EP3602057A1 - Method for determining bisphenol a in biological material, diagnostic device for detection of bisphenol a in biological material, diagnostic kit for detection of bisphenol a in biological material - Google Patents

Method for determining bisphenol a in biological material, diagnostic device for detection of bisphenol a in biological material, diagnostic kit for detection of bisphenol a in biological material

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Publication number
EP3602057A1
EP3602057A1 EP18770405.1A EP18770405A EP3602057A1 EP 3602057 A1 EP3602057 A1 EP 3602057A1 EP 18770405 A EP18770405 A EP 18770405A EP 3602057 A1 EP3602057 A1 EP 3602057A1
Authority
EP
European Patent Office
Prior art keywords
bpa
antibodies against
detection
zone
membrane
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18770405.1A
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German (de)
French (fr)
Other versions
EP3602057A4 (en
Inventor
Aleksandra RUTKOWSKA
Aleksandra KONIECZNA
Jacek NAMIESNIK
Blazej KUDLAK
Jan Krzysztof LUDWICKI
Katarzyna GORALCZYK
Pawel STRUCINSKI
Andrzej MILEWICZ
Szymon GRACZYK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Narodowy Instytut Zdrowia Publicznego - Panstwowy Zaklad Higieny
Politechnika Gdanska
Medical Uniwersity of Gdansk
Original Assignee
Narodowy Instytut Zdrowia Publicznego - Panstwowy Zaklad Higieny
Politechnika Gdanska
Medical Uniwersity of Gdansk
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Filing date
Publication date
Priority claimed from PL420935A external-priority patent/PL235548B1/en
Priority claimed from PL424955A external-priority patent/PL242100B1/en
Application filed by Narodowy Instytut Zdrowia Publicznego - Panstwowy Zaklad Higieny, Politechnika Gdanska, Medical Uniwersity of Gdansk filed Critical Narodowy Instytut Zdrowia Publicznego - Panstwowy Zaklad Higieny
Publication of EP3602057A1 publication Critical patent/EP3602057A1/en
Publication of EP3602057A4 publication Critical patent/EP3602057A4/en
Pending legal-status Critical Current

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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
    • G01N33/53—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
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • 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
    • 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
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G01N33/54389—Immunochromatographic test strips based on lateral flow with bidirectional or multidirectional lateral flow, e.g. wherein the sample flows from a single, common sample application point into multiple strips, lanes or zones
    • 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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • 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

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Endocrinology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Method for determining bisphenol A in biological material that relies on binding BPA from biological material with antibodies against bisphenol A and subsequent quantitative determining of BPA content, characterized in that, a migration membrane is being prepared to determine BPA from the migrating biological material so that the membrane is divided into at least a zone of binding with a carrier, a detection zone and a control zone. Color medium covered with monoclonal or polyclonal antibodies against BPA is placed at the zone of binding with the carrier. The detection zone is divided into at least two detection areas while at the first detection area a selected concentration of antibodies against BPA is adsorbed so that BPA can be detected in the range of 0.5 ng/mL to 50 ng/mL and on the second detection area selected concentration of antibodies against BPA is adsorbed so that BPA can be detected in the 0.5 ng/mL to 50 ng/mL. On the control zone monoclonal or polyclonal antibodies against monoclonal or polyclonal antibodies against BPA. Analysed biological material is being applied at the zone of binding with carrier wherein in case of BPA presence, the BPA is being bound with color medium and thus the created complexes of BPA-color medium are directed into the detection zone in which the complexes of BPA-color medium are bound to the antibodies against BPA while the color medium not bound with BPA are directed to the control zone where the color medium is bound with the antibodies against antibodies against BPA. BPA presence is determined by color indication in at least one detection area of the detection zone and by determination of color at the control zone.

Description

Method for determining bisphenol A in biological material, diagnostic device for detection of bisphenol A in biological material, diagnostic kit for detection of bisphenol A in biological material
The object of an invention is the method of determining bisphenol A - BP A in biological material and diagnostic device to detect bisphenol A - BPA in biological material and diagnostic kit to detect bisphenol A - BPA in biological material.
Bisphenol A - BPA is an organic chemical belonging to phenols group, considered to be endocrine active compound, structure of which is similar to steroid hormones, especially oestrogens. Due to this similiarity of structures it can interact with oestrogen, androgen or thyroid hormones receptors increasing risk of health disorders e.g. obesity, diabetes, endocrinopathies, fertility disorders, hormone-dependent cancers. Koch CA, Diamanti- Kandarakis E. Introduction to Endocrine Disrupting Chemicals— is it time to act? Reviews in endocrine & metabolic disorders. 2015;16(4):269-70.
BPA is commonly used in production of plastic materials. Its physical and chemical properties have been found valuable all over the world and its global production reached >6mln tones annually. Goods produced with BPA are transparent, light, easy to use and resistant to damage. BPA derivative are commonly used in production of everyday objects e.g. plastic bottles and food packaging plastics like CD or DVD, electronic parts, polyester polysulphone and polyacryxlic resins, inner covers of metal cans, thermal paper,. It is also present in cosmetics, eye-lenses, dental fillings, toys, pacifiers, bottles and plates for kids. Konieczna A, Rutkowska A, Rachon D. Health risk of exposure to bisphenol A (BPA). Rocz PZH 2015, 66(1): 5-11.
