QUANTITATIVE ASSAY FOR HEAT SHOCK PROTEIN 70 (HSP70) PROTEIN IN BODY FLUIDS
20230116051 · 2023-04-13
Inventors
Cpc classification
G01N33/577
PHYSICS
International classification
Abstract
Provided is a novel kit and assay for free and lipid-bound (exosomal) Hsp70. In particular, an ELISA is described for determining the level of Hsp70 in sample derived from a body fluid of a subject, characterized in that the level of Hsp70 is determined by an anti-Hsp70 antibody.
Claims
1. An in vitro method for assaying free and lipid-bound (exosomal) heat shock protein 70 (Hsp70) in a sample derived from a body fluid of a subject, characterized in that the level of Hsp70 is determined by an anti-Hsp70 antibody which is antibody cmHsp70.1 as produced by hybridoma cmHsp70.1 (Accession Number DSM ACC2629), or cmHsp70.2 as produced by hybridoma cmHsp70.2 (Accession Number DSM ACC2630), or a HSP70-binding fragment or biotechnological or synthetic derivative thereof.
2. The method of claim 1, wherein the body fluid is blood and the sample comprises serum or plasma, or urine, preferably wherein the serum is separated from blood by centrifugation after allowing to clot.
3. The method of claim 1, wherein an elevated level of Hsp70 compared to a control sample derived from a healthy subject is indicative for a tumor or an inflammatory disease, preferably multiple sclerosis (MS).
4. The method of claim 1, further comprising the steps of (i) diagnosing a tumor in a subject or whether a cancer patient is amenable to the treatment with an anti-tumor agent, in particular Hsp70 specific agent, respectively, wherein an elevated level of Hsp70 compared to a control sample is indicative for the tumor and possibility for the treatment with the agent, respectively; or (ii) monitoring the treatment of a tumor in subject with an anti-tumor agent or determining the therapeutic utility of a candidate anti-tumor agent, comprising determining the level of Hsp70 in the sample obtained from the subject following administration of the agent to the subject, wherein a reduced level of Hsp70 in the sample of the subject compared to a control indicates progress in the treatment and therapeutic utility of the agent, respectively, or (i) diagnosing multiple sclerosis (MS) in a subject or whether a MS patient is amenable to the treatment with an anti-multiple sclerosis agent, in particular Hsp70 specific agent, respectively, wherein an elevated level of Hsp70 compared to a control sample is indicative for multiple sclerosis (MS) and possibility for the treatment with the agent, respectively; or (ii) monitoring the treatment of multiple sclerosis (MS) in subject with an anti-multiple sclerosis agent or determining the therapeutic utility of a candidate anti-neoplastic agent, comprising determining the level of Hsp70 in the sample obtained from the subject following administration of the agent to the subject, wherein a reduced level of Hsp70 in the sample of the subject compared to a control indicates progress in the treatment and therapeutic utility of the agent, respectively.
5. The method of claim 1, wherein the anti-Hsp70 antibody comprises a detectable label, preferably wherein the detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorophore, a heavy metal, a tag or a ligand.
6. The method of claim 5, wherein the anti-Hsp70 antibody is biotinylated.
7. The method of claim 1, wherein the level of Hsp70 is determined by ELISA, preferably sandwich ELISA.
8. The method of claim 7, wherein the anti-Hsp70 antibody is used as a detection reagent and antibody against recombinant Hsp70 protein, preferably polyclonal antibody as a capture reagent.
9. Use of an anti-Hsp70 antibody as defined in claim 1 for assaying free and exosomal Hsp70 in a test sample, preferably the sample is derived from the body fluid of a subject, preferably wherein the sample comprises serum or plasma.
10. An assay kit adapted to carry out the method of claim 1 for determining the presence, progression and/or relapse of a tumor in a subject, comprising means for assaying a serum or plasma sample from the subject for free and liposomal Hsp70 and means for comparing the measured level of Hsp70 to a set of reference standard and/or a control.
11. An assay kit for determining the presence of free and exosomal Hsp70 in a test sample comprising: (i) a detection reagent comprising an anti-Hsp70 antibody as defined in one of claims 1 to 6, optionally conjugated to a detectable label, ligand or tag; (ii) a capture reagent for Hsp70 which is different from the detection reagent of (i); (iii) optionally a detectable label conjugated to a ligand-binding tag; preferably (iv) a calibrated immunoassay standard or control of recombinant human Hsp70; and optionally (v) recommendations for microplates, buffers, diluents, substrates and/or solutions as well as instructions how to perform the assay of any one of claims 1 to 8.
12. The kit of claim 11, wherein (i) the detection anti-Hsp70 antibody is conjugated to a ligand, preferably biotin; (ii) the capture reagent comprises a polyclonal antibody directed against human recombinant Hsp70; (iii) the ligand-binding tag is streptavidin conjugated to an enzyme, preferably horseradish peroxidase (HRP); and/or (iv) the standard comprises a serial dilution of Hsp 70 including at least a high standard of ≥15 ng/ml, preferably ≥25 ng/ml and more preferably ≥50 ng/ml.
13. The kit of claim 10, comprising: (i) a biotinylated detection antibody; (ii) an antibody-coated ELISA well plate with a capture antibody; (iii) concentrated HRP-conjugated streptavidin; (iv) calibrated immunoassay standard recombinant human Hsp70 (v) washing buffer; (vi) assay/sample diluent buffer(s); (vii) ELISA colorimetric reagent, preferably 3, 3¢, 5, 5¢-tetramethylbenzidine (TMB) in buffer solution; and (viii) ELISA stop solution, preferably 0.2 M sulfuric acid.
14. A therapeutic agent for use in the treatment of a patient suffering from a tumor, characterized in that a serum or plasma sample derived from the patient's blood, compared to a control shows an elevated level of Hsp70 as determined in accordance with the method of claim 1, preferably wherein the serum level of Hsp70 is higher than about 7.7 ng/ml, preferably higher than 10 ng/ml.
