WO2008100161A1 - Essai de résonance de plasmon de surface pour stéroïdes - Google Patents

Essai de résonance de plasmon de surface pour stéroïdes Download PDF

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Publication number
WO2008100161A1
WO2008100161A1 PCT/NZ2008/000021 NZ2008000021W WO2008100161A1 WO 2008100161 A1 WO2008100161 A1 WO 2008100161A1 NZ 2008000021 W NZ2008000021 W NZ 2008000021W WO 2008100161 A1 WO2008100161 A1 WO 2008100161A1
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Prior art keywords
antibody
chain
steroid
assay
cortisol
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WO2008100161A9 (fr
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John Stanton Mitchell
Timothy Edward Lowe
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Horticulture and Food Research Institute of New Zealand Ltd
New Zealand Institute for Bioeconomy Science Ltd
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Horticulture and Food Research Institute of New Zealand Ltd
New Zealand Institute for Plant and Food Research Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance

Definitions

  • the present invention provides a method for the measurement of steroids in an animal.
  • Steroid hormones are important regulators of the metabolism. Altered levels of these hormones are found in metabolic disorders, for example Cortisol is elevated in Cushing's syndrome and reduced in Addison's disease. Steroid hormones levels also vary in mammals with no such disorders. For example Cortisol levels show diurnal variation. In addition steroid hormone levels may reflect other factors such as stress. Physical and emotional stress increase serum Cortisol.
  • Radioimmunoassays for steroid hormones in blood plasma or serum are well known. While these are valuable in diagnosis of hormone-related disorders, they are not practicable for regular measurements of the hormones in healthy individuals.
  • the invention provides a method for measurement of the level of a steroid hormone in a biological sample comprising:
  • the steroid hormone is selected from Cortisol, testosterone and dehydroepiandrosterone.
  • the immobilised steroid hormone is joined to the linker through carbon-4 of the steroid.
  • the macromolecule or nanoparticle is a protein.
  • proteins preferred are metal-binding proteins, for example haemoproteins.
  • metal-binding proteins for example haemoproteins.
  • horseradish peroxidase Use of horseradish peroxidase has been found to have the advantage of providing reduced non-specific binding.
  • the macromolecule or nanoparticle is a gold particle.
  • antibody also includes not only intact immunoglobulin molecules but also the well- known active fragments F(ab') 2 , and Fab.
  • F(ab') 2 Fab fragments which lack the Fc fragment of intact antibody, Fv, single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity.
  • binding partner binds to the hapten of interest without substantial cross reactivity to other species in the sample to enable a meaningful detection result to be obtained.
  • the surface to which the steroid is bound may be the surface of any suitable substantially insoluble bulky support forming part of a sensor that permits attachment of a linker.
  • the surface may include but is not limited to a chip surface, gels (e.g. cross-linked chromatography gels) and a solid support as well as any other support well known in the art.
  • suitable immobilisation substrates suitable for the practice of the present invention include:
  • insoluble polymeric materials such as polystyrene, polypropylene, polyester, polyacrylonitrile, polyvinyl chloride, polyvinylidene, polysulfone, polyacrylamide, cellulose, cellulose nitrate, cross-linked dextrans, fluorinated resins, agarose, crosslinked agarose, and polysaccharides etc;
  • metal gold, silver or platinum
  • metal strips and metal beads
  • test tubes test tubes, microtiter plates, dipsticks, lateral flow devices, resins, PVC, latex beads and nitrocellulose.
  • the senor is based around a surface of an optical biosensor chip.
  • a preferred sensor chip is a BIAcore CM5 chip.
  • a further preferred sensor chip is a Spreeta chip, available, for example, from ICx Technologies.
  • the linker is typically 4 to 50 atoms in length, preferably 10 to 50, more preferably 10 to 30 atoms in length excluding any bridging groups.
  • linkers suitable for the practice of the present invention are preferably (a) a carbon-based chain; (b) carbon-chain containing one or more heteroatoms such as N, S, O; (c) carbon-chain with substituted groups; (d) an amino acid chain, amino acid fragments incorporated into the chain, or multiple amino-acid fragments chain by for example homologation; (e) a polyethylene glycol chain; (f) a chain have one or more sites of unsaturation such as alkenyl; (g) a nucleic acid chain; or (h) a polysaccharide chain etc.
  • the chain can be made hydrophobic or hydrophilic by including fewer or more groups respectively that are more polar or ionic in the chain.
  • the linker can be selected from different molecular types and lengths. It has been found that the best performance is obtained when the linker is selected to ensure that non-bulky groups are proximal the hapten. It is preferred that the chain be carbon-based.
  • the carbon-based chain may comprise one or more heteroatoms selected from N, S, and O. Side chain substituent groups may also be provided.