Results of studies show, that at room temperature BPA migrates from polycarbon bottles to food and is detected in concentrations from 0,2 to 0,3 mg/1, while in alluminium covered bottle the concentration ranged from 0.08 to 1.9 mg/1. Cooper JE, Kendig EL, Belcher SM. Assessment of bisphenol A released from reusable plastic, aluminium and stainless steel water bottles. Chemosphere 2011, 85(6): 943-947. Contact of surfaces containing BPA with products of acidic or basic pH, with increased content of fat, salt or sugar, alcohol, or heated the package increases migration rate of BPA to food and cosmetics. BPA is being also released from devices and materials to home dust. There are three ways of human exposure dermally, orally and with air inhaled. Its content is detected in body fluids - serum, urea, saliva, amniotic fluid or fatty tissue. Szybiak A, Rutkowska A, Wilczewska K, Wasik A, Namiesnik J Rachon D. Daily diet containing canned products significantly increases serum concentrations of endocrine disrupter bisphenol A in young women. Polish archives of internal medicine. 2017;127(4):278-80.
Human exposure to BPA increases the risk of hormonal disorders leading to endocrinopathies and civilization diseases namely obesity, diabetes, cardiovascular diseases, infertility, polycystic ovary syndrome (PCOS), hormone dependent cancers e.g. breast cancer, prostate cancer, testicles cancer.
There are known diagnostic methods of determining BPA in biological material, including instrumental ones e.g. liquid chromatography, gas chromatography, hyphenated often to mass spectrometers (MS), time of flight analyser (TOF), ions mass analysers, biological tests like CALUX, (z ang. Chemically Activated LUciferase gene expression), E-SCREEN (assessing potential of estrogenic activity of chemicals), XenoScreen YES/YAS (test to detect substances of endocrine potential) or immunoanalytical tests based on detection and quantitative determination of selected analytes including those of EDC group as a result of binding by mono- and polyclonal antibodies. Depending on test format, quantitative determination is connected with performing enzymatic, fluorescent or radiochemical reaction of secondary antibodies.
There is still a necessity to find new diagnostic methods to perform quickly and precisely determination of BPA in biological material in qualitative, semi-quantitative or quantitative, especially in out-of-laboratory conditions.
The invention is the new method of BPA determination in biological material which is based on the migration membrane to quantify BPA from migrating material, which is divided into at least "zone of binding with carrier", "detection zone" and control zone, while at the "zone of binding with carrier" color medium covered with polyclonal or monoclonal antibodies against BPA were added.
"Detection zone", area within the membrane, is divided into at least two detection areas, while at the first detection area selected concentration of antibodies against BPA is adsorbed so that BPA can be detected in the range of 0.5 ng/mL to 50 ng/mL. On the second detection area selected concentration of antibodies against BPA is adsorbed so that BPA can be detected in the 0.5 ng/mL to 50 ng/mL of BPA. At the "control zone", area within the membrane, monoclonal or polyclonal antibodies against monoclonal or polyclonal antibodies against BPA are imposed. Then biological material is being applied at the "zone of binding with carrier" - area within the membrane. If the BPA is present in the sample, it binds to color medium - carrier that is colored and that can bind to BPA and migrate with a complex in a form BPA-medium, and is being subsequently directed in the form of complexes BPA-color carrier to the "detection zone" where the BPA-color carrier complexes are caught by antibodies against BPA. Color medium-carriers without BPA bound are directed to the control zone, where the color medium is caught by antibodies against antibodies anti-BPA. Presence of BPA in sample of biological material is detected and determined by indicating colored area in at least one detection area of detection zone and by simultaneously colored area in the "control zone".
Preferably "the binding zone with carrier" consists of a carrier membrane and/or BPA- free reaction vessel, where color carriers covered with monoclonal or polyclonal antibodies against BPA are added.
Preferably, at the migration membrane additional membrane is added to separate blood morphologic elements from serum. It may be made from woven fibers that show high absorptivity of water in range starting from 70 mg/cm3.
Preferably the "detection zone" is divided into three detection areas while at the first detection area selected concentration of antibodies against BPA is adsorbed so that BPA can be detected in the 0.5 ng/mL to 50 ng/mL and on the second detection area selected concentration of antibodies against BPA is adsorbed so that BPA can be detected in the 0.5 ng/mL to 50 ng/mL while on the third detection area selected concentration of antibodies against BPA is adsorbed so that BPA can be detected in the 0.5 ng/mL to 50 ng/mL.
Preferably, the detection zone is divided into detection areas while at every detection area different maximum amount of antibodies against BPA is adsorbed.
Preferably, migration membrane is composed of nitrocellulose and/or cotton and/or glass fibers and/or polyester fibers and/or their composites.
Preferably, color spatial structures made of BPA-free materials, especially latex, India rubber, silicone, polystyrene, gold with diameter of single structure >0.1 nm are used as color carriers.
Preferably, nitrocellulose and/or cotton and/or glass fibers and/or polyester fibers and/or their composites membranes can be used as the carrier membrane. In this invention device to determine bisphenol BPA in the biological material contains "migration membrane" to determine BPA in the biological material migrating on the membrane, while this "migration membrane" has "zone of binding to carrier", "detection zone" and "control zone". Zone also means a separated area within the membrane. "Zone of binding with carrier" contains color carriers covered with monoclonal or polyclonal antibodies against BPA while "detection zone" has at least two detection areas. At the first detection area selected concentration of antibodies against BPA is adsorbed so that BPA can be detected in the 0.5 ng/mL to 50 ng/mL and on the second detection area selected concentration of antibodies against BPA is adsorbed so that BPA can be detected in the 0.5 ng/mL to 50 ng/mL. The control zone contains monoclonal or polyclonal antibodies against monoclonal or polyclonal antibodies against BPA.