15. The method of claim 1, the use of claim 9, the kit of claim 10 or the agent of claim 15, wherein the tumor is selected from the group consisting of head and neck cancer, lung cancer, colorectal carcinoma, pancreatic cancer, glioblastoma and hematological malignancies or wherein the disorder in which free and lipid-bound (exosomal) heat shock protein 70 (Hsp70) is detected is selected from the group consisting of an infectious or inflammatory disorder.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0084] The present invention generally relates to a novel assay for the assessment of blood-borne biomarkers for the detection and diagnosis of tumors, including, but not limited to, human head and neck cancer, lung cancer, colorectal carcinoma, pancreatic cancer, glioblastoma and hematological malignancies as well as infectious or inflammatory disorders such as multiple sclerosis (MS). In accordance with the present invention, the practice of the detection and diagnosis of tumors, infections or inflammation is indicated by the presence and localization of certain markers in diseased tissue or cells. In particular, the present invention relates to a method of diagnosing a disease or condition associated with free and lipid-bound (exosomal) Hsp70, for example lipid-associated Hsp70 in exosomes and in the plasma membrane of living tumor cells, which in accordance with the present invention is reflected by an increased levels of lipid-bound (exosomal) Hsp70 and a specific conformational epitope of lipid-bound (exosomal) Hsp70, respectively, in a body fluid, in particular plasma or serum of the subject affected with the disease or condition. The findings of the present invention that the herein used anti-Hsp70 antibodies recognize a specific conformational epitope of lipid-bound (exosomal) Hsp70 further let to the development of a novel method for assaying free and lipid-bound (exosomal) Hsp70 in a sample derived from a body fluid of a subject, preferably an anti-Hsp70 antibody comprising determining the level of free and lipid-bound (exosomal) Hsp70 in a sample derived from a body fluid, preferably plasma or serum of the subject, wherein the increased level of Hsp70 in the sample of the subject compared to a control indicates the presence of or provides the opportunity to monitor a tumor, respectively, wherein the method is characterized in that the level of free and lipid-bound (exosomal) Hsp70 is determined by way of detecting a particular conformational epitope of Hsp70.
[0085] The above disclosure generally describes the present invention. Unless otherwise stated, a term as used herein is given the definition as provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0 19 850673 2. Several documents are cited throughout the text of this specification. Full bibliographic citations may be found at the end of the specification immediately preceding the claims. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application including the background section and manufacturer's specifications, instructions, etc.) are hereby expressly incorporated by reference; however, there is no admission that any document cited is indeed prior art as to the present invention. Furthermore, for the avoidance of any doubt the technical content of the prior art referred to in the background section form part of the disclosure of the present invention and may be relied upon for any embodiment claimed herein. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention.
EXAMPLES
[0086] The Examples 1 to 7 which follow and corresponding
[0087] Blood-borne biomarkers have potential utility for the detection of tumors, monitoring tumor growth and assessing the outcome of anti-tumor therapies [34-36], but many have been criticized for their lack of specificity and selectivity [37-39]. Although heat shock (stress) proteins are commonly considered as being intracellular molecules, elevated levels of Hsp70 have been detected in the supernatants of cultured tumor cells [16] and also in the peripheral circulation of patients with cancer and other diseases [40]. Levels of circulating heat shock proteins, including Hsp70 might therefore serve as useful biomarkers for disease in a number of clinical settings. Extracellular Hsp70 exists either as a free protein, as a protein in association with lipid vesicles such as exosomes [15] and lysosomal endosomes [41] or in the context of cholesterol-rich microdomains [42]. The minor part of extracellular Hsp70 is free Hsp70, which is mostly derived from dying cells. Only combined treatment modalities such as radiation plus hyperthermia have been shown to increase the release of free Hsp70 by dying cells [43]. The major proportion of extracellular Hsp70, which is derived from living, metabolically active tumor cells, is bound to small lipid vesicles such as exosomes, which are actively released by a large variety of human tumor cell types [8, 15]. Lipid-bound, exosomal Hsp70 could therefore be an interesting novel biomarker, which might better reflect the presence and size of viable tumor masses in patients and their response to treatment. However, most commercially available Hsp70 ELISA systems are optimized and validated for the analysis of free Hsp70 protein in buffer systems, and it is therefore not too surprising that different “in-house” and commercially available Hsp70 assays have reported different Hsp70 levels [17-21]. A prerequisite for measuring the absolute levels of Hsp70 in patient blood is therefore an assay that reliably detects both free and liposomal Hsp70. The inventors of the present invention have previously reported on the development and validation of a mouse monoclonal antibody, which is able to bind to a form of Hsp70 that is selectively located in the plasma membrane of viable tumor cells (cmHsp70.1) [29], but also detects free Hsp70 in Western blots. This antibody was validated as a detection reagent for liposomal and free Hsp70 in the lipHsp70 ELISA, which is described in the following Examples. The lipHsp70 ELISA allows the quantification of Hsp70 in serum and plasma, and is less susceptible to matrix effects that are often caused by serum components as demonstrated in Examples 2-4 as well as in
[0088] In summary, the reliability and robustness of the method of the present invention, in particular the lipHsp70 ELISA together with its ability to detect higher levels of Hsp70 in the circulation of patients with cancer and multiple sclerosis (MS) as demonstrated in the Examples and confirmed by Breuninger el al., J. Clin. Cell Immunol. 5 (2014), 264; Gunther el al., Front. Immunol. 6 (2015), 556 makes this method a promising tool for monitoring the presence and size of viable tumor mass, as well as therapeutic outcomes.
[0089] Detailed descriptions of conventional methods, such as those employed herein can be found in the cited literature; see also “The Merck Manual of Diagnosis and Therapy” Seventeenth Ed. ed. by Beers and Berkow (Merck & Co., Inc., 2003). The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. For further elaboration of general techniques useful in the practice of this invention, the practitioner can refer to standard textbooks and reviews in cell biology and tissue culture; see also the references cited in the Examples. General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Non-viral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplitt & Loewy eds., Academic Press 1995); Immunology Methods Manual (Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998). Reagents, cloning vectors and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.