  • Other preferred chains are selected from the group comprising amino acids, a polyethylene glycol, alkyl, alkenyl, nucleic acid, and polysaccharide.
  • the chain can have one or more sites of unsaturation. Multiple amino-acid fragments may be provided by homologation.
  • the use of hybrid peptide-nucleic acid fragments as linkers is also contemplated.
  • each linker provides a chain of length 0.5-1 OOnm, preferably most preferably 1-5 nm.
  • a preferred synthesis of a steroid derivative to use in the present invention is controlled and performed by inserting a polyethylene glycol (PEG) chain of appropriate length as a linker and immobilising the hapten derivative onto the sensor surface direcdy (Reaction Scheme 1).
  • PEG polyethylene glycol
  • Such a steroid derivative having a PEG unit as a linker has some distinctive advantages such as 1) PEG chain as a linker can make hapten derivative more water-soluble, and therefore the hapten derivative can be easily in-situ or on-line immobilized onto the sensor surface, which is convenient in real time for process monitoring and quality control in terms of reproducibility performance of immobilization.
  • Use of a PEG chain as a linker can also provide hydrophilic molecular layers to reduce non-specific binding and create more space and a favourable binding medium between the chip surface and the immuno-complex for better antibody binding.
  • immobilisation techniques for immobilising the steroid to be immobilised on a sensor surface is by a covalent coupling reaction (e.g. to an amine, a carboxyl or sulfhydryl group on the surface), nucleic acid hybridisation, or ligand interaction. Immobilisation on the sensor surface may be also by passive adsorption, or via a ligand interaction, such as an avidin/biotin complex (US Patent: 4,467,031).
  • a thioether or ether bridging group preferably a thioether group, generally through their mono-bromide intermediate compounds.
  • nanoparticles refers to the particles used to provide sensitivity through mass labels and are solid particles ranging widely in the size of nanoscale, which includes metal particles (colloidal gold), non-metal particles (latex beads), or any other suitable nanoparticles used as mass labels for signal enhancement.
  • Macromolecule refers to a molecule with a molecular weight of at least 20 kD. Macromolecules for use as signallers in this invention are preferably of molecular weight 50 kD, more preferably at least 100 kD.
  • immunogold particles are used because they are inexpensive and relatively stable.
  • rapid on-line regeneration is used to completely remove hapten conjugates to allow multiple measurements. This may be carried out by injection of regeneration solutions that may include sodium hydroxide and acetonitrile.
  • a standard curve may be prepared from solutions with a series of known analyte concentrations, and the concentrations of analyte in unknown samples may then be derived from the standard curve.
  • the assays of the present invention are optical biosensor-based competitive immunoassays particularly surface plasmon resonance (SPR)-based immunoassays for steroids.
  • This SPR-based immunoassay format method typically comprises the steps:
  • regeneration buffer preferentially composed of sodium hydroxide and acetonitrile
  • steps (b), (c), (d), and (e) are repeated three times or more for reproducibility.
  • kits comprising a first and a second moiety with their various attachments as described above in separate containers with or without instructions for their use.
  • Biological samples for use in the invention include blood, serum, plasma, interstitial fluid and saliva.
  • Saliva is a preferred sample type for use in the invention.
  • Figure 1 shows a salivary Cortisol surface plasmon resonance immunoassay standard curve (percentage bound versus Cortisol (pg/ml)).
  • Figure 2 shows plot of response versus primary antibody concentration for both the secondary antibody enhanced and primary antibody only plots.
  • Figure 3 shows monoclonal antibody Binding Plot with IgG-GoId Enhancement in a testosterone assay.
  • Figure 4 shows assay curve for testosterone saliva assay rapid format using IgG-gold enhancement.
  • Figure 5 shows correlation plot of SPR results vs. radioimmunoassay results for saliva samples taken from 20 healthy male volunteers.
  • Figure 6 shows an assay standard curve referenced to blanks with reference flow cell subtraction for testosterone in buffer.
  • Figure 7 shows an assay standard curve for testosterone referenced to blanks using abbreviated assay format in buffer.
  • Figure 8 shows art assay standard curve for testosterone in human saliva using the total response — mAb response in triplicate.
  • Figure 9 shows an assay standard curve for Cortisol in saliva with IgG-HRP enhancement, reference subtraction and with the full data.
  • Figure 10 shows: (a) schematic diagram of the structure studied, showing gold hole array layer with period, P, and hole diameter, D; and (b) SEM image of a fabricated substrate showing Au film through-holes on glass substrate.
  • Figure 12 shows peak shift for primary antibody (mAb), followed by secondary antibody, or by secondary antibody + gold nanoparticle binding enhancement, and for comparison the nonspecific binding response of secondary antibody and secondary antibody-gold in the absence of the primary antibody.
  • the reactions were all performed using analytical grade solvent.
  • the DMF was dried over magnesium sulfate and then stored over molecular sieves.