Preferably, "the binding zone with carrier" is an additional carrier membrane and/or BPA-free reaction vessel, where color carriers covered with monoclonal or polyclonal antibodies against BPA were added. Preferably the carrier membrane is a membrane made of nitrocellulose and/or cotton and/or glass fibers and/or polyester fibers and/or their composites.
Preferably at the migration membrane also additional membrane may be added to separate blood morphologic elements from serum, and the additional membrane can be made of woven fibers that show high absorptivity of water in range starting from 70 mg/cm3.
Preferably the "detection zone" is divided into three detection areas while at the first detection area selected concentration of antibodies against BPA is adsorbed so that BPA can be detected in the range from 0.5 ng/mL to 50 ng/mL of BPA and on the second detection area selected concentration of antibodies against BPA is adsorbed so that BPA can be detected in the range from 0.5 ng/mL to 50 ng/mL while on the third detection area selected concentration of antibodies against BPA is adsorbed so that BPA can be detected in the range from 0.5 ng/mL to 50 ng/mL.
Preferably, the detection zone is divided into detection area while at every indication field different maximum amount of antibodies against BPA is adsorbed.
Preferably, as a migration membrane nitrocellulose and/or cotton and/or glass fibers and/or polyester fibers and/or their composites are used.
Preferably, color spatial structures made of BPA-free materials, especially latex, India rubber, silicone, polystyrene, gold with diameter of single structure >0.1 nm are used as color carriers - medium. Another invention is the diagnostic kit for the detection of bisphenol A BPA in a biological material that contains:
- a carrier capable of absorption and/or migration of biological material such as a membrane or reaction vessel or a multi-well plate or capillary,
- polyclonal and/or monoclonal antibodies against BPA, polyclonal and/or monoclonal antibodies against anti-BPA antibodies,
- colored carriers coated with polyclonal or monoclonal antibodies against BPA,
- β-glucuronidase.
Preferably, the carrier contains at least one or more separable detection areas separate from each other and at least one dedicated control zone and preferably the termination zone in the test variants: strip or capillary or diaper.
Preferably, the detection area enables detection and quantifying of the concentration of BPA in the biological material through the binding of BPA by adsorbed polyclonal or monoclonal antibodies against BPA, applied in an appropriate concentration.
Preferably, the control zone comprises adsorbed polyclonal or monoclonal antibodies against anti-BPA antibodies.
Preferably, the migrating biological material is absorbed in the termination zone.
Preferably, the colored carriers are coated with polyclonal or monoclonal antibodies that bind BPA present in the biological material in an unbound form.
Preferably, β-glucuronidase sprayed on the bottom and/or walls of the reaction vessel or added to the membrane or added to the biological material in the form of a tablet or pellet or capsule or powder or solution converts the bound form of BPA into an unbound form.
Preferably, the carrier is a membrane with at least one or more separable detection areas separate from each other that enable the detection and determination of BPA concentration in a minimal BPA concentration range of 1-15 ng/mL.
Preferably, the carrier is a membrane with at least one detection area that enables the detection and determination of the BPA concentration at a minimum BPA content of 0.5 ng/mL in case of an addition of β-glucuronidase to biological material.
Preferably, the carrier is a membrane with at least one detection area that enables the detection and determination of the BPA concentration at a minimum BPA content of 0.1 ng/mL.
Preferably, the carrier is a reaction vessel with at least one detection area that allows detection and determination of the BPA concentration at a minimum BPA content of 1 ng/mL. Preferably, the carrier is a multi-well plate with at least one or more separable detection areas separate from each other that enable the detection and determination of BPA concentration in a minimal BPA concentration range of 1-15 ng/mL.
Preferably, the carrier is a microcapillary or a set of microcapillares with at least one or more separable detection areas separate from each other that enable the detection and determination of BPA concentration in a minimal BPA concentration range of 1-15 ng/mL.
Preferably, the kit contains:
- a set of membranes enabling the absorption and migration of biological material and colored carriers coated with polyclonal or monoclonal antibodies against BPA,
- at least one separated detection area that enables detecting and determining the BPA content through an adequate concentrations of polyclonal or monoclonal antibodies against BPA adsorbed in this area, where a membrane with at least one detection area enables the detection and determination of BPA concentration in a minimal BPA concentration range of 1-15 ng/mL,
- at least one control zone containing adsorbed polyclonal or monoclonal antibodies against anti- BPA antibodies,
- the termination zone where migrating biological material and unbound colored carriers coated with antibodies are absorbed,
- β-glucuronidase sprayed on the bottom and/or walls of the reaction vessel or added to the membrane or added to the biological material in the form of a tablet or pellet or capsule or powder or solution.