[0090] Material and Methods
[0091] Collection of Plasma and Serum Samples
[0092] Blood samples (9 ml) were taken from 114 healthy human volunteers and patients with head and neck cancer (n=23), lung cancer (n=22), colorectal cancer (n=44), pancreatic cancer (n=46), gliobastoma (n=30) or hematological malignancies (n=32), multiple sclerosis (MS, n=60) with relapsing remitting multiple sclerosis (RRMS, n=32), non-relapsing remitting multiple sclerosis (non-RRMS, n=19), SPMS (n=12), other inflammatory neurological diseases (OIND, n=18) non-inflammatory neurological diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease (NIND, n=22) who provided informed, written consent. Patient characteristics are summarized in tables 2 and 3. Blood was collected in one EDTA KE tube and one serum separator tube (S-Monovette, Sarstedt, Nümbrecht, Germany) and mixed by gently inverting the tube. For plasma separation, EDTA blood was centrifuged at 1,500 g for 15 min. For serum collection, blood was allowed to clot for 15 min at room temperature and serum was separated by centrifugation at 750 g for 10 min. Serum from leukemia patients was allowed to clot for two to three hours and separated by centrifugation at 380 g for 5 min. Serum and plasma were stored in 150 μl aliquots at −80° C. Approval of the study was obtained by the Ethics Committees of the universities that are involved in the study. All procedures were in accordance with the Helsinki Declaration of 1975 as revised in 2008. To validate the ELISA, the interference factors food intake of the blood donor, repeated freezing and thawing and hemolysis of the serum samples were tested. To test the influence of food intake, serum samples were collected from healthy human individuals before and 2 h after intake of a high-fat diet. Repeated freezing and thawing procedures of up to ten cycles were performed on the serum samples. In order to study the impact of hemolysis on the assay precision, erythrocytes were isolated from the blood of healthy donors by density gradient centrifugation using LSM1077 (PAA, Colbe, Germany). Erythrocytes were lysed by applying shear stress and the corresponding serum samples were spiked with increasing amounts of the lysed erythrocytes. The hemoglobin content of the spiked serum samples was analyzed by measuring the absorbance of the samples at 562 nm, 578 nm and 598 nm. The hemoglobin concentration was calculated as described elsewhere [30].
[0093] Recombinant Hsp70
[0094] His-tagged Hsp70 protein was isolated from a Sf9 insect cell system (Orbigen, San Diego, Calif., USA). Briefly, Sf9 cells were transfected with baculovirus containing cDNA coding for human Hsp70 protein with a His-tag on the N terminus (Orbigen, San Diego, Calif., USA). Cell lysates were loaded on Ni-sepharose columns (GE Healthcare, Chalfont St. Giles, UK) in binding buffer (20 mM sodium phosphate, 0.5 M NaCl, pH 7.4). His-tagged Hsp70 was eluted with increasing concentration of elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4). Fractions containing high amounts of Hsp70 were pooled and the buffer was exchanged using PD-desalting columns (GE Healthcare, Chalfont St. Giles, UK). Protein amount was determined using a BCA protein kit (Pierce, Thermo, Rockford, Ill., USA) and aliquots were stored at −20° C. As a further control, recombinant Hsp70 without His-tag was purchased from Stressgen (ADI-NSP-555, Enzo Life Sciences, Farmingdale, N.Y., USA)
[0095] Antibody Biotinylation
[0096] The monoclonal mouse antibody cmHsp70.1 (multimmune, Munich, Germany) was biotinylated using EZ-link sulfo NHS-LC-biotin (Thermo, Rockford, Ill., USA). The antibody was incubated with a 40-fold molar excess of biotin for 1 h at room temperature. The remaining free biotin was removed using Zeba spin desalting columns (Thermo). Protein concentration was determined with a BCA protein kit (Pierce, Thermo) and aliquots were stored at 4° C.
[0097] Preparation of Lipid Vesicles
[0098] Lipid vesicles were prepared as described previously [31]. Briefly, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, Avanti Polar Lipids, Alabaster, Ala., USA) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS, Avanti Polar Lipids), dissolved in chloroform, were mixed in a molar ratio of 8:2 and dried under nitrogen gas. Lipids were rehydrated in 25 mM Tris/HCl pH 7.4, 250 mM NaCl at 10 mg lipid per 1 ml of buffer for 1.5 h at room temperature. The Mini Extruder (Avanti Polar Lipids) was assembled according to the manufacturer's instructions and the lipid suspension was pushed through a 100 nm polycarbonate membrane 13 times to achieve uniformly sized vesicles. Vesicle concentration was adjusted to 1 mg/ml with 1 mM Bis/Tris buffer pH 7.4 and 100 μl of the suspension were incubated with 1 μg of recombinant Hsp70 protein for 30 min at room temperature. After addition of an equal amount of ultrapure water the vesicle suspension was centrifuged at 200,000 g for 2 h at 4° C. in an ultracentrifuge. The pellet was resuspended in PBS and stored at 4° C. Hsp70-containing lipid vesicles and empty control vesicles were used for further tests within 24 hours.
[0099] SDS-PAGE and Western Blot Analysis
[0100] Lysed Hsp70-containing vesicles and defined concentrations of recombinant Hsp70 were loaded onto a 10% acrylamide gel under reducing conditions and blotted onto nitrocellulose membranes. The protein was detected using the monoclonal antibody cmHsp70.1 (multimmune, Munich, Germany). Bound antibodies were visualized using a horseradish peroxidase-conjugated secondary antibody (Dako, Glostrup, Denmark) and a chemiluminescence developing kit (Pierce, Thermo, Rockford, Ill., USA). The Hsp70 protein content of the vesicles was quantified by densitometry and compared to the signals generated by defined amounts of a recombinant Hsp70 protein that were run on the same gel.