  • the chloroform and triethylamine were dried over molecular sieves.
  • the Cortisol (Q 3880-000) and testosterone (A6950-000) were obtained from Steraloids (Newport, RI, USA).
  • the di-tertbutyl-dicarbonate and 4,7,10-triox-l,13- tridecanediamine were purchased from Fluka Chemie (Buchs, Germany) and triethylamine from BDH (Poole, UK). All other reagents were purchased from Aldrich Chemical Company (Milwaukee, WI, USA) and were used without further purification.
  • % Purity mean raw sample area x 100 mean standard area
  • the column used was a Sphereclone 3 ⁇ m C8 150 x 4.6 mm and the oven temperature was 40 0 C and the flow rate 1 ml/min unless otherwise stated.
  • PEG-short 4,7,10-trioxa-l,13-tridecanediamine (PEG-short) (1.9g) was dissolved in 20ml of molecular sieves dried methanol in a round bottom flask (50ml).
  • Triethylamine (ImI) was added to the vigorously stirred PEG- short solution under septa.
  • the DBDC solution was then added drop-wise under septa to the vigorously stirred solution via syringe. The solution was stirred overnight. The solvent was removed and the sample dried in vacuo.
  • Cortisol (362.5mg, l.Ommol) was partially dissolved in methanol (13ml) and ethanol (5ml) and chilled to 0 0 C.
  • Sodium hydroxide solution (10%w/v in distilled water, ImI) was added followed by 30% hydrogen peroxide solution (400 ⁇ l).
  • the reaction was kept stirring at 0 0 C on ice for three hours.
  • the reaction mixture was then raised to room temperature; any remaining solid was filtered off using a sintered glass funnel.
  • the filtrate pH was carefully adjusted to 7.0 using acetic acid and the resulting solution dried in vacuo to yield a clear, colourless oil.
  • This sample was then constituted in distilled water (30ml) and extracted with 3 x 30ml of ethyl acetate.
  • Cortisol epoxide (586.8mg, 1.559mmol) was dissolved in ethanol (dried over molecular sieves, 5ml). A solution of potassium hydroxide (25%w/v in distilled water, 730 ⁇ l) was added to a small flask and stirred whilst 3-mercaptopropionic acid (224 ⁇ l) was added. The stirring solution then had the epoxide solution added dropwise and was immediately placed under nitrogen and stirred at room temperature for four hours. Distilled water (30ml) was added. The aqueous phase was then extracted with diethyl ether (3 x 30ml) before adjusting the pH of the aqueous phase to 1.5 with IM HCl.
  • aqueous phase was then extracted with 3 x 30ml of ethyl acetate.
  • the organic phase was then dried over sodium sulfate and the liquor decanted and solvent removed and sample dried in vacuo.
  • the sample was then column separated using chloroform, 15:1 chloroform: methanol and methanol eluent. The sample was then dried to yield a clear, colourless oil. Yield: 479.9mg (66%).
  • R f 0.42 (5:1 chloroform: methanol).
  • Cortisol acid (479.9mg, 1.029mmol) was dissolved in dry DMF (4ml, dried over molecular sieves) and DCC (275.9mg, 1.337mmol, in 1ml dry DMF) was added dropwise to die stirring steroid solution. This was followed by NHS (153.9mg, 1.337mmol, in 1ml dry DMF) also added dropwise. The reaction was stirred overnight at room temperature in the dark. The white solid formed was then filtered off and washed with dry DMF and the filtrate solvent removed in vacuo. The sample was then column separated using chloroform, 15:1 chloroform: methanol, 10:1 chloroform: methanol to yield a pale yellow semi-solid.
  • Cortisol ester (486.9mg, 0.864mmol) was dissolved in dry DMF (3.5 ml, dried over molecular sieves). To the stirring steroid solution, was added mono-Boc PEG (416.0 mg, 1.296 mmol, in 1.25ml of dry chloroform (dried over molecular sieves)) dropwise, with an additional 2 x 250 ⁇ l of dry chloroform used to wash. The stirring solution had dry triethylamine added (750 ⁇ l, dried over molecular sieves). The reaction was then stirred at room temperature in the dark for 60 hours. After 12 hours, another ImI of dry DMF was added to aid solubility.
  • iV-hydroxysuccinimide (1.00 g, 8.7 mmol) was stirred in dichloromethane (500 mL) over a period of 30 min to yield a clear solution to which 3-mercaptopropionic acid (MPA) (0.92 g, 8.7 mmol) in dichloromethane (50 mL) was added.