Preferably, the kit contains:
- a set of membranes enabling the absorption and migration of biological material and colored carriers s coated with polyclonal or monoclonal antibodies against BPA,
- at least one detection area that enables the detection and determination of a specific concentration of BPA through an adequate concentration of polyclonal or monoclonal antibodies against BPA adsorbed in this area, where a membrane with at least one detection area enables the detection and determination of BPA concentration at a minimum BPA content of 0.5 ng/mL,
- at least one control zone containing associated polyclonal or monoclonal antibodies against anti-BPA antibodies,
- the termination zone where migrating biological material and unbound colored carriers coated with antibodies are absorbed,
- β-glucuronidase enzyme added to the absorption membrane. Preferably, the kit contains:
- a set of membranes enabling the absorption and migration of biological material and colored carriers coated with polyclonal or monoclonal antibodies against BPA,
- at least one detection area that enables the detection and determination of a specific concentration of BPA through an adequate concentration of polyclonal or monoclonal antibodies against BPA adsorbed in this area, where a membrane with at least one detection area enables the detection and determination of BPA concentration at a minimum BPA content of 0.1 ng/mL,
- at least one control zone containing associated polyclonal or monoclonal antibodies against anti-BPA antibodies,
- the termination zone where migrating biological material and unbound colored carriers coated with antibodies are absorbed
Preferably, the kit contains:
- reaction vessel as a carrier for the detection and control zone,
- at least one detection area that enables the detection and determination of a specific concentration of BPA through an adequate concentration of polyclonal or monoclonal antibodies against BPA adsorbed in this area, where a reaction vessel with at least one detection area enables the detection and determination of BPA concentration at a minimum BPA content of 1 ng/mL,
- at least one control zone containing associated polyclonal or monoclonal antibodies against anti-BPA antibodies,
- β-glucuronidase sprayed on the bottom and/or walls of the reaction vessel or added to the biological material in the form of a tablet or pellet or capsule or powder or solution and color carriers coated with polyclonal or monoclonal antibodies against BPA.
Preferably, the kit contains:
- reaction plate with wells that are detection areas
- at least one separate detection area that enables the detection and determination of a specific concentration of BPA through an adequate concentration of polyclonal or monoclonal antibodies against BPA adsorbed in this area, where a reaction plate with at least one detection area enables the detection and determination of BPA concentration in a minimal BPA concentration range of 1-15 ng/mL.
- at least one control zone containing associated polyclonal or monoclonal antibodies against anti-BPA antibodies, - β-glucuronidase sprayed on the bottom and/or walls of the reaction vessel or added to the biological material in the form of a tablet or pellet or capsule or powder or solution and color carriers coated with polyclonal or monoclonal antibodies against BPA.
Preferably, the kit contains:
- microcapillaries as a carrier, enabling the migration of biological material
- at least one separated detection area that enables the detection and determination of a specific concentration of BPA through an adequate concentration of polyclonal or monoclonal antibodies against BPA adsorbed in this area/areas, where a microcapillary and/or a set of microcapillaries contains at least one detection area that enables the detection and determination of BPA concentration in a minimal BPA concentration range of 1-15 ng/mL.
- at least one control zone containing adsorbed polyclonal or monoclonal antibodies against anti- BPA antibodies,
- β-glucuronidase sprayed on the bottom and/or walls of the reaction vessel or added to the biological material in the form of a tablet or pellet or capsule or powder or solution and color carriers coated with polyclonal or monoclonal antibodies against BPA.
The invention enables the rapid determination of bisphenol A in a quantitative or semiquantitative manner in out-of-laboratory conditions even at the level of sensitivity of instrumental methods. Due to the use of at least two detection areas in the detection zone with a selected value of BPA detection, the invention enables the semi-quantitative and even quantitative determination of the concentration of BPA in the biological material. Due to the use of a migration membrane divided into further zones in which migrating BPA in biological material is bound by antibodies on colored carriers, then by adsorbed antibodies and is determined in individual detection area in the detection zone and then using the control zone, the invention enables fast, sensitive and specific determination of BPA. The present invention enables the determination of BPA in biological material, especially in whole blood and serum of patients with both very low and very high exposure to BPA.
The method and device of the invention is based on the use of one or a combination of membranes, a colored medium - carriers, antibodies against BPA and antibodies against anti- BPA antibodies. The diagnostic kit of the invention consists of polyclonal or monoclonal antibodies against BPA, and polyclonal or monoclonal antibodies against anti-BPA antibodies, colored carrier beads made especially of material, e.g. latex or silicon and/or colloidal gold, coated with antibodies against BPA and carriers in the form of membranes or vessels or plates or glass capillaries depending on the variant of the kit. An additional element of the set is a reaction vessel for a sample of the patient's biological material (made of BPA-free material and its derivatives) and the β-glucuronidase enzyme, necessary for the hydrolysis of the binding in BPA glucuronate, a product of conversion of BPA in the urine. As part of the kit, several test variants were distinguished, depending on the type of carrier for migration of the biological sample: strip test, diaper test: without the addition of β-glucuronidase enzyme, with the addition of β- glucuronidase enzyme, cup test, plate test, capillary test.
In the method and device of the invention, the area of application of the biological sample, especially serum or whole blood, is a migration membrane, on which additionally a carrier membrane and/or absorbent membrane made of fibers with average water absorption 70-100 mg / cm3 may be added, which allows to separate blood cells from the serum and retention of blood cells, allowing free passage of the water-protein phase - serum for BPA determination. The colored medium carriers made of materials such as latex, silicone or colloidal gold are coated with polyclonal or monoclonal antibodies against BPA and are placed on a carrier membrane or in a BPA-free reaction vessel. The migration membrane allows free migration of a sample of biological material and carriers coated with antibodies against BPA, and associated carrier-BPA complexes.