[0101] Liphsp70 ELISA
[0102] 96-well MaxiSorp Nunc-Immuno plates (Thermo, Rochester, N.Y.) were coated overnight with 2 μg/ml rabbit polyclonal antibody (Davids, Biotechnologie, Regensburg, Germany), directed against human recombinant Hsp70, in sodium carbonate buffer (0.1 M sodium carbonate, 0.1 M sodium hydrogen carbonate, pH 9.6). After washing three times with phosphate buffered saline (PBS, Life Technologies, Carlsbad, Calif., USA) with 0.05% Tween-20 (Calbiochem, Merck, Darmstadt, Germany), the wells were blocked with 2% milk powder (Carl Roth, Karlsruhe, Germany) in PBS for 1.5 h at 27° C. Following another washing step, serum samples diluted 1:5 in CrossDown Buffer (Applichem, Chicago, Ill., USA) were added to the wells for 2 h at 27° C. Then the wells were washed again and incubated with 4 μg/ml of the biotinylated mouse monoclonal antibody cmHsp70.1 (multimmune, Munich, Germany) in 2% milk powder in PBS for 2 h at 27° C. Finally, after another washing step, 0.2 μg/ml horseradish peroxidase-conjugated streptavidin (Pierce, Thermo, Rockford, Ill., USA) in 1% bovine serum albumin (Sigma-Aldrich, St. Louis, Mo., USA) was added for 1 h at 27° C. Binding was quantified by adding substrate reagent (R&D Systems, Minneapolis, Minn., USA) for 30 min at 27° C. and absorbance was read at 450 nm, corrected by absorbance at 570 nm, in a Microplate Reader (BioTek, Winooski, Vt., USA). An Hsp70 eight point standard was included into each ELISA test using 0-50 ng/ml recombinant Hsp70 diluted in CrossDown Buffer. As a control, Hsp70 serum levels were also determined using the DuoSet® IC Human/Mouse/Rat Total Hsp70 ELISA (R&D Systems, Minneapolis, Minn., USA) following the manufacturer's protocol.
[0103] ELISA Validation
[0104] Linearity was evaluated according to the Clinical Laboratory Standards Institute (CLSI) guideline EP6-A. Briefly, six solutions of different Hsp70 concentrations were analyzed with the ELISA and their relative concentration was plotted against the system output (concentration according to ELISA measurement). First-, second- and third-order models were then fitted to the data and a t-test was applied to the non-linear coefficients of the second- and third-order models using SigmaPlot 12.5 software. If none of the non-linear coefficients were significant (p>0.05), the dataset was considered linear. To determine intra-assay precision, control serum samples from two different donors were run in 20 replicates on a single plate. Inter-assay precision was assessed by running control serum samples in duplicate on three different days. The concentration was determined for each sample and the Co-efficients of Variation (CVs) were calculated. The Limit of Detection (LoD) was established according to the Clinical Laboratory Standards Institute (CLSI) guideline EP17-A as summarized by Armbruster and Pry [32]. Briefly, OD values of 36 blank samples and 36 samples with a low Hsp70 concentration (0.63 ng/ml) were converted to concentrations by back-calculating against the standard curve. The Limit of Blank (LoB) was calculated according to the following equation: LoB=μ+1.645 σ, where μ and σ are the mean and standard deviation of the blank measurements, respectively. Finally, the Limit of Detection (LoD) was calculated according to the following equation: LoD=LoB+1.645 σS, where σS is the standard deviation of the low sample measurements. Recovery was assessed by spiking defined amounts of the respective standard Hsp70 into 1:5 diluted serum samples. The Hsp70 concentration of the serum alone was subtracted from the measured value and recovery was calculated as the ratio of observed concentration versus expected.
[0105] Deposit of Hybridoma Cell Lines
[0106] The material listed below was deposited with the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7b formerly Mascheroder Weg lb, D-38124 Braunschweig, Germany on Nov. 14, 2003, and assigned Accession Number DSM ACC2629 and DSM ACC2630. The deposits were made under the provisions of the Budapest Treaty on the International Recognition of Deposited Microorganisms for the Purposes of Patent Procedure and Regulations thereunder (Budapest Treaty). Maintenance of a viable culture is assured for thirty years from the date of deposit. The hybridomas have been made available by the DSMZ under the terms of the Budapest Treaty, and subject to an agreement between the applicant and the DSMZ which assures unrestricted availability of the deposited hybridomas and progeny thereof to the public upon issuance of a pertinent US and European patent; see EP 1 706 423 B1/B8, EP 2 070 947 B1, U.S. Pat. No. 7,700,737 B2 and U.S. Pat. No. 8,440,188 B2. The applicant of the present application has agreed that if a culture of the material(s) on deposit should die or be lost or destroyed when cultivated under suitable conditions, to replace the material(s) promptly upon notification with another of the same.
[0107] i. Hybridoma: cmHsp70.1, Date: Nov. 14, 2003, Accession Number DSM ACC2629
[0108] ii. Hybridoma: cmHsp70.2, Date: Nov. 14, 2003, Accession Number DSM ACC2630
Example 1: Hsp70 ELISA Validation: Calibration Curve, Intra- and Inter-Assay Precision, Limit of Detection and Recovery
[0109] Hsp70 is frequently overexpressed in tumor cells and can be actively released in lipid vesicles by viable tumor cells. Therefore, elevated Hsp70 serum levels have potential utility as biomarkers for the detection of viable tumor mass and to measure the response to therapeutic interventions. However, commercially available Hsp70 ELISAs are neither optimized for the measurement of serum Hsp70 nor of lipid-associated Hsp70. Therefore, for the novel lipHsp70 ELISA, blocking reagents, antibody combinations and serum diluent had to be optimized. A 1:5 dilution of the serum samples was found to be optimal to reduce unfavorable matrix effects, which are observed with undiluted serum. As an internal control, a commercial ELISA was used in parallel. A representative calibration curve with eight measuring points and the fitting equation are shown in
TABLE-US-00001 TABLE 1 Assay performance characteristics of the lipHsp70 ELISA. Parameters Performance Linear range (ng/ml) 0.36-17.41 Intra-assay precision (% CV) 5.2-8.1 Inter-assay precision (% CV) 1.0-18.0 Recovery (%): Buffer/Serum 101 ± 3/78 ± 3 Limit of Detection (ng/ml) 0.31
Example 2: Hsp70 Serum Levels in Healthy Human Volunteers
[0110] Serum samples from 114 healthy human volunteers at different ages (age range 20-74, Table 2) were analyzed to determine the basal Hsp70 levels in blood. To minimize matrix effects, serum was diluted 1:5 in CrossDown Buffer prior to analysis. Significantly higher basal levels (6.4±2.7 ng/ml) could be detected with the lipHsp70 ELISA compared to the control ELISA (2.8±1.3 ng/ml) (
Example 3: Comparison of the Detection of Hsp70 in Serum and Plasma
[0111] To test whether the lipHsp70 ELISA is suitable for measuring Hsp70 levels in both serum and plasma, samples were taken in parallel from four healthy donors and measured using the lipHsp70 ELISA. For this experiment, donors with different basal levels of Hsp70 were chosen. For all four donors, the Hsp70 levels in plasma did not differ significantly from those in the corresponding serum samples. These data indicate that both serum and plasma can be used to measure Hsp70 levels with the lipHsp70 ELISA (
Example 4: Influence of Interference Factors on the Detection of Hsp70
[0112] Different factors in the donor's lifestyle or in the sample preparation could have an impact on the measurement of Hsp70 in serum [33]. In order to determine the robustness of the lipHsp70 ELISA, the influence of food intake of the donor, repeated freezing and thawing of the serum sample and hemolysis was tested. Serum samples from seven healthy individuals were taken before and two hours after intake of a high-fat diet. In all donors, Hsp70 serum levels did not differ significantly before and after food intake (
Example 5: Detection of Lipid-Bound Hsp70
[0113] To test the ability of both ELISA tests to measure lipid-bound Hsp70, artificial POPS/POPC lipid vesicles were produced and loaded with recombinant Hsp70. The amount of lipid-associated Hsp70 that was determined with the lipHsp70 ELISA showed an excellent correlation with the Hsp70 concentration that was determined by Western blotting (data not shown). However, a comparison of the levels of liposomal Hsp70 using the lipHsp70 ELISA and the control ELISA revealed large differences. The recovery of liposomal Hsp70 using the lipHsp70 ELISA was 76±5%, whereas that of the control ELISA was only 7±1%. These data indicate that the detection of lipid-associated Hsp70 was more than 10-fold better with the lipHsp70 ELISA than with the control ELISA (
Example 6: Hsp70 Serum Levels in Patients with Different Tumor Entities
[0114] Hsp70 levels were measured in the serum of patients with head and neck cancer (n=23), lung cancer (n=22), colorectal cancer (n=44), pancreatic cancer (n=46), glioblastoma (n=30) or hematological malignancies (n=32) (Table 3A-F) and compared to the Hsp70 levels in healthy donors (n=114). The mean Hsp70 serum levels in patients of all tumor entities were significantly higher than those of the healthy donors (
Example 7: Hsp70 Serum Levels in Patients with Multiple Sclerosis, Non-Inflammatory Neurological Diseases and Other Inflammatory Neurological Diseases
[0115] Hsp70 levels were measured in the serum of patients with Multiple Sclerosis (MS, n=53), non-inflammatory neurological diseases (NIND; n=21) and other inflammatory neurological diseases (OIND, n=16) (Table 4A-C) and compared to the Hsp70 levels in healthy donors (n=100) using a commercially available Hsp70 ELISA as internal control and the lipHsp70 ELISA. The mean Hsp70 serum levels in patients of MS and OIND were significantly higher than those of the patients with NIND and the healthy donors. Furthermore, a comparison of the levels of liposomal Hsp70 using the lipHsp70 ELISA and the control ELISA revealed large differences. The recovery of liposomal Hsp70 using the lipHsp70 ELISA in patients with MS NIND and with OIND were 85±5%, whereas that of the control ELISA was only 32±9%, (
TABLE-US-00002 TABLE 2 Age, gender and Hsp70 levels of healthy donors and patients. Head & Healthy neck Lung Colorectal Pancreatic Hematological donors cancer cancer carcinoma cancer Glioblastoma malignancies Number (n) 114 23 22 44 46 30 32 Gender (M/F) 67/47 21/2 16/6 26/18 26/20 14/16 25/7 Age Mean 42.9 62.5 66.1 64.2 69.8 56.2 41.7 Range 20-74 36-83 48-88 29-81 44-90 25-77 19-64 SD 14.6 12.2 10.3 13.0 40.6 14.1 12.2 Median 41.5 61.0 66.5 67.5 73.0 59.0 42.0 lipHsp70 Mean Hsp70 6.4 12.4 16.8 11.0 14.8 67.6 11.1 ELISA (ng/ml) SD 2.7 6.1 16.2 5.2 7.3 37.5 9.0 ROC AUC (CI 95%) — 0.80 0.78 0.81 0.86 0.92 0.62 p-value — <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.03 Sensitivity (%) — 78 73 70 85 90 41 Specificity (%) — 75 75 75 75 75 75 Abbreviations: AUC: Area Under the Curve; M: Male; F: Female; CI: Confidence Interval; ROC: Receiver Operating Characteristic; SD: Standard Deviation
TABLE-US-00003 TABLE 3A Clinico-pathological characteristics of head and neck cancer patients. Patient # Tumor location Histology Staging Grading 1 hypopharynx SCCHN T1 N0 M0 G2 2 hypopharynx SCCHN T3 N2b M0 G3 3 hypopharynx SCCHN T1 N0 M0 G3 4 larynx SCCHN T3 N2 M0 G2 5 larynx SCCHN T3 N0 M0 G3 6 larynx SCCHN T4 N1 M0 G3 7 larynx SCCHN T3 N0 M0 G2 8 larynx SCCHN T4a N0 M0 G3 9 naso/oro/hypo pharynx SCCHN T4c N2c M0 G2 10 naso/oro/hypo pharynx SCCHN T3-4a N2c M0 G3 11 nasopharynx SCCHN T2 N0 M0 G1 12 oral cavity SCCHN T1 N0 M0 G2 13 oro/hypopharynx SCCHN T2 N2b M0 G3 14 oro/hypopharynx SCCHN T2 N2a M0 G2 15 oropharynx SCCHN T4a N2c M0 G3 16 oropharynx SCCHN T3 N0 M0 G3 17 oropharynx SCCHN T1 N2b M0 G2 18 oropharynx SCCHN T1a N0 M0 G2 19 oropharynx SCCHN T4 N0 M0 G2 20 oropharynx SCCHN T2 N2b M0 G3 21 oropharynx SCCHN T4 N0 M0 G3 22 oropharynx SCCHN T2 N1 M0 G3 23 paranasal sinus SCCHN T2 N0 M0 G3 Abbreviations: SCCHN: Squamous Cell Carcinoma of the Head and Neck
TABLE-US-00004 TABLE 3B Clinicopathological characteristics of lung cancer patients. Patient # Histology Histology Staging Grading 1 NSCLC adeno SCCHN T2 N2 M1 G3 2 NSCLC adeno SCCHN T4 N2 M0 G2 3 NSCLC adeno SCCHN T2 N2 M0 G3 4 NSCLC adeno SCCHN T3 N3 M1 G3 5 NSCLC adeno SCCHN T4 N2 M1 G3 6 NSCLC adeno SCCHN T2 N0 M0 G2 7 NSCLC adeno SCCHN T1 N3 M0 G3 8 NSCLC adeno SCCHN T2 N2 M1 G2 9 NSCLC squamous SCCHN T3 N3 M1 G3 10 NSCLC squamous SCCHN T3 N3 M1 G3 11 NSCLC squamous SCCHN T4 N0 M0 G2 12 NSCLC squamous SCCHN T1 N0 M0 G3 13 NSCLC squamous SCCHN T4 N2 M0 G3 14 NSCLC squamous SCCHN T4 N3 M1 G3 15 NSCLC squamous SCCHN T3 N3 M0 G3 16 NSCLC squamous SCCHN T3 N2 M1 G3 17 NSCLC squamous SCCHN T4 N2 M1 G3 18 NSCLC squamous SCCHN T3 N2 M1 G2 19 SCLC SCCHN limited — 20 SCLC SCCHN extensive — 21 SCLC S( C HN extensive — 23 SCLC SCCHN extensive — Abbreviations: NSCLC: Non-Small Cell Lung Cancer; SCLC: Small Cell Lung Cancer
TABLE-US-00005 TABLE 3C Clinico-pathological characteristics of colorectal carcinoma patients. Abbreviations: C: Staging by Clinical Examination; u: Staging by Ultrasonography Patient # Staging Grading 1 uT3 uN+ MX G2 2 uT3 uN0 cM0 G2 3 cT3 cN+ cM0 G2 4 cT3 cN1 cM0 G2 5 cT3 cN1 cM1 G2 6 cT3 cN0 cM0 G2 7 cT3 cN0 cM0 G2 8 uT3 uN0 cM0 G2 9 cT3 cN2 cM0 G2 10 cT3 uN0/cN+ M0 11 cT3 cN+ cM0 G2 12 uT3 cNX cM0 G2 13 cT3 cN1 cM0 G2 14 uT3 uN1 cM0 G2 15 uT3 cN0 cM0 G2 16 cT2 cN1 cM0 G2 17 cT3 cN2 MX G2 18 cT3 cN2 cM0 G2 19 cT3a cN2 cM0 20 cT3-4 cN+ cM0 G2 21 cT3 cN0 cM0 G2 22 cT3 cN1 cM0 G3 23 uT3c N0 cM0 G2 24 uT3 uN0 cM0 G2 25 cT4 cN1 cM0 G2 26 uT3 uN2 cM0 G2 27 uT3 uN1 cM0 G2 28 uT3 uN1 cM0 G2 29 uT3 N+ cM0 G2 30 cT3 cN0 cM0 31 cT3 cN1 cM0 G2 32 cT3 cN2 cM1 G2 33 T3 N2 M0 G2 34 cT3 cN0 M0 G2 35 uT3 uN0 cM0 36 cT3 cN+ cM0 37 cT3 cN0 cM0 38 cT2 uN0 M0 G2 39 cT3 cN+ cM0 G2 40 uT3 uN1 cM0 G2 41 uT3b-4 cN+ cM0 42 uT3 uN1 cM0 G2 43 cT3 cN+ cM0 G2 44 cT3 uN1 cM0 G2
TABLE-US-00006 TABLE 3D Clinico-pathological characteristics of pancreatic cancer patients. Patient # Tumor Location Staging Grading 1 body T3 N0 M0 G2 2 body/tail T2 N1 M1 — 3 body/tail T4 N2 M1 — 4 body/tail T4 N2 M1 — 5 body/tail T1 N1 M1 — 6 body/tail T4 N2 M1 — 7 body/tail T3 N1 M0 — 8 body/tail T4 N2 M1 — 9 body/tail T4 N3 M1 — 10 head T3 N1 M0 G2 11 head T4 N1 M0 G2 12 head T3 N0 M0 G2 13 head T3 N1 M0 G1 14 head T3 N1 M0 G2 15 head T3 N0 M0 G1 16 head T3 N1 M0 G2 17 head T1 N0 M0 G3 18 head T3 N1 M0 G3 19 head T3 N1 M0 G3 20 head T3 N1 M0 G3 21 head T4 N2 M1 — 22 head T3 N1 M0 — 23 head T3 N2 M1 — 24 head T3 N2 M1 — 25 head T2 N1 M0 — 26 head T3 N2 M1 — 27 head T2 N1 M1 — 28 head T2 N0 M0 — 29 head T3 N1 M0 — 30 head T2 N0 M0 — 31 head T2 N1 M0 — 32 head T4 N2 M1 — 33 head T2 N1 M1 — 34 head T3 N1 M1 — 35 head/body/tail T4 N2 M1 — 36 head/tail T1 N0 M0 G1 37 head/uncinate process T2 N0 M0 — 38 head/uncinate process T3 N2 M1 — 39 head/uncinate process T2 N0 M0 — 40 head/uncinate process T4 N2 M1 — 41 tail T4 N1 M0 G3 42 tail T3 N1 M0 G2 43 tail T3 N0 M0 G2 44 tail T4 N3 M1 — 45 uncinate process T3 N1 M0 — 46 uncinate process T2 N0 M0 —
TABLE-US-00007 TABLE 3E Clinico-pathological characteristics of glioblastoma patients. Patient # Tumor Origin Grading 1 primary G4 2 primary G4 3 primary G4 4 primary G4 5 primary G4 6 primary G4 7 primary G4 8 primary G4 9 primary G4 10 primary G4 11 primary G4 12 primary G4 13 primary G4 14 primary G4 15 primary G4 16 primary G4 17 primary G4 18 primary G4 19 primary G4 20 primary 21 secondary G4 22 secondary G4 23 secondary G4 24 secondary G4 25 secondary G4 26 secondary G4 27 secondary G4 28 secondary G4 29 secondary G4 30 secondary
TABLE-US-00008 TABLE 3F Clinico-pathological characteristics of patients with hematological malignancies. Abbreviations: ALL: Acute Lymphoid Leukemia; AML: Acute Myeloid Leukemia; BAL: Biphenotypic Acute Leukemia; CML: Chronic Myeloid Leukemia; MDS: Myelodysplastic Syndrome; MPS: Myeloproliferative Syndrome; NHL: Non-Hodgkin Lymphoma. Patient # Hematological disease 1 ALL 2 AML 3 AML 4 AML 5 AML 6 AML 7 AML 8 AML 9 AML 10 AML 11 AML 12 BAL 13 CML 14 Hodgkin lymphoma 15 Hodgkin lymphoma 16 Hodgkin lymphoma 17 MDS & MPS 18 Multiple myeloma 19 Multiple myeloma 20 Multiple myeloma 21 MDS 22 MDS 23 MDS 24 MDS 25 MDS 26 MDS 27 MDS 28 NHL 29 NHL 30 NHL 31 NHL
TABLE-US-00009 TABLE 4A Clinico-pathological characteristics of patients with MS (n = 53; 16 males, 37 females). Subtype of MS Numbers Clinico-pathological characteristics n = 29; 6 males, 23 females of patients with relapsing remitting Multiple Sclerosis (RRMS) Clinico-pathological characteristics n = 11; 6 males, 5 females of patients with non-relapsing-remitting Multiple Sclerosis (non RRMS) Clinico-pathological characteristics n = 13; 4 males, 9 females of patients with clinically isolated syndrome (CIS)
TABLE-US-00010 TABLE 4B Clinico-pathological characteristics of patients with non-inflammatory neurological diseases (NIND). Total n = 21: 7 males, 14 females. Symptoms Numbers Headache n = 15 Facial pain n = 1 Pseudo tumor n = 3 Acute lumboischialgy n = 2
TABLE-US-00011 TABLE 4C Clinico-pathological characteristics of patients with other inflammatory neurological diseases (OIND). Total n = 16; 9 males, 7 females. Symptoms Numbers Encephalitis n = 11 Meningitis (bacterial viral) n = 9 Meningoencephalitis n = 6
REFERENCES
[0116] 1. Lindquist S, Craig E A (1988) The heat-shock proteins. Annu Rev Genet 22: 631-677. [0117] 2. Hartl F U (1996) Molecular chaperones in cellular protein folding. Nature 381: 571-579. [0118] 3. Daugaard M, Rohde M, Jäättelä M (2007) The heat shock protein 70 family: Highly homologous proteins with overlapping and distinct functions. FEBS Lett 581: 3702-3710. [0119] 4. De Maio A (1999) Heat shock proteins: facts, thoughts, and dreams. Shock 11: 1-12. [0120] 5. Jäättelä M (1999) Escaping cell death: survival proteins in cancer. Exp Cell Res 248: 30-43. [0121] 6. MulthoffG, Botzler C, Wiesnet M, Müller E, Meier T, et al. (1995) A stress-inducible 72-kDa heat-shock protein (HSP72) is expressed on the surface of human tumor cells, but not on normal cells. Int J Cancer 61: 272-279. [0122] 7. Gehrmann M, Liebisch G, Schmitz G, Anderson R, Steinem C, et al. (2008) Tumor-specific Hsp70 plasma membrane localization is enabled by the glycosphingolipid Gb3. PLoS One 3: e1925. [0123] 8. Vega V L, Rodriguez-Silva M, Frey T, Gehrmann M, Diaz J C, et al. (2008) Hsp70 translocates into the plasma membrane after stress and is released into the extracellular environment in a membrane-associated form that activates macrophages. J Immunol 180: 4299-4307. [0124] 9. Gehrmann M, Marienhagen J, Eichholtz-Wirth H, Fritz E, Ellwart J, et al. (2005) Dual function of membrane-bound heat shock protein 70 (Hsp70), Bag-4, and Hsp40: protection against radiation-induced effects and target structure for natural killer cells. Cell Death Differ 12: 38-51. [0125] 10. Arispe N, Doh M, Simakova O, Kurganov B, De Maio A (2004) Hsc70 and Hsp70 interact with phosphatidylserine on the surface of PC12 cells resulting in a decrease of viability. FASEB J 18: 1636-1645. [0126] 11. Botzler C, Li G, Issels R D, MulthoffG (1998) Definition of extracellular localized epitopes of Hsp70 involved in an NK immune response. Cell Stress Chaperones 3: 6-11. [0127] 12. Multhoff G, Hightower L E (2011) Distinguishing integral and receptor-bound heat shock protein 70 (Hsp70) on the cell surface by Hsp70-specific antibodies. Cell Stress Chaperones 16: 251-255. [0128] 13. Hantschel M, Pfister K, Jordan A, Scholz R, Andreesen R, et al. (2000) Hsp70 plasma membrane expression on primary tumor biopsy material and bone marrow of leukemic patients. Cell Stress Chaperones 5: 438-442. [0129] 14. Pfister K, Radons J, Busch R, Tidball J G, Pfeifer M, et al. (2007) Patient survival by Hsp70 membrane phenotype. association with different routes of metastasis. Cancer 110: 926-935. [0130] 15. Gastpar R, Gehrmann M, Bausero M A, Asea A, Gross C, et al. (2005) Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells. Cancer Res 65: 5238-5247. [0131] 16. De Maio A (2011) Extracellular heat shock proteins, cellular export vesicles, and the Stress Observation System: a form of communication during injury, infection, and cell damage. It is never known how far a controversial finding will go! Dedicated to FerruccioRitossa. Cell Stress Chaperones 16: 235-249. [0132] 17. Lichtenauer M, Zimmermann M, Nickl S, Lauten A, Goebel B, et al. (2014) Transient hypoxia leads to increased serum levels of heat shock protein-27, -70 and caspase-cleaved cytokeratin 18. Clin Lab 60: 323-328. [0133] 18. Lebherz-Eichinger D, Ankersmit H J, Hacker S, Hetz H, Kimberger O, et al. (2012) HSP27 and HSP70 serum and urine levels in patients suffering from chronic kidney disease. Clin Chim Acta 413: 282-286. [0134] 19. Fredly H, Reikvam H, Gjertsen B T, Bruserud O (2012) Disease-stabilizing treatment with all-trans retinoic acid and valproic acid in acute myeloid leukemia: serum hsp70 and hsp90 levels and serum cytokine profiles are determined by the disease, patient age, and anti-leukemic treatment. Am J Hematol 87: 368-376. [0135] 20. Pockley A G, Shepherd J, Corton J M (1998) Detection of heat shock protein 70 (Hsp70) and anti-Hsp70 antibodies in the serum of normal individuals. Immunol Invest 27: 367-377. [0136] 21. Njemini R, Demanet C, Mets T (2005) Comparison of two ELISAs for the determination of Hsp70 in serum. J Immunol Methods 306: 176-182. [0137] 22. Bausero M A, Gastpar R, Multhoff G, Asea A (2005) Alternative mechanism by which IFN-gamma enhances tumor recognition: active release of heat shock protein 72. J Immunol 175: 2900-2912. [0138] 23. Mathivanan S, Ji H, Simpson R J (2010) Exosomes: extracellular organelles important in intercellular communication. J Proteomics 73: 1907-1920. [0139] 24. Théry C, Ostrowski M, Segura E (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9: 581-593. [0140] 25. Record M, Subra C, Silvente-Poirot S, Poirot M (2011) Exosomes as intercellular signalosomes and pharmacological effectors. Biochem Pharmacol 81: 1171-1182. [0141] 26. Anand P K (2010) Exosomal membrane molecules are potent immune response modulators. CommunIntegr Biol 3: 405-408. [0142] 27. Kharaziha P, Ceder S, Li Q, Panaretakis T (2012) Tumor cell-derived exosomes: a message in a bottle. Biochim Biophys Acta 1826: 103-111. [0143] 28. Botzler C, Schmidt J, Luz A, Jennen L, Issels R, et al. (1998) Differential Hsp70 plasma-membrane expression on primary human tumors and metastases in mice with severe combined immunodeficiency. Int J Cancer 77: 942-948. [0144] 29. Stangl S, Gehrmann M, Riegger J, Kuhs K, Riederer I, et al. (2011) Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody. Proc Natl Acad Sci USA 108: 733-738. [0145] 30. Kahn S E, Watkins B F, Bermes E W Jr (1981) An evaluation of a spectrophotometric scanning technique for measurement of plasma hemoglobin. Ann Clin Lab Sci 11: 126-131. [0146] 31. Schilling D, Gehrmann M, Steinem C, De Maio A, Pockley A G, et al. (2009) Binding of heat shock protein 70 to extracellular phosphatidylserine promotes killing of normoxic and hypoxic tumor cells. FASEB J 23: 2467-2477. [0147] 32. Armbruster D A, Pry T (2008) Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev 29 Suppl 1: S49-52. [0148] 33. Wenk R E (1998) Mechanism of interference by hemolysis in immunoassays and requirements for sample quality. Clin Chem 44: 2554. [0149] 34. Madu C O, Lu Y (2010) Novel diagnostic biomarkers for prostate cancer. J Cancer 1: 150-177. [0150] 35. HiralesCasillas C E, Flores Fernandez J M, Padilla Camberos E, Herrera López E J, Leal Pacheco G, et al. (2014) Current status of circulating protein biomarkers to aid the early detection of lung cancer. Future Oncol 10: 1501-1513. [0151] 36. Alvarez-Chaver P, Otero-Estevez O, Paez de la Cadena M, Rodriguez-Berrocal F J, Martinez-Zorzano V S (2014) Proteomics for discovery of candidate colorectal cancer biomarkers. World J Gastroenterol 20: 3804-3824. [0152] 37. Loeb S (2014) Guideline of guidelines: prostate cancer screening. BJU Int 114: 323-325. [0153] 38. Grunnet M, Sorensen J B (2012) Carcinoembryonic antigen (CEA) as tumor marker in lung cancer. Lung Cancer 76: 138-143. [0154] 39. Hayes J H, Barry M J (2014) Screening for prostate cancer with the prostate-specific antigen test: a review of current evidence. JAMA 311: 1143-1149. [0155] 40. Pockley A G, Henderson B, and Multhoff G (in press) Extracellular cell stress proteins as biomarkers of human disease. Biochem Soc Trans [0156] 41. Mambula S S, Calderwood S K (2006) Heat shock protein 70 is secreted from tumor cells by a nonclassical pathway involving lysosomal endosomes. J Immunol 177: 7849-7857. [0157] 42. Broquet A H, Thomas G, Masliah J, Trugnan G, Bachelet M (2003) Expression of the molecular chaperone Hsp70 in detergent-resistant microdomains correlates with its membrane delivery and release. J Biol Chem 278: 21601-21606. [0158] 43. Schildkopf P, Frey B, Ott O J, Rubner Y, Multhoff G, et al. (2011) Radiation combined with hyperthermia induces HSP70-dependent maturation of dendritic cells and release of pro-inflammatory cytokines by dendritic cells and macrophages. Radiother Oncol 101: 109-115. [0159] 44. Brown D A, London E (2000) Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem 275: 17221-17224.