  • MPA 3-mercaptopropionic acid
  • Testosterone 807.5mg, 2.8mmol was dissolved in methanol (45ml). The solution was stirred and cooled to O 0 C on ice, after which 10%w/v sodium hydroxide was added (3.4ml in distilled water), followed by 30% hydrogen peroxide (3.7ml). The reaction was then stirred at 0 0 C for four hours. The reaction solution was then raised to room temperature and the pH adjusted to 7.0 with 2M acetic acid and the solvent removed in vacuo before drying. The resulting clear, colourless semi-solid was partially dissolved in distilled water (30ml) and then extracted with ethyl acetate (3 x 30ml).
  • Testosterone epoxide (517.5mg, 1.7mmol) was dissolved in ethanol (5 ml, dried over molecular sieves). In a 20ml flask, 25%w/v potassium hydroxide (0.8 ml in distilled water) was added with 3- mercaptopropionic acid (244 ⁇ l, 2.8mmol). The epoxide solution was then added slowly to the stirring MPA solution and the sample immediately placed under nitrogen and stirred for four hours. Distilled water (30ml) was then added which immediately precipitated a white solid.
  • Testosterone acid 642.3mg, 1.637mmol was dissolved in dry DMF (5ml, dried over molecular sieves).
  • DCC 416.4mg, 2.128mmol, in 1ml dry DMF
  • NHS 232.1mg, 2.128mmol, in 1ml of dry DMF
  • the solution was stirred at room temperature for 48 hours in the dark.
  • the white solid formed was filtered off and washed thoroughly with dry DMF. The filtrate had solvent removed and sample dried in vacuo.
  • the sample was then column separated using chloroform and 15:1 chloroform: methanol as eluents yielding a white semi-solid.
  • Testosterone ester (658.9mg, 1.347mmol) was dissolved in dry DMF (3.5ml) and stirred whilst a solution of mono-Boc-PEG was added dropwise (646.2mg, 2.021mmol, in 1.5ml of dry chloroform) followed by a chloroform rinse (250 ⁇ l). Triethylamine (750 ⁇ l) was then added to the stirring solution and the solution stirred at room temperature in the dark for 60 hours. The solvent was then removed and sample dried in vacuo and the sample column separated using chloroform, 15:1 chloroform: methanol and 10:1 chloroform: methanol as eluents to yield an orange oil. Yield 724.5mg (77%).
  • Testosterone-PEG-Boc (103.5mg) was dissolved in formic acid (4 mL) and stirred at room temperature for 4 hours before removing the solvent in vacuo to yield an orange oil.
  • R f 0.12 (10:1 MeOH:Acetic acid).
  • the surface of a commercially available BIAcore sensor chip (dextran CM5) is in-situ modified by attachment of new Cortisol - PEG-NH 2 (from Example 3) using covalent amine mediated coupling as shown in Scheme 1.
  • a 100 mg/mL solution is prepared in DMF and diluted to 1 mg/mL with PBS/T pH 9.0 and 100 ⁇ L is quick injected at 5 ⁇ L/min.
  • the carboxylated surface of the dextran chip is first activated with EDC and NHS coupling reagents and then the free amine-bearing Cortisol derivative is flowed over the surface. Un-used activated binding points are deactivated with ethanolamine.
  • the immobilised chip surface was then applied in a competitive immunoassay format with primary antibody (US Biological, Mouse anti-human C7904-02) and labelling with secondary antibody — horse radish peroxidase conjugate (Sigma A9044) in a BIAcore 2000 instrument.
  • the computer program transfers 70 ⁇ L of primary antibody (50 ng/mL) to a microplate well followed by 70 ⁇ L of the human saliva sample or standard and the two are mixed.
  • 60 ⁇ L of the mix is then injected over the surface at 20 ⁇ L/min followed immediately by secondary antibody - HRP (1 /25 dilution) (60 ⁇ L at 20 ⁇ L/min) and then regeneration with 0.2 M NaOH 20%v/v acetonitrile (20 ⁇ L, 20 ⁇ L/min). The process is then repeated for the desired number of replicates.
  • the saliva samples are collected into polypropylene tubes and frozen at — 20 0 C and then thawed and centrifuged at 14,600 x g for 15 min. The supernatant is then used for the assay. No other pre- treatment and no pre-dilution is needed.
  • the standards are prepared by taking human saliva and adsorbing out the Cortisol with activated charcoal to produce a blank sample and then making up standards from a concentrated buffer solution (1 ⁇ g/mL).
  • Figure 1 shows the salivary Cortisol SPR immunoassay standard curve.
  • IC50 inhibitory concentration at 50% bound Dynamic range: the concentration range covered by 20-80% bound antibody
  • Linear range the concentration range over which linear response can be obtained
  • Slope sensitivity the amount of signal change (in response units RU) that is observed per unit concentration change.
  • the limit of detection is quite sensitive enough to detect concentrations down past the lowest end of the physiological range (about 70 pg/mL).
  • the active region of the assay curve can be adjusted by adjusting antibody concentrations to cover the entire desired analytical range. In this case the assay may be made less sensitive to embrace the higher values expected above 1 ng/mL. Indeed, there are very seldom any values below 100 pg/mL and so the detection limit could be raised to 100 pg/mL.
  • Figure 2 shows plot of response vs. primary antibody concentration for both the secondary antibody enhanced and primary antibody only plots.
  • the total assay time is 13.7 min per cycle, making it practical for use in dynamic hormone monitoring.
  • the assay will soon be correlated against mass spectrometry and radioimmunoassay technology.
  • the assay can then be applied to the BIAcore Q instrument and used as a routine assay technology.
  • the overall binding format is summarised in Scheme 2.
  • the total assay time may be able to be reduced further to 9.7 min upon adjustment of the active range of the assay to higher concentrations.
  • the surface of a commercially available BIAcore sensor chip (dextran CM5) is in-situ modified by attachment of new testosterone-PEG-NH 2 (from Example 3) using covalent amine mediated coupling shown in Scheme 1.
  • a 100 mg/mL solution is prepared in DMF and diluted to 1 mg/mL with PBS/T pH 9.0 and 100 ⁇ L is quick injected at 5 uL/min.
  • the carboxylated surface of the dextran chip is first activated with EDC and NHS coupling reagents and then the free amine-bearing testosterone derivative is flowed over the surface. Un-used activated binding points are deactivated with ethanolamine.
  • the immobilised chip surface was then applied in a competitive immunoassay format with mouse anti-testosterone antibody (US Biological T2950-18A) and labelling with gold labelled secondary antibody in a BIAcore 2000 instrument.
  • 25 nm gold colloid was prepared according to the citrate reduction method and the anti-mouse IgG (Sigma M 7023) was conjugated at 3mg/mL starting concentration (300 ⁇ g/mL final concentration) according to JS Mitchell, Y Wu, CJ Cook, and L Main Anal. Biochem. 343, 125-135 (2005).
  • UV- Vis suggests a final colloid size of about 40 nm.
  • the gold colloid was used as a signal enhancement agent in SPR by injecting first mAb (60 ⁇ L, 50 ng/mL) followed by gold sol (60 ⁇ L, 10 ⁇ L/min) at dilutions of 0.5, 0.25 and 0.125 in deionised water with a total 10%v/v PEG-400. Non-specific binding at each concentration was also tested by simple injection of the gold alone. In all cases it was found that a single pulse of 20 mM NaOH 20%v/v MeCN (20 ⁇ L) was sufficient to completely regenerate the surface. The results are summarized in Table 3.
  • the enhancement plot showed clearly 12.5 fold enhancement of the mAb binding response through use of gold labelled secondary antibody. From this curve one can see that a suitable concentration to use for the assay is 9 ng/mL final mAb concentration as this gives the 100 RU of specific binding required.
  • a saliva assay was carried in the rapid format with no incubation, all quickinjects, no extraclean and IgG-gold done at 20 ⁇ L/min.
  • the 2.5%w/v total BSA was used in the mAb which was at 9 ng/mL going to 4.5 ng/mL on injection.
  • the assay curve is given below in Figure 4.
  • the testosterone standards were prepared by taking human saliva and adsorbing out the testosterone with activated charcoal to produce a blank sample and then making up standards from a concentrated solution.
  • Figure 4 shows assay curve for testosterone saliva assay rapid format using IgG-gold enhancement.
  • the linear ranges are good but the reduction in mAb concentration has led to a reduction in signal sensitivity as the curve has not shifted to lower concentration enough to compensate for die loss of total signal.
  • the assay is now capable of covering die full physiological range of 25-250 pg/mL between 87.5 — 54.3 % bound on the modified assay. This assay is acceptable in terms of both rapidity and sensitivity and will likely serve the desired purpose.
  • Cortisol surface plasmon resonance BIAcore assay correlates linearly with results obtained by conventional radioimmunoassay when measuring the same saliva samples collected from 20 male volunteers.
  • Saliva is collected from 20 male volunteers upon signing die required consent form. Samples were taken at various times throughout the working day. The subjects chewed on sugar free gum (1/2 a stick) to stimulate saliva production and then salivated into a polypropylene tube after first discarding the first 30s of saliva production. Each subject donated between 8-10 mL of saliva. The saliva was then frozen at -20° C before moving to a -80° C freezer for storage. Closer to the time of use the samples were thawed and aliquoted into sub samples of 1.5 mL and then re-frozen immediately. Before use the samples were thawed and then measured according to the protocols for each method. The RIA kit used was Diagnostic Systems Laboratory kit (DSL-2000) adapted for use with saliva according to the following protocol.
  • DSL-2000 Diagnostic Systems Laboratory kit
  • Run six standards in duplicate (0, 50, 250, 500, 1000, 2500 pg/mL) and four singly (25, 100, 10,000, 25,000 pg/mL).
  • Run three QC samples (high, medium, low singly).
  • Stripped saliva is prepared by donating 10 mL of saliva according to the usual methods and then freezing it. On the day before use it is thawed and centrifuged (4620 x g, 15min) and 7.5 mL of supernatant is removed and added to another polypropylene sample tube with 75 mg of activated charcoal. The tube is vortex mixed at maximum setting for 30 s and then shaken at 560 rpm overnight. Next day the tube is centrifuged (4620 x g, 15 min) and clear supernatant removed. Centrifugations are repeated as necessary to remove residual activated charcoal.
  • HBS-EP HEPES buffered saline with Tween-20 pH 7.4
  • mAb monoclonal antibody
  • a fresh batch of testosterone-PEG-NH 2 was prepared according to the method of Example 3.
  • a new SensiQ sensor chip (ICx Technologies) had a 1:1 JV-(3-Dimethylamino ⁇ ropyl)-N'- ethylcarbodiimide hydrochloride (EDC) : ⁇ T-Hydroxysuccinimide (NHS) solution (10 ⁇ L of 1:1 0.4 M EDC and 0.1 M NHS) applied to the gold surface ex-situ. After 10 min the sensing surface was rinsed with dH 2 O.
  • Testosterone-PEG-NH 2 solution (1 mg/mL in l%v/v DMF in PBS pH 9, 10 ⁇ L) was applied to the gold surface and allowed to dry over 60 min at room temperature. Ethanolamine (1 M, 10 ⁇ L) was applied for 10 min and then washed away with dH 2 O.
  • a buffer assay for testosterone was constructed as follows: 80 ⁇ L of mAb (300 ng/mL) was mixed with 80 ⁇ L of testosterone standard in running buffer (0, 2.5, 10, 25, 50, 100, 250, 500 pg/mL, 1, 10 ng/mL) and incubated at room temperature for 5 min before injection of 125 ⁇ L at 25 ⁇ L/min (60 s lead delay), 90 s delay before injection of IgG-HRP (1/25, 50 ⁇ L, 25 ⁇ L/min), 90 s delay, injection of 50 mM NaOH 20%v/v MeCN (20 ⁇ L, 20 ⁇ L/min), 100 s delay, buffer injection to purge from loop (25 ⁇ L at 100 ⁇ L/min) and then 59 s end delay. Each concentration was done for at least four replicates.
  • the assay parameters are within the required specifications for a testosterone assay. It is now best to try and reduce time by abbreviating the assay. The assay was repeated in buffer as before but without the 5 min incubation and without the flushing injection and its associated 90 s program delay. The assay standard curve is given below in Figure 7.
  • the flow rates are always 25 ⁇ L/min unless otherwise stated.
  • the buffer loop was rinsed out between runs though with 3 x air and 3 x running buffer alternately.
  • Mixing of mAb and standards involved adding 80 ⁇ L of mAb to a PCR tube and then 80 ⁇ L of standard and vortex mixing. All the saliva measurements were reference flow cell subtracted.
  • FIG 10 shows the schematic of the structure used in this study.
  • the Au nanohole arrays were prepared on a BK7 glass substrate 31 (microscope cover slips 22 mm x 22 mm x 0.20 mm). After solvent (acetone, methanol, and IPA) cleaning, a layer of poly(methyl methacrylate) (PMMA) was spun onto the substrate. A nanodot-array pattern was then defined onto the PMMA by means of electron beam lithography (EBL). Next, the exposed PMMA was removed with an aqueous solution of methyl isobutyl ketone (MIBK) in isopropyl alcohol (IPA).
  • MIBK methyl isobutyl ketone
  • NiCr layer was to improve the adhesiveness of Au on the substrate.
  • Au nanohole arrays were created on the substrate through the removal of the PMMA nanodots via solvent assisted lift-off process. A SEM image of a fabricated Au nanohole array is shown in Fig. Ib.
  • a series of 0.5 mm x 0.5 mm Au nanohole array patterns was fabricated on each individual coverslip, on which the periods of the nanhole array, P, range from 500 nm to 600 nm with an increment of 20 nm, and the diameter of the holes 34, D, varied from 70 nm to 200 nm. Up to nine discrete regions (with a particular hole and period geometry) were produced on each cover slip. 8.2 Transmission-mode spectrophotometry of mounted substrate
  • Optical transmission measurements were performed on the nanohole array substrates using a modified Shimadzu 1605 dual beam spectrophotometer.
  • the optical path was modified by replacing the standard sample holders with a precisely machined aperture (0.70 mm in diameter) and flow cell platform as shown in Figure 12.
  • the flow cell 41 was custom designed and manufactured using 3D photo-cure printing techniques and materials (Rl 1 Perfactory Resin, StrataTec, Wales, New Zealand) to provide a 30 ⁇ L laminar flow volume (internal dimensions of the flow channel are 20 x 1.5 x 1 mm, Figure 12) for multiple array regions.
  • the nanohole array substrate 42 was mounted to one side of die flow cell 41 widi a second microscope slide 43 sealing die flow chamber whilst allowing light transmission.
  • a white light source 44 was projected through an aperture 45 on to the nanohole array and transmission measured over a wavelength range from 400 nm to 1100 nm.
  • Sample and buffer flow was controlled using a peristaltic pump (P625/900.133, Instech Laboratories, Plymouth Meeting, PA) 45 operated in vacuum mode to avoid cross contamination between samples and buffer reagents. Unless otherwise stated, binding procedures were conducted at a constant flow rate of 5.5 ⁇ L min 1 with an increased rate of 140 ⁇ L min 1 for buffer flushes and purges.
  • die flow cell volume temperature was controlled using a resistive heater element attached to the glass slide to provide a good thermal conduction path.
  • sucrose solutions were measured at random to ensure objective data.
  • the nanohole array substrate was first cleaned with absolute ethanol (six rinses) and was air dried.
  • An ethanolic solution of cortisol-OEG-SH (Example 2.8) (5 ⁇ L, 10 mg mL "1 ) was applied to the gold nanoarray surface and incubated at room temperature for 16 h in the dark. Additional 5 ⁇ L volumes were applied twice for a further 5 h each.
  • the slide was then rinsed with ethanol and mounted and bound into the flow cell.
  • a further ethanolic solution of cortisol-OEG-SH 100 ⁇ L, 1 mg mL "1 ) was flowed over the flow cell in situ and allowed to pass through over a 1 h period.
  • anti-mouse IgG secondary antibody gold 40 nm diameter conjugate (BA.GAM 40, British Biocell, Cambridge, UK) (2 x 25 ⁇ L undiluted) or anti-mouse IgG secondary antibody (M7023, Sigma, St Louis, USA, 2 x 25 ⁇ L, 400 ⁇ g mL 1 ). All spectral peak shifts stated were after the flow cell had been returned to running buffer. All experiments used HEPES buffered saline with EDTA and Tween 20 pH 7.4 (HBS-EP) running buffer. Four replicates of each binding were performed.
  • Non-specific binding tests for both secondary antibody-gold and secondary antibody were performed by injecting the secondary antibody (2 x 25 ⁇ L at the above concentrations) without first injecting primary antibody. All surfaces were regenerated with 20 mM NaOH (2 x 25 ⁇ L injections).
  • the in-air nanohole array transmission response to hole period from P 500 nm to 600 nm in 20 nm increments included a broad transmission peak centered at 510 nm due to general light transmission of the relatively thin 40 nm gold layer used, and between 700 and 1100 nm two additional peaks that can be attributed to plasmonic effects.
  • the approximate position of these peaks can be determined by the equation:
  • Equation (1) tends to predict resonance peak positions that are lower than those achieved experimentally due to neglecting presence of interaction between LSPR and SPPs, even though the spectral positions of the transmission peaks are dominated by the SPP modes.
  • the cortisol-OEG-SH compound (Example 2.8) is designed to achieve maximum projection of the steroid into the aqueous mobile phase by use of an oligoethylene glycol linker linked at the 4- position.
  • the thiol terminus allows convenient attachment to a gold (or silver) surface by the formation of a dative bond.
  • This compound enables convenient one-step immobilizations without the need to modify intermediate SAMs.
  • This approach is well suited to inhibition biosensor assays, and is particularly well suited for small molecule analysis, that is for molecules with a molecular weight less than 2000 g mol "1 .
  • a SensiQ SPR biosensor was primed twice into dH2O (filtered 0.45 ⁇ m and degassed) and l%w/v sucrose (100 ⁇ L, 25 ⁇ L/min) was injected both in discrete and co-inject modes.
  • the phase change of ⁇ 1300 RU was in line with that expected for the refractive index change from dH2O to l%w/v sucrose and occurred in about 5 s after the injection period began and so is within specifications.
  • the pinch valve change produced a rise in the baseline in Fc2 but not in FcI at die beginning of the injection and a correction once the pinch valve changed again at the end of the injection. This did not occur during co-injection.
  • mAb binding tests were performed using mAb (InM 1:1 with either blank buffer or Cortisol buffer standard (100 pg/mL). Regeneration was with 50 mM NaOH and then later 50 mM NaOH 20%v/v MeCN.
  • a SensiQ assay was then conducted in running buffer using the following protocol: 60 s lead delay, mAb mixed 1:1 Cortisol standard (no pre-incubation) and 125 ⁇ L injected (25 ⁇ L/min flow rate diroughout) 71 s delay, 50 mM NaOH 20%v/v MeCN (20 ⁇ L, 20 ⁇ L/min) and 79 s end delay. Standards of 0, 10, 25, 50, 100, 250, 500, 1000, 5000, 10000 pg/mL Cortisol were used. Analysis was done widi four replicates of each point. The assay standard curves were prepared (not shown, but this assay was subsequently found not to be useful for saliva samples because of fluctuations in the response, caused by high mass molecules in saliva).
  • Flow rate was 25 ⁇ L/min unless otherwise stated. No pre-incubation was performed (samples injected as soon as mixed) and there was no buffer pulse switching. The buffer loop was rinsed out between runs though with 3 x air and 3 x running buffer alternately.
  • Mixing of mAb and standards involved adding 80 ⁇ L of mAb to a PCR tube and then 80 ⁇ L of standard and vortex mixing.
  • the above examples are illustrations of practice of the invention. It will be appreciated by those skilled in the art that the invention can be carried out with numerous modifications and variations. For example the haptens, the linkers, the antibodies and the concentrations used may all be varied.

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Abstract

L'invention concerne le niveau d'une hormone stéroïdienne, choisie parmi le cortisol, la testostérone et la déshydroépiandrostérone, dans un échantillon biologique qui est mesuré par un procédé comprenant les étapes suivantes consistant à : (a) fournir un échantillon biologique; (b) fournir un premier anticorps capable de se lier à l'hormone stéroïdienne; (c) fournir un écoulement de a) et b) séparément ou ensemble à une hormone stéroïdienne immobilisée liée à une surface par l'intermédiaire d'un lieur; (d) fournir un écoulement d'un second anticorps capable de se lier au premier anticorps, où le second anticorps est lié à une macromolécule ou une nanoparticule; et (e) détecter la quantité d'anticorps primaire lié à l'hormone stéroïdienne immobilisée par un système de détection de résonance de plasmon de surface.
PCT/NZ2008/000021 2007-02-12 2008-02-12 Essai de résonance de plasmon de surface pour stéroïdes Ceased WO2008100161A1 (fr)

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CN103513042A (zh) * 2013-09-23 2014-01-15 中国科学院动物研究所 用于预测或早期诊断妊娠高血压疾病的试剂盒
CN103543109A (zh) * 2013-08-12 2014-01-29 苏州大学 用于测定汞离子的lspr传感膜及其制备方法
JP2017142241A (ja) * 2016-02-05 2017-08-17 協和メデックス株式会社 検体中のステロイドホルモンの測定方法
US20170370950A1 (en) * 2014-12-08 2017-12-28 Calbiotech, Inc. Biotin conjugates of analytes containing amino, hydroxyl, or thiol functional groups for use in immunodiagnostic assays
CN109666057A (zh) * 2018-12-12 2019-04-23 郑州安图生物工程股份有限公司 一种4-位含有羧基的类固醇衍生物合成方法
CN111896755A (zh) * 2020-08-24 2020-11-06 广东工业大学 一种脱氢表雄酮快速检测试纸条以及脱氢表雄酮检测方法
US20250327819A1 (en) * 2022-05-09 2025-10-23 Aveta Life, Inc. Tagged Compounds for Detection and Assay of Small Molecules

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DE102013011304A1 (de) * 2013-07-02 2015-01-22 Technische Universität Dresden Verfahren und Anordnung zur Erfassung von Bindungsereignissen von Molekülen

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101887016A (zh) * 2010-06-07 2010-11-17 北京理工大学 一种利用表面等离子体共振传感器检测睾酮的方法
CN103543109A (zh) * 2013-08-12 2014-01-29 苏州大学 用于测定汞离子的lspr传感膜及其制备方法
CN103513042A (zh) * 2013-09-23 2014-01-15 中国科学院动物研究所 用于预测或早期诊断妊娠高血压疾病的试剂盒
US20170370950A1 (en) * 2014-12-08 2017-12-28 Calbiotech, Inc. Biotin conjugates of analytes containing amino, hydroxyl, or thiol functional groups for use in immunodiagnostic assays
JP2017142241A (ja) * 2016-02-05 2017-08-17 協和メデックス株式会社 検体中のステロイドホルモンの測定方法
CN109666057A (zh) * 2018-12-12 2019-04-23 郑州安图生物工程股份有限公司 一种4-位含有羧基的类固醇衍生物合成方法
CN109666057B (zh) * 2018-12-12 2021-04-02 郑州安图生物工程股份有限公司 一种4-位含有羧基的类固醇衍生物合成方法
CN111896755A (zh) * 2020-08-24 2020-11-06 广东工业大学 一种脱氢表雄酮快速检测试纸条以及脱氢表雄酮检测方法
US20250327819A1 (en) * 2022-05-09 2025-10-23 Aveta Life, Inc. Tagged Compounds for Detection and Assay of Small Molecules

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