for the control parameters.

[0089] In the second trial, the control parameters were compared with Rg23, Rg24, Rg25, and Rg26. These parameters are listed in Table 1, and the data are shown in Table 3.

[0090] For the Rg25 and Rg26 parameters, the amount of polyhapten delivered was not changed, but bubbles were removed and replaced with different amounts of tracking water. These parameters were prepared to simulate the delivery of the polyhapten reagent, where bubbles collapse and are partially or completely replaced with tracking water. Dilution of the reaction mixture is expected to result in a lower HbA1c signal. This lower signal will result in a higher HbA1c analyte result. Since the reaction volume was not corrected in calculations, the HbA1c signal did not decrease as much as expected. As a result, the signal was While there was a decrease compared to the control parameters, it was not as significant as expected. HbA1c results for these parameters were elevated compared to the control parameters. This data is shown in Table 3. The polyhapten delivery check values ​​for these parameters were lower than those for the control parameters.

[0091] For the Rg23 parameter with the polyhapten content reduced by half, the presence of less polyhapten is expected to result in less aggregation, which leads to a lower HbA1c signal. A lower HbA1c signal is expected to result in a higher HbA1c analyte. Simultaneously, the total reaction volume was reduced to 14 μl. This lower volume resulted in high aggregation and a high HbA1c signal, leading to a lower HbA1c analyte. In this situation, two factors (less polyhapten and lower reaction volume) compete.

[0092] Consideration of competitive factors For the Rg21 parameter, the HbA1c% result showed an increase of approximately 0.8%. For the Rg25 and Rg26 parameters, the HbA1c% result for Rg26 was approximately 0.5% lower than that for Rg25, which contained an additional 5 μL of tracking water (depending on the HbA1c% level). The difference in polyhapten amount between Rg23 and the control parameter was 14 μL. This would result in a decrease of approximately 1.4% in HbA1c%. Since both effects were present in the Rg23 parameter, the result showed a decrease in HbA1c%. This decrease in the result differed depending on the analyte level.

[0093] [Table 3] [Table 4]

[0094] Figure 3 is a graph showing R2 polyhapten delivery check data using assay parameters designed to simulate problems related to polyhapten reagent delivery. Each data point represents the average value calculated from 2–5 tests / replicas. Samples contained various HbA1c analytes (QCL1, QCL2, MDP1–4).

[0095] Figure 4 is a graph showing the delivery check data for R2 polyhapten in the samples compared to the result monitoring limit. Each data point represents the value calculated for each individual test. The samples were MDP1-4, which contained various HbA1c analytes.

[0096] Based on the data from these two tests, the delivery check thresholds for R2 polyhapten were set at -15% and +12% compared to the average value of this delivery check.

[0097] Conclusion: Based on the data from these studies, the mean -15% and mean +12% threshold values ​​(i.e., “established flag constants”) for R2 polyhapten delivery checks were set to flag outcomes that may have clinically significant effects due to polyhapten reagent delivery problems.

[0098] However, these flag constants are established for illustrative purposes only and are not intended to limit this disclosure. Any other flag constant values ​​calculated in accordance with the methods described herein or otherwise intended may also be available in accordance with this disclosure and therefore may be included within its scope.

[0099] Accordingly, methods, compositions, kits, and apparatus for use herein have been provided that fully satisfy the purposes and advantages set forth herein. Although this disclosure has been described in conjunction with the specific drawings, experiments, results, and language set forth herein, it is obvious that many alternatives, modifications, and variations will be apparent to those skilled in the art. Therefore, it is intended to encompass all such alternatives, modifications, and variations that fall within the spirit and broad scope of the currently disclosed inventive concept.

Claims

1. A method for detecting abnormal results caused by insufficient delivery of polyhapten reagents used in immunoassays, (A) A step of reacting a biological sample suspected to contain the target analyte with a target analyte-specific binding partner in a reaction cuvette, thereby forming a soluble analyte / specific binding partner complex, (B) A step of adding a polyhapten reagent to the reaction cuvette, wherein the polyhapten reagent reacts with an excess of the target analyte-specific binding partner to form an insoluble polyhapten / target analyte-specific binding partner complex, (C) A step of irradiating the reaction cuvette with light, (D) A step of measuring absorbance values ​​at at least three wavelengths at multiple time points after the addition of the polyhapten reagent, wherein the first wavelength detects the insoluble polyhapten / target analyte-specific binding partner complex by turbidimetry, the second wavelength detects the protein, and the third wavelength functions as a blank, (E) A step of estimating the absorbance value at the time of delivery of the polyhapten reagent by using the regression of absorbance values ​​measured at the second and third wavelengths at two time points after the addition of the polyhapten reagent, (F) If the estimated absorbance value of the polyhapten reagent upon delivery differs from the predicted value by an established flag constant, the step of flagging the concentration value of the target analyte obtained by another algorithm as unacceptable. The method, including the method described above.

2. The method according to claim 1, wherein the target analyte is selected from the group consisting of glycated hemoglobin (HbA1c), albumin, human chorionic gonadotropin (hCG), ferritin, growth hormone, prolactin, thyroglobulin (Tg), C-reactive protein (CRP), rheumatoid factor (RF), gentamicin, tobramycin, CRP, digoxin, amikacin, caffeine, carbamazepine, digitoxin, disopyramide, ethosuxamide, lidocaine, lithium methotrexate, NAPA, phenobarbital, phenytoin, primidone, procainamide, quinidine, theophylline, tobramycin, valproic acid, and vancomycin.

3. The method according to claim 1, wherein the target analyte-specific binding partner is an antibody against the target analyte.

4. The method according to claim 3, wherein the target analyte is glycated hemoglobin (HbA1c), the target analyte-specific binding partner is an anti-HbA1c antibody, and the polyhapten reagent comprises a plurality of HbA1c epitopes.

5. The method according to claim 1, wherein the first wavelength is in the range of about 300 nm to about 650 nm, the second wavelength is in the range of about 190 nm to about 300 nm, and the third wavelength is in the range of about 650 nm to about 850 nm.

6. The method according to claim 5, wherein the first wavelength is approximately 340 nm, the second wavelength is approximately 293 nm, and the third wavelength is approximately 700 nm.

7. The method according to claim 1, wherein in step (E), the first of two time points after the addition of the polyhapten reagent is approximately 7.2 seconds after the addition of the polyhapten reagent, and the second of the two time points is approximately 7.2 seconds after the first time point.

8. The method according to claim 1, wherein the biological sample is selected from the group consisting of urine, whole blood or any part thereof, whole blood cells or lysed blood cells, saliva, sputum, cerebrospinal fluid, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, tears, mucus, bladder lavage fluid, semen, and combinations thereof.

9. A method for detecting abnormal results caused by insufficient delivery of polyhapten reagents used in glycated hemoglobin (HbA1c) immunoassays, (A) A step of reacting a biological sample suspected to contain a target analyte containing HbA1c with an anti-HbA1c antibody against the target analyte in a reaction cuvette, thereby forming a soluble HbA1c-antibody complex, (B) A step of adding a polyhapten reagent to the reaction cuvette, wherein the polyhapten reagent reacts with an excess of anti-HbA1c antibody to form an insoluble polyhapten / target analyte-specific binding partner complex, (C) A step of irradiating the reaction cuvette with light, (D) A step of measuring absorbance values ​​at at least three wavelengths at multiple time points after the addition of the polyhapten reagent, wherein the first wavelength detects the insoluble polyhapten / target analyte-specific binding partner complex by turbidimetry, the second wavelength detects the protein, and the third wavelength functions as a blank, (E) A step of estimating the absorbance value at the time of delivery of the polyhapten reagent by using the regression of absorbance values ​​measured at the second and third wavelengths at two time points after the addition of the polyhapten reagent, (F) If the estimated absorbance value of the polyhapten reagent upon delivery differs from the predicted value by an established flag constant, the step of flagging the concentration value of the target analyte obtained by another algorithm as unacceptable. The method comprising the above.

10. The method according to claim 9, wherein the first wavelength is in the range of about 300 nm to about 650 nm, the second wavelength is in the range of about 190 nm to about 300 nm, and the third wavelength is in the range of about 650 nm to about 850 nm.

11. The method according to claim 10, wherein the first wavelength is approximately 340 nm, the second wavelength is approximately 293 nm, and the third wavelength is approximately 700 nm.

12. The method according to claim 9, wherein in step (E), the first of two time points after the addition of the polyhapten reagent is approximately 7.2 seconds after the addition of the polyhapten reagent, and the second of the two time points is approximately 7.2 seconds after the first time point.

13. The method according to claim 9, wherein the biological sample is selected from the group consisting of urine, whole blood or any part thereof, whole blood cells or lysed blood cells, saliva, sputum, cerebrospinal fluid, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, tears, mucus, bladder lavage fluid, semen, and combinations thereof.

14. A method for detecting abnormal results caused by insufficient delivery of polyhapten reagents used in glycated hemoglobin (HbA1c) immunoassays, (A) A step of reacting a biological sample suspected to contain a target analyte containing HbA1c with an anti-HbA1c antibody against the target analyte in a reaction cuvette, thereby forming a soluble HbA1c-antibody complex, (B) A step of adding a polyhapten reagent to the reaction cuvette, wherein the polyhapten reagent reacts with an excess of anti-HbA1c antibody to form an insoluble polyhapten / target analyte-specific binding partner complex, (C) A step of irradiating the reaction cuvette with light, (D) Absorbance values ​​at at least three wavelengths at multiple time points after the addition of the polyhapten reagent The measurement process, (i) The first wavelength is detected by turbidimetry as the insoluble polyhapten / target analyte-specific binding partner complex is in the range of approximately 300 nm to approximately 650 nm. (ii) The second wavelength detects proteins and is in the range of approximately 190 nm to 300 nm, and (iii) the third wavelength functions as a blank and is in the range of approximately 650 nm to 850 nm. The above process, (E) A step of estimating the absorbance value at the time of delivery of the polyhapten reagent by using the regression of absorbance values ​​measured at the second and third wavelengths at two time points after the addition of the polyhapten reagent, (F) If the estimated absorbance value of the polyhapten reagent upon delivery differs from the predicted value by an established flag constant, the step of flagging the concentration value of the target analyte obtained by another algorithm as unacceptable. The method comprising the above.

15. The method according to claim 14, wherein the first wavelength is approximately 340 nm, the second wavelength is approximately 293 nm, and the third wavelength is approximately 700 nm.

16. The method according to claim 14, wherein in step (E), the first of two time points after the addition of the polyhapten reagent is approximately 7.2 seconds after the addition of the polyhapten reagent, and the second of the two time points is approximately 7.2 seconds after the first time point.

17. The method according to claim 14, wherein the biological sample is selected from the group consisting of urine, whole blood or any part thereof, whole blood cells or lysed blood cells, saliva, sputum, cerebrospinal fluid, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, tears, mucus, bladder lavage fluid, semen, and combinations thereof.

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