The migration membrane contains a separate detection and a control zone. The detection zone contains at least one or more detection areas that contain associated polyclonal antibodies or monoclonal antibodies against BPA. The variable number of detection areas and the variable content of antibodies against BPA in detection area enable the determination of a wide range of BPA concentrations in biological material in the range of 0.5-150.0 ng/mL depending on the number of detection areas and the saturation of the area with adsorbed antibodies against BPA. Therefore, a result with sensitivity of instrumental methods is obtained, but in out-of-laboratory conditions and without the need for additional analytical or detection equipment. The obtained color reaction is observable within a few minutes from applying a sample of biological material.
The control zone contains adsorbed polyclonal or monoclonal antibodies against anti-BPA antibodies and binds colored carriers coated with antibodies, indicating the correction of the migration of the biological sample and thus, the correctness of the method. If more than one detection area is placed in the detection zone in the migration membrane, the area located the closest to the area of applied biological sample enables detection of BPA concentration in the biological material corresponding to the lowest BPA concentration for which the detection parameters were determined in each area, and combined with further detection areas - gives the result of the total BPA concentration in the biological material.
The invention is shown in the embodiment and in drawings in which in figure 1 the general schematic placement of the carrier, detection and control zones on the migration membrane is shown; in figure 2, an example of the arrangement of the carrier zone, the detection zone with two detection areas and the control zone is shown, whereas in figure 3 a solution with three detection areas is shown. It may contain more areas depending on the variant of solution as shown in the tables below for indicative variants of determination.
In sketch, figures 1 to 9 apply to the method and device to BPA determination where Fig 1 : Schematic distribution of the carrier, detection and control zones on the migration membrane in the case of isolation of only 1 detection area; Fig. 2: Schematic distribution of the carrier, detection and control zone on the migration membrane in case of separation of two detection areas; Fig. 3: Schematic distribution of the carrier, detection and control zone on the migration membrane in case of separation of three detection areas; Fig. 4. Schematic test construction in case of replacement of a carrier membrane with a reaction vessel; Fig. 5: Schematic test construction in case of a solution enabling determination of BPA in venous whole blood; Fig. 6: An example of the test implementation according to the scheme of Fig. 2 using 2 detection areas that detect BPA at a concentration of 0.5 ng/mL each. Strip assay without glucuronidase, nitrocellulose migration membrane, example "b", sample 10; Fig. 7: An example of the test implementation according to the scheme of Fig. 3 using 3 detection areas that detect BPA at a concentration of 0.5 ng/mL, 2 and 5 ng/mL counting from the edge of the carrier membrane. Strip assay without glucuronidase, migration membrane of glass fibers, example "d", sample 17; Fig. 8: An example of the test implementation according to the scheme of Fig. 4 using 1 detection area that detects BPA at a concentration of 2 ng/mL and replacing the carrier membrane with a reaction vessel. Strip assay without glucuronidase, migration membrane of nitrocellulose fiber, sample 15; Fig. 9: An example of the test implementation according to the scheme of Fig. 1 using 1 detection area that detects BPA at a concentration of 30 ng/mL. A glucuronidase test strip, a cotton fiber migration membrane, example "b", sample 18. Fig. 10-33 shows a diagnostic kit according to the invention where: Fig. 10: A detailed diagram of the strip test structure; Fig.l l : Scheme of diaper test construction; Fig. 12: Scheme of the distribution of antibodies against BPA in the detection zone and antibodies against anti-BPA antibodies in the control zone; Fig. 13: Scheme of the cup test structure; Fig. 14: Diagram of the cup test operation; Fig. 15: Scheme of the plate test structure; Fig.16: Diagram of the plate test operation; Fig.17: Scheme of the capillary test structure; Fig. 18: Diagram of an operation of the strip test and exemplary test results at various concentrations of BPA in biological samples; Fig. 19-22 - strip test result, Fig. 23: Scheme of diaper test construction and negative test result; Fig. 24: positive test result; Fig. 25: negative test result; Fig. 26: positive test result; Fig. 27: positive test result; Fig. 28: Positive test result; Fig. 29 Positive result of a plate test; Fig. 30: Positive result of a plate test; Fig. 31 : Kit included in the capillary test; Fig. 32: Migration of a urine sample containing colored latex beads coated with antibodies against BPA; Fig. 33-34: Positive result of a capillary test; Figure 35: Simultaneous use of capillaries for measuring BPA concentration in a biological sample; Fig. 36: A calibration curve for determining the concentration of BPA in urine; Fig 37: An example of LC-MS / MS chromatogram analysis of a urine sample.
Example la
Venous blood was drawn after overnight fast to a glass tube deprived from BPA and its derivatives. The tubes with blood were then centrifuged at 2500 rpm for 15 minutes. The separated serum was collected using a transfer pipette into glass microvials. The biological material was either used immediately to determine the BPA content or frozen and stored at -20° C for further analyses.
The isolation of serum step can be omitted by applying a whole blood sample to the test if an absorption membrane is placed within the migration membrane, before or on a carrier membrane. Absorption membrane needs to be made of a mix of natural and artificial fibers, which water absorption capacity is of an average 70-100 mg/cm3, but separate the blood cells, allowing the water-protein-serum phase to pass freely to the further test structure.
Construction of a device to determine BPA - a test strip
As shown in Fig. 2, a migration membrane was prepared, as the material, nitrocellulose membrane of thickness of 240-280 μπι was selected. Alternative fiber compositions for this membrane were cotton, glass, polyester fibers and their composites., The binding zone with carriers, the detection zone with the specified detection areas and the control zone were separated on this migration membrane. The detection area was adsorbed with rabbit polyclonal antibodies against BPA previously placed in a phosphate-buffered saline (PBS) with the addition of 2% BSA (Bovine Serum Albumin). The amount of applied antibodies corresponded to the detection of different concentrations of BPA depending on the use of combination of number and value of detection areas - saturation with antibodies.
The minimum of detected BPA concentration in the biological material was 0.5 ng/mL for 1 detection area, if two detection areas were used, it was 1 ng/mL. In the control zone on the migration membrane, goat polyclonal antibodies against rabbit antibodies against BPA were adsorbed, which was determined as the control area of the test, indicating the correctness of the test. Carrier membrane was chosen as binding zone with carrier, onto which a colored medium is applied - carriers coated with polyclonal antibodies against BPA. In an alternative design, the zone of binding with carrier is an additional carrier membrane.
A glass fiber was chosen for the carrier membrane that constitutes the binding zone, which was impregnated with a PBS solution with the addition of 2.5% BSA. It is also possible to use nitrocellulose, cotton, glass, polyester fibers and their composites. After 24 hours of drying the carrier membrane, 40 μΐ of 1-3% solution of a colored medium - carriers coated with rabbit antibodies against BPA in a stabilizing buffer was applied. Prepared migration and carrier membranes made of e.g. nitrocellulose, cotton, glass or polyester fibers or their composites, were dried at room temperature for 24 hours. The number of colored carriers (per 1 ml of solution), depending on the used material and the carrier size, ranged from 6- 104 for carriers with an average radius of 45 μηι, to 4- 1013 for carriers with an average radius of 0.05 μηι. The concentration of antibodies against BPA used to coat the carriers was 1 mg/ml.
In an alternative design of the zone of binding with carrier, into a vial made of materials free of BPA and its derivatives, a small volume of patients' blood serum was collected and then 100 μΐ of serum was transferred with a micropipette to the carrier membrane. The free BPA from the serum sample was bound by antibodies against BPA placed on colored carriers located on the carrier membrane or in the reaction vessel. The BPA-carrier complexes migrated through the carrier membrane to the detection zone. The migration speed of the carriers, in accordance with membrane parameters, was 100s/4 cm. The maximum amount of antibodies applied corresponded to the detection of BPA concentrations in the range 0.5-50.0 ng/mL for a particular detection area depending on the design shown in the table below. The following combinations of number and variants of detection area were used in the test examples:
The test result, being a determination of the total BPA concentration in the biological material, was the sum of the values of the BPA concentrations detected in each colored detection area in the detection zone. The presence of BPA in serum is indicated by: at least one colored detection area - for min. BPA concentration of 0.5 ng/mL; 2 colored detection areas - for min. BPA> 1.0 ng/mL; and 3 areas- for BPA> 1.5 ng/mL. Depending on the chosen amount of antibodies against BPA for specific detection areas as well as the number of detection areas, the patient's exposure to BPA can be estimated. In the "a" example, one detection area can detect min. BPA concentration of 0.5 ng/mL, in the example "b" the test detects BPA concentrations in the range of 0.5-1 ng/mL, whereas 0.5 ng/mL is detected when the first detection area is colored and 1 ng/mL when both detection areas are colored.
In the example "e", the test can detect BPA concentration in the range of 1-8 ng/mL and the staining of the first area indicates a concentration of 1 ng/mL, two areas of 3 ng/mL, all 3 areas > 8 ng/mL. Low exposure occurred in the absence or an appearance of a maximum one colored detection area detecting BPA concentrations in the range of 0.5-2.0 ng/mL. The patient BPA exposure was considered as average in the case of the staining of detection areas determining the BPA concentration in the range 2.0-10.0 ng/mL. The appearance of detection areas in the case of applying the antibodies that enable the detection of BPA concentration of > 10.0 ng/mL determines high exposure of a given person to this compound. A properly functioning test (migration of colored carriers correctly coated with antibodies against BPA) was confirmed by the appearance of the blue color in the control zone. Described method and obtained examples of design indicate new possibilities of precise determination of BPA in biological material, on the level of analytical sensitivity of instrumental methods, but without the need to use highly specialized laboratory equipment. It allows getting the results in less than 5 minutes and can be interpreted with eyesight. The modification of the number of detection areas and the BPA concentrations detected by them allows the method to be adapted to various applications, including assessment of the exposure of children for whom BPA may lead to negative health effects, even in very low concentrations. The use of this method could contribute to reducing the exposure to BPA in the population and thus, reduce the exposure to environmental factors.
Example lb
Construction of a device to determine BPA in a strip form
The device is constructed as described in example 1 and is shown in Fig. 1 ; however, the carrier zone is not a separate carrier membrane but a reaction vessel made of a BPA-free material containing a 1-3% solution of a colored carrier medium coated with antibodies against BPA, as shown in FIG. 4. The methods of device preparation and its operation were identical to those described in example 1, whereas 100 μΐ of serum was transferred not to the carrier zone but to the reaction vessel with the colored medium and then directly to the migration membrane. Below is an example of comparison of the obtained results of BPA concentrations in samples of biological material analyzed by the strip test with the results of a standardized liquid chromatography method combined with mass spectrometry. Figures 6, 7, 8 illustrate pictures of tests results with the use of different numbers of detection areas and various types of migration membranes for samples 10, 15 and 17.
Table. Results of instrumental BPA determinations in blood samples not treated with β- glucuronidase
Samples BPA ng/mL Samples BPA ng/mL Samples BPA ng/mL
1 0.057 5 0.491 9 0.957
2 0.034 6 0.535 10 1.025
3 0.097 7 0.528 11 0.978
4 0.079 8 0.505 12 0.798 13 1.69 17 8.22
14 2.12 18 54.06
15 2.59 19 18.7
16 3.45 20 25.93
Example 2
The device to determine BPA in a strip form - with an addition of β-glucuronidase
The device is constructed as described in example 1 and is shown in Fig. 1 ; however, a migrating membrane is made of glass fibers with a thickness of 420 μιη, 75-85 g/m2. Alternative compositions of fibers for the membrane were nitrocellulose, cotton, polyester fibers and their composites. An additional absorption membrane made of mixed natural and artificial fibers has been placed, which water absorption capacity of an average 70-100 mg/cm3, to separate the blood cells, allowing the water-protein-serum phase to pass freely to the further test structure.
Additionally, on the migration membrane the termination zone of the test was placed - a membrane absorbing the migrating serum sample, which was a membrane of cotton fibers (290 g/m2, 830 μηι). Although this was not a key element for correctness of the test, it significantly improved the test finishing.
200 μΐ of venous blood were collected in a vial made of materials free of BPA and its derivatives, 25 μΐ of β-glucuronidase (of activity > 20,000 units/g of protein) was added and then incubated for 2 hours in a 37° C water bath. The micropipette was then used to transfer 100 μΐ of the prepared sample to the absorption membrane (separating the blood cells), from where the sample migrated to the carrier membrane with colored carriers and then directly to the migration membrane. The free BPA from the serum sample was bound by antibodies against BPA adsorbed on colored carriers located on the carrier membrane. The BPA-carrier complexes migrated through the carrier membrane to the detection zone. The migration speed of the carriers, in accordance with membrane parameters, was 10s I A cm. The amount of antibodies applied corresponded to the detection of BPA concentrations in the range 0.5-50.0 ng/mL for a particular detection area depending on the design. The following combinations of number and variants of indicator fields were used:
The test result, being a determination of the total BPA concentration in the biological material, was the sum of the values of the BPA concentrations detected in each colored detection area in the detection zone. The presence of BPA in serum is indicated by: at least one colored detection area- for min. BPA concentration of 0.5 ng/n L; 2 colored detection areas - for min. BPA> 1.0 ng/mL; and 3 detection areas - for BPA> 1.5 ng/mL. In the "b" example, one detection area can detect min. BPA concentration of 50 ng/mL, in the example "c" the test detects BPA concentrations in the range of 0.5-3,5 ng/mL, whereas 0.5 ng/mL is detected when the first detection area is colored, 1.5 ng/mL when two, and 3.5 ng/mL when three detection areas are colored. In the example "g" test enables detection of BPA in the range of 0,5-18.5 ng/mL, and the staining of the first detection area indicates a concentration of 0.5 ng/mL, two detection areas 1.5 ng/mL, 3 detection areas 3.5 ng/mL, 4 detection areas 8.5 ng/mL, and 5 detection areas >15.5 ng/mL.
Depending on the chosen amount of antibodies against BPA for specific detection area as well as the number of detection areas, the patient's exposure to BPA can be estimated. Low exposure occurred in the absence or appearance of a maximum of one colored detection area detecting BPA concentrations in the range of 0.5-2.0 ng/mL. The patient BPA exposure was considered as average in the case of the staining of detection areas determining the BPA concentration in the range 2.0-10.0 ng/mL. The appearance of detection areas in the case of applying the antibodies that enable the detection of BPA concentration of > 10.0 ng/mL determines high exposure of a given person to this compound. A properly functioning test (migration of colored carriers correctly coated with antibodies against BPA) was confirmed by the appearance of the blue color in the control zone. The results of analyses using chromatography are given below. Fig. 9 shows a photo of a stripe test detecting BPA concentration in the sample 18.
Table. Results of instrumental BPA determinations in blood samples treated with β- glucuronidase
Example 3
Checking the integrity of the device according to the invention
Blood serum was collected from volunteers of both genders. The BPA content in each of the samples was determined using liquid chromatography coupled to tandem mass spectrometry. Detailed parameters of BPA detection, necessary to confirm the test, are presented below.
Devices and instruments used:
Liquid chromatograph and tandem mass spectrometer:
1) Shimadzu tandem spectrometer (LCMS-8060, Shimadzu, Japan) with electrospray (ESI) source operating in negative mode (formation of deprotonated ions). Compound analysis mode used: ion transition monitoring (MRM) for:
BPA: 227.3 -> 212.1 m z, collision energy: 20V, preloads on quadrupole Ql and Q3 respectively: 11 and 13 V
The operating parameters of the ion source are as follows: Nebulizing gas: nitrogen 3 L/min Heating gas: air 10 L/min Source temperature: 300°C Temperature of the desolvation line: 250°C Heating block temperature: 400°C Drying gas: nitrogen 10 L/min
2) UPLC Nexera X2 liquid chromatograph system (Shimadzu, Japan) consisting of: two LC- 30AD pumps, DGU-20A5R eluent degassers, CBM-20A controler, SIL-30AC autosampler and CTO-20AC thermostat.
3) Parameters of chromatographic analysis
A mobile phase consisting of methanol (B) and water (A) was used. Both ingredients were used without any additives. Isocratic elution: 50% B. Column: Ascentis® Express (CI 8 15cm x 2.1mm, 2.7μπι) with a pre-column of the same packing (0.5cm x 2.1mm, 2.7μιη). Volume flow of the mobile phase stream: 0.55 mL/min. Column thermostat temperature: 50 ° C. Injection volume: 5.0 μL·.
4) Standards and reagents
BPA and BPA standards were purchased from Sigma-Aldrich (St. Louis, USA). Internal standard 13C-labeled BPA (ring-13C12) was supplied by Cambridge Isotope Laboratories Inc. (UK). Methanol of LC-MS grade was obtained from Merck KGaA (Darmstadt, Germany), deionized water was prepared using the HPL5 device.
5) Results of analyses
5.1). Calibration curves for BPA and BPS were prepared based on prepared solutions with concentrations: 0.05; 0.1 ; 0.25; 0.5; 1.0; 2.5 and 5.0 ng/mL. The internal standard concentration was 25.0 ng/mL.
5.2 Equations of calibration curves:
BPA y=0.092x+0.0015 (LOD = 0.0093 ng/mL, LOQ = 0.028 ng/mL) BPS y=0.065x+0.017 (LOD = 0.022 ng/mL, LOQ = 0.067 ng/mL)
Example 4 The diagnostic kit for the detection of bisphenol A in a biological material that contains:
- a carrier capable of absorption and/or migration of biological material such as a membrane or reaction vessel or a multi-well plate or capillary,
- polyclonal and/or monoclonal antibodies against BPA, polyclonal and/or monoclonal antibodies against anti-BPA antibodies,
- colored carriers coated with polyclonal or monoclonal antibodies against BPA, In that design it contains β-glucuronidase.
Operating principle of the diagnostic kit for detecting BPA in biological material:
- as a result of treating with β-glucuronidase, the BPA glucuronate (a bound form of BPA) present in the sample of biological material releases the unbound form of BPA, which conjugates with colored carriers coated with polyclonal or monoclonal antibodies against BPA,
- then as a result of the migration of samples of biological material, the colored complexes of the carriers- antibody-unbound BPA are captured in the detection areas by the antibodies against BPA associated in these areas,
- the presence of unbound BPA in the biological material captured by carriers coated with anti- BPA antibodies causes the appearance of at least one or more separate coloured detection areas depending on the concentration of BPA,
- the correctness of the test is proved by at least one control zone that is colored, regardless of the presence of BPA in the sample of biological material, at the moment of an arrival of colored carriers into this zone and their binding by antibodies against antibodies against BPA applied in this zone,
- biological material and unbound colored carriers in the detection zone and/or control zone, may be caught in the termination zone in the case of strip, diaper or capillary test.
Kit - first variant: strip test - fig.10.
Diagnostic kit - set is based on using combination of membranes, color carriers covered with antibodies against BPA and polyclonal/monoclonal antibodies against antibodies against BPA. Detailed scheme of test construction is presented in fig. 10.
Area - place 1 of putting the biological sample e.g. urine so called sample pad is a membrane that shows high sample absorptivity (e.g. of glass fibers) on which the small volume of biological material is added. Just below sample pad (overlapping) there is a conjugate pad 2 - area (membrane) at which color carriers are placed (e.g. latex, silicone or made of colloidal gold) covered with monoclonal or polyclonal antibodies against BPA. Layer 3 is a membrane enabling free migration of sample of biological material and carriers covered with antibodies against BPA, also of those bound BPA present in sample. Membrane 3 contains at least one or more detection areas 5 that contains polyclonal or monoclonal antibodies against BPA and at least one separate control zone containing bound polyclonal or monoclonal antibodies against antibodies against BPA - control line 6. If more than one detection area is placed at the membrane than the detection area being in closest vicinity to sampling pad enables detection of BPA at concentration enabling reflecting the low risk of BPA exposure in given group of patients, while in combination with next detection area - concentration ranges for medium exposure. Coloring of all three subsequently placed detection areas reflects high exposure to BPA.
Control zone(s) designated as control line contains antibodies against antibodies against BPA and its (their) color as a result of putting biological material confirms proper action of the test Migration of the biological material and unbound carriers covered with antibodies against BPA is terminated in the zone of termination 4 so called absorbent pad. Backing free of BPA and its derivatives 7 constitutes an additional element (not necessary for test to run) holding membrane up and enabling performing test in non-laboratory conditions.
Kit: variant second: diaper test
Set in this variant is based on using combination of membranes, color carriers covered with antibodies against BPA and polyclonal/monoclonal antibodies, and polyclonal/monoclonal antibodies against antibodies against BPA.
Detailed scheme of construction of diaper test is presented in fig. 11. In fig. 12 the scheme of placing antibodies against BPA in the detection zone is presented and of antibodies against antibodies against BPA in the control zone.
Area (place) 1 of putting the biological sample e.g. urine so called sample pad is a membrane that shows high sample absorptivity and will absorb urine of child from diaper. Just below sample pad (overlapping) there is a conjugate pad 2 - membrane at which color carriers are placed (e.g. latex, silicone or made of colloidal gold) covered with monoclonal or polyclonal antibodies against BPA. Layer 3 is a membrane enabling free migration of sample of biological material and carriers covered with antibodies against BPA, also of those bound BPA present in sample. Membrane 3 is evenly covered with monoclonal or polyclonal antