Method for Correction for Sample Volume

Abstract

A method for the analysis of one or more non-invasive sample biomarkers. Biomarkers within a normal concentration range may indicate not only the lack of disease, but also general health or lack of disease. These non-invasive sample biomarkers are corrected based on excretion of another non-invasive sample biomarker reflecting changes to the sample volume in a normal concentration range lacking disease. Method allows for indicating the existence of diseases, such as renal insufficiency, infections or other disease biomarkers by analyzing the concentration of biomarkers.

Claims

1. A method of determining concentration of at least one biomarker in a non-invasive sample comprising: introducing a sample into a well, wherein the sample comprises at least one biomarker; capturing the at least one biomarker; and determining that the at least one biomarker is indicative of lack of health.

2. The method of claim 1, further comprising determining concentration of the at least one biomarker indicating non-invasive sample volume and comparing the concentration of the at least one biomarker to a normal range.

3. The method of claim 1, wherein a concentration of a first biomarker is corrected by a concentration of a second biomarker to as to rates of excretion of the first biomarker.

4. The method of claim 3, wherein concentrations of the first or second biomarkers outside of the normal concentration range are used to indicate a disease.

5. The method of claim 1, wherein the sample is chosen from a group comprising human serum albumin, hydroxynonnel to human serum albumin, malondialedehyde to human serum albumin, uristatin, or bikunin.

6. The method of claim 1, further comprising determining a concentration of the biomarker, wherein a concentration of albumin indicates the concentration of the biomarker.

7. The method of claim 3, wherein concentrations of the first or second biomarkers inside of the normal concentration range are used to indicate lack of disease or health.

8. The method of claim 3, wherein concentrations of the first or second biomarkers inside of the normal concentration range are used to indicate a degree of health or progression to disease.

9. The method of claim 1, further comprising determining a concentration of the biomarker in non-invasive sample fluids, like urine, saliva tears, sweat, nasal lavage, interstitial fluid, and other samples, are used to estimate the biomarker concentrations in the blood without having to directly sample blood.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 shows a schematic view of the analysis of biomarkers in non-invasive sample according to a non-limiting embodiment of the invention.

[0032] FIG. 2 shows a schematic view of a set of wells for capture and detection a biomarker according to a non-limiting embodiment of the invention.

[0033] FIG. 3 shows four immunoassays for urinary biomarkers according to a non-limiting embodiment of the invention.

DESCRIPTION OF THE INVENTION

[0034] No aspect, component, element, structure, act, step, function, instruction, and/or the like used herein should be construed as critical or essential unless explicitly described as such. Also, as used herein, the articles “a” and “an” are intended to include one or more items and may be used interchangeably with “one or more” and “at least one.” Furthermore, as used herein, the term “set” is intended to include one or more items (e.g., related items, unrelated items, a combination of related and unrelated items, and/or the like) and may be used interchangeably with “one or more” or “at least one.” Where only one item is intended, the term “one” or similar language is used. Also, as used herein, the terms “has,” “have,” “having,” or the like are intended to be open-ended terms. Further, the phrase “based on” is intended to mean “based at least partially on” unless explicitly stated otherwise.

[0035] FIGS. 1 to 3 illustrate an embodiment of the present disclosure, where immunoassays are able to determine concentration levels of one or non-invasive sample biomarkers indicating the presence of disease while those inside the normal concentration range indicate the lack of disease or general health. Such can be indicated by levels of one or more additional non-invasive sample biomarkers that indicate changes in the non-invasive sample concentration when measured against concentration levels in the normal range of the additional non-invasive sample biomarkers.

[0036] FIG. 1 shows a schematic view of the present disclosure for analysis of biomarkers in non-invasive samples where the analyte (1) is captured in a well (2) by an affinity agent (3) either directly attached to the linkage arm (4) or bound to the linkage arm (4) via a high affinity label and capture agent (5). The analyte (1), such as a biomolecule or cell is captured by an affinity agent (3) after the addition of a sample (6) to the well. A second affinity agent (7) for a biomarker may be added and may attach to a reagent capable of generating a signal (8) after the removal of sample waste (23) from the well (2).

[0037] FIG. 2 shows a schematic view of a set of wells (10) able to capture and detect a biomarker, or an analyte (1), by addition of an affinity agent (3) for specific capture and detection (9). In a non-limiting example, a first biomarker may be a neutravidin microparticle (11) that is captured in the well (2). A first biomarker, such as a cell or biomolecule, is captured by affinity agent (3) and capture surface of the well (2) after the addition of reagents (3 and 7) and sample (6) to well (2) and followed by removal of waste (23) and by generation of signal (8) for determination of the first biomarker. One or more other wells (12) can be used for determination of a second biomarker via affinity agents and capture surface specific to the second biomarker.

[0038] FIG. 3 shows four immunoassays for urinary biomarkers in accordance with a non-limiting embodiment of the invention as panel of biomarkers that indicate the presence of disease while those inside the normal concentration range indicate the lack of disease or general health. The biomarker (1) for measuring changes in the non-invasive sample concentration is shown by human serum albumin (HSA) (13) being bound by capture (14) and detection antibodies (15) against human serum albumin (HSA) (13). A first biomarker indicating the presence of disease or general health is the adduct of hydroxynonenal to human serum albumin (HNE-HSA) (16) being bound by capture agent (14) against human serum albumin (HSA) (13) and detection antibodies (17) against hydroxynonenal (HNE). A second biomarker indicating disease or general health is the adduct of malondialdehyde (MDA) to human serum albumin (MDA-HSA) (18) being bound by capture (14) against human serum albumin (HSA) and detection antibodies (19) against malondialdehyde (MDA). A third biomarker indicating health or lack of health is urinary trypsin inhibitor (Uristatin or Bikunin) (21) shown being bound by capture agent (20) and detection antibodies (22) against urinary trypsin inhibitor.

[0039] In some non-limiting embodiments of the present disclosure, the first step determines if the biomarker concentrations of the first and second biomarkers are within measurable range and able to produce valid ratio results. The next step determines if the second biomarker concentration is within the normal range and not at a level indicative of disease and able to produce valid a non-invasive sample concentration. Assuming results are reportable and not indicative of disease, the final step takes a ratio of the first biomarker with the second biomarker and reports the ratio results, which accurately reflects the actual rates of excretion of the first biomarker at levels indicative of lack of disease or general health. In some non-limiting embodiments of the present disclosure, there may be multiple first biomarkers, which indicate inflammation, oxidative stress, and other signs of general lack of lack of disease or general health.

[0040] In some non-limiting embodiments of the present disclosure, when the concentration levels of the first or second biomarkers outside the measurable ranges are measured, the limit of measurement ranges are reported. In some non-limiting embodiments of the present disclosure, if the second biomarker is at a level indicative of disease, then the ratio is not performed and the indication of disease is reported based on the second biomarker. In some non-limiting embodiments of the present disclosure, if the first biomarker is at a level indicative of disease, then the ratio is not performed and the indication of disease is reported based on the first biomarker. In some non-limiting embodiments of the present disclosure, the second biomarker may be an albumin, which may be indicative of disease, and the ratio is not performed.

[0041] In some non-limiting embodiment of the present disclosure, immunoassays are able to determine concentration levels of biomarkers by sandwich or competitive assay within dynamic ranges that allow measurement of concentrations determining disease or health. In practice, the invention can make use of optical immunoassay (OP-IA), electrochemical immunoassay (EC-IA) and mass spectrometric immunoassays (MS-IA) as an example previously described discussed in Pugia et al 63/006,833, 63/089,286, and 63/089,308, which are incorporated herein reference in their entireties.

Example 1: Accurate Method to Correct the Urinary Concentration of Biomarkers to Reflect the Actual Rates of Excretion

[0042] To demonstrate the present disclosure, quantitative optical immunoassays (OP-IA) were developed for adduct of 4-Hydroxynonenal (HNE-HSA) to human serum albumin (HSA), adduct of Malondialdehyde (MDA) to HSA, and urinary trypsin inhibitor (also known as Uristatin or Bikunin) for lack of disease or general health. These biomarkers when above the normal range indicate a lack of health as important indicators of oxidative stress, as discussed in, for example, Akbari, M., et al., The Effects of Vitamin D Supplementation on Biomarkers of Inflammation and Oxidative Stress Among Women with Polycystic Ovary Syndrome: A Systematic Review and Meta-Analysis of Randomized Controlled Trials. Horm Metab Res, 2018. 50(4): p. 271-279 (hereinafter “Reference 12”), Mansournia, M. A., et al., The Effects of Vitamin D Supplementation on Biomarkers of Inflammation and Oxidative Stress in Diabetic Patients: A Systematic Review and Meta-Analysis of Randomized Controlled Trials. Horm Metab Res, 2018. 50(6): p. 429-440 (hereinafter “Reference 13”), Nasri, K., et al., The effects of vitamin D and evening primrose oil co-supplementation on lipid profiles and biomarkers of oxidative stress in vitamin D-deficient women with polycystic ovary syndrome: A randomized, double-blind, placebo-controlled trial. Endocr Res, 2018. 43(1): p. 1-10 (hereinafter “Reference 14”), Razavi, M., et al., The Effects of Vitamin D-K-Calcium Co-Supplementation on Endocrine, Inflammation, and Oxidative Stress Biomarkers in Vitamin D-Deficient Women with Polycystic Ovary Syndrome: A Randomized, Double-Blind, Placebo-Controlled Trial. Horm Metab Res, 2016. 48(7): p. 446-51 (hereinafter “Reference 15”), and Zheng, H. J., et al., The effect of probiotic and synbiotic supplementation on biomarkers of inflammation and oxidative stress in diabetic patients: A systematic review and meta-analysis of randomized controlled trials. Pharmacol Res, 2019. 142: p. 303-313 (hereinafter “Reference 16”) or inflammatory disease, as discussed in, for example, Pugia, M. J., et al., Immunological evaluation of urinary trypsin inhibitors in blood and urine: role of N- & O-linked glycoproteins. Glycoconj J, 2007. 24(1): p. 5-15 (hereinafter “Reference 17”), and Sasaki, M., et al., Measurement of the albumin content of urinary protein using dipsticks. J Clin Lab Anal, 1999. 13(5): p. 246-50 (hereinafter “Reference 18”) are elevated outside of their normal ranges to a level indicative of disease, as shown in Table 1. A second biomarker, namely human serum albumin (HSA), is used to illustrate a non-limiting embodiment of the present disclosure where this additional second biomarker indicates changes in the urine concentration when measured against the normal range. Concentration levels of first or second biomarkers outside a measurable range are not measurable other than to be reported at their limits of range by <= or >=.

[0043] Detection and capture affinity agents were made from HNE-HSA, MDA-HSA, uTi and HSA assays (See Materials below). Samples of urine were diluted 25-fold in PBS at pH 9 and a 100 μL sample added to a separate wells of polypropylene sample plate for each of four assays. Both detection and capture affinity agents were added at 0.5 to 2.0 μg/well to bind to HNE-HSA, MDA-HSA, uTi and HSA in samples using separate wells for each, and were incubated for 60 min at 37° C. The bound immunoassay complexes were transferred to a streptavidin coated high binding capacity microplates (Pierce) after blocked for 24 h with the blocking agent as described (Pugia Anal Chem 2021). The immunoassay complexes were bound to streptavidin coated microplates using separate wells for each sample by incubating for 5 min at 25° C. ELISA wells were then washed five with 200 μL TBS-T(0.05% Tween-20) in a EL406 plate washer (BioTek) and read on optical readers (BioTek) after addition of para-nitro phenyl phosphate as the signal. In this method, alkaline phosphatase is used to generate para-nitro phenol as the optical reporter from para-nitro-phenyl phosphate.

TABLE-US-00001 TABLE 1 Measurement ranges, normal ranges and levels indicative of disease for biomarkers Levels Measurable Normal indicative Assay Range range of disease MDA-HSA 0.5 to 8.0 mg/dL 0.5 to 8.0 mg/dL >4.0 mg/dL HNE-HSA 0.5 to 8.0 mg/dL 0.5 to 8.0 mg/dL >4.0 mg/dL Uristatin 0.5 to 12.0 mg/L  0.5 to 7.5 mg/L  >7.5 mg/L  HSA 0.5 to 80.0 mg/L  0.5 to 40 mg/L  >40 mg/L 

Materials:

[0044]

TABLE-US-00002 Detection Polyclonal antibodies for recognizing Bikunin (LMX Med Tech MI), affinity malondialdehyde (MDA) (Abcam) and for recognizing 4-hydroxy-nonenal agents (HNE) (Abcam) were conjugated to ALP using the lightning link reagent (Abcam, MA). Capture Polyclonal antibodies recognizing Bikunin (LMX Med Tech MI), were affinity used for Bikuinin capture and those recognizing HSA were used for HSA, agents HNE-HSA and MDA-HSA capture (Bethyl Laboratories, PA) and each were conjugated to biotin-PEG4 using the EZ-Link NHS-conjugation kits (Thermo Fisher Scientific). The resultant antibody conjugates were stored at 4° C. Antigens Urinary Trypsin Inhibitor (SCPIC product code P205-1) and HSA (fraction V, Thermo Fisher) were used. Both malondialdehyde (MDA) and 4- hydroxy-nonenal (HNE) were reacted with human serum albumin to form the adduct of hydroxynonenal to human serum albumin (HNE-HSA) and the adduct of malondialdehyde to human serum albumin (MDA-HSA) followed by FPLC purification (AKTA prime, GE life sciences)

[0045] Unless otherwise noted all other materials were purchased from Sigma Aldrich or Thermo Fisher Scientific.

[0046] All four methods were able to produce accurate and quantitative results in the measurable range and were also able to indicate when the biomarkers were outside the normal range, thus indicating disease such as shown in Table 1. Inside the normal range, lower biomarker values for MDA-HSA, HNE-HSA and Uristatin indicate a lack of disease or general health, with little inflammation or oxidative stress. Inside the normal range, lower HSA values indicate a lower urine concentration or low specific gravity (SG), and higher HSA values indicate a higher urine concentration or high specific SG value.

[0047] To demonstrate the correction for urine concentration according a non-limiting embodiment of the invention, the concentration of the MDA-HSA, HNE-HSA, HSA and uTi biomarkers were measured in low specific gravity (SG) (SG=1.005), medium SG (SG=1.010) and high SG (SG=1.030) urines. These changes in SG reflect changes in the excretion rate of biomolecules and urine volume, with high SG indicating 200% decrease in the excretion rate or 2× concentration due to reduced urine volume compared to medium SG urine concentration and low SG indicating 50% increase in excretion rate or 2× dilution due to increased urine volume compared to medium SG urine concentration.

[0048] Human serum albumin (HSA) values measured in the three urine pools of different SG were found to be 0.8 mg/L for low SG of 1.004, 1.7 mg/L for med SG of 1.011 and 5.2 mg/L for high SG of 1.019. Human serum albumin (HSA) was selected as an example the second biomarker for correction as it predicted urine concentration within its normal range of 0.5 to 40 mg/L. It is well known that HSA values less than 40 mg/L indicate health in the patient or a lack or albuminuria, while HSA values greater than 40 mg/L indicate disease, such as kidney disease or hypertension.

[0049] The uTi values in these pools were measured in three pools and found to be 5.0 mg/L for low SG, 4.8 mg/L for med SG and 10.2 mg/L for high SG. The uristatin levels of >7.5 mg/L indicate disease, e.g. infection. Therefore, uristatin (uTi) cannot be selected as, for example, the second biomarker for correction, as it did not predict urine concentration within its normal range of 0.5 to 7.5 mg/L. The MDA-HSA and HNE-HSA values were measured in three pools and also did not correlate to urine concentration (e.g. SG), and therefore could not be selected as an example of a second biomarker for urine correction. They were all within their normal range of 0.5 to 8.0 mg/L.

[0050] Using HSA ratioing of three biomarkers of panel assays of HNE-HSA, MDA-HSA and uTi could be corrected for urine concentration (e.g. SG) according to a non-limiting embodiment of the invention. This was performed by first confirming the biomarker concentrations of the first biomarkers (HNE-HSA, MDA-HSA and uTi) and the second biomarker (HSA) where all inside measurable ranges were able to produce a valid ratio result. If the second biomarker, HSA in this example, had concentrations outside the normal range indicating disease (>40 mg/L), then the disease level would be reported to indicate that sample was not from a healthy person and not able to produce valid ratio results. The final step is to ratio each of first biomarkers (HNE-HSA, MDA-HSA and uTi) to the second biomarker (HSA) and to report ratio results (mg biomarker 1/mg biomarker 2), which accurately reflect the actual rates of excretion of the first biomarker at levels indicative of disease or health.

[0051] To demonstrate the invention, eight levels of each analyte were added to three urine pools spanning the SG range to provide contrived samples that spanned the measurable ranges HNE-HSA, MDA-HSA and uTi assays (Table 1). The ratios of HNE-HSA to HSA (mg/mg), MDA-HSA to HSA(mg/mg) and uTi to HSA(mg/mg) were determined for each of eight levels. The sample bias between expected and reported concentrations was <18% for all ratio levels and within the acceptable limits for producing accurate and quantitative ratio results in contrived urine samples. This compared favorably to the sample bias between expected and reported concentration levels without ratioing of <21% across all samples.

[0052] For verification of the improvement, the panel assays were ratioed with 87 urine specimens collected from patients with diseases and healthy patients. All urine specimens were measured for specific gravity by TS meter to 0.001 SG and found to range from SG 1.004 to 1.030. Of these 87 samples, 21 had value of 40 mg/L HSA indicative of disease. These 21 samples had HSA values independent of urine SG as shown by a correlation coefficient of zero as they were samples of disease. Whereas the 66 samples below 40 mg/L HSA, had HSA values dependent on the urine as shown by a coefficient of R>0.8 to SG.

[0053] The impact of the albumin ratio according to the invention was measured by reducing the sample bias for MDA-HSA, HNE-HSA, and uTi across range of urine concentrations (SG 1.004 to 1.030). After performing the HSA ratio, the sample bias was improved to 19%, 11%, and 15% for HNE-HSA to HSA (mg/mg), MDA-HSA to HSA(mg/mg), and uTi to HSA(mg/mg) respectively. This is significantly improved from the bias observed without ratioing, which were 35%, 31% and 34% for HNE-HSA (mg/dL), MDA-HSA to HSA(mg/mg) and uTi to HSA(mg/mg) respectively. Additionally, without removing samples with 40 mg/L HSA indicative of disease, the ratio could not improve the sample bias for these samples SG 1.004 to 1.030.

[0054] The correction for urine volume for using urine biomarkers indicates changes in the urine concentration when measured against the normal range. It was somewhat un-expected that biomarkers known to be impacted by disease, such as HSA, could reduce the impact of urine concentration on other urine biomarkers when used in the normal range. While not bound to a mechanism of such reaction, it is believed that normal albumin excretion (e.g. low levels) is generally constant and changes in the normal range primarily reflect change in urine concentration (e.g. SG).

Example 2: Accurate Method to Correct the Saliva Concentration of Biomarkers to Reflect the Actual Rates of Excretion

[0055] To demonstrate the present disclosure as a correction for saliva volume measures the adduct of 4-Hydroxynonenal (HNE-HSA) to human serum albumin (HSA) and human serum albumin (HSA) were used.

[0056] Human serum albumin (HSA) was picked from over 500 measurable proteins have found in human saliva as the vast majority are impacted in patients with cancer (Paper 4). The impact of cancer excludes these proteins from use as biomarker for correction sample volume. Human serum albumin (HSA) and amylase were the most abundant proteins. Both are in the 50-kDa molecular weight range ideal for measuring of sample volume due to vascular permeation of blood into saliva. However, amylases' main function is to hydrolyze the glycosidic bonds in starch molecules, converting complex carbohydrates to simple sugars, and its secretion is greatly impacted by diet, prohibiting it uses.

[0057] Human serum albumin (HSA) was selected as an example the second biomarker for correction as it predicted saliva concentration as it normal range is 0.1 to 0.4 g/L in saliva (Paper 5). HSA ratioing of HNE-HSA, was done to demonstrate the expected expect impact on correction from of saliva samples with volume SG of 1.005, 1.015 and 1.030 according to a non-limiting embodiment of the invention. These sample had observed HSA values of 0.03 g/L. 0.1 g/L and g/L respectively.

[0058] To demonstrate the invention, four levels of HNE-HSA, were added to these three salvia pools spanning at SG of 1.005, 1.015 and 1.030. The 1.030 sample had 2 fold higher concentration levels of HNE-HSA then the 1.015 sample which had 2 fold higher concentration levels of HNE-HSA than the 1.005 samples. All samples spanned the measurable ranges HNE-HSA, and HSA. The ratios of HNE-HSA to HSA (mg/mg), were determined all twelve levels. The values varied by 211% without correction by HSA The values varied with correction by HSA ranged compared favorably eliminating sample bias between expected and reported concentration levels with ratioing at <11% across all samples.

[0059] The correction for saliva volume for using saliva biomarkers indicates changes in the saliva concentration can be correct by HSA. Again, it was somewhat un-expected that biomarkers known to be impacted by disease, such as HSA, could reduce the impact of saliva concentration on other saliva biomarkers when used in the normal range. While not bound to a mechanism of action, it is believed that normal albumin excretion (e.g. in the absence of tissue damage) is generally constant and changes primarily reflect change in saliva concentration (e.g. SG) due to vascular permeability, allowing a better estimation of blood values into non-invasive fluid.

[0060] The impact of the albumin ratio according to the invention was measured by reducing the sample bias for MDA-HSA, HNE-HSA, and uTi across range of urine concentrations (SG 1.004 to 1.030). After performing the HSA ratio, the sample bias was improved to 19%, 11%, and 15% for HNE-HSA to HSA (mg/mg), MDA-HSA to HSA(mg/mg), and uTi to HSA(mg/mg) respectively. This is significantly improved from the bias observed without ratioing, which were 35%, 31% and 34% for HNE-HSA (mg/dL), MDA-HSA to HSA(mg/mg) and uTi to HSA(mg/mg) respectively. Additionally, without removing samples with 40 mg/L HSA indicative of disease, the ratio could not improve the sample bias for these samples SG 1.004 to 1.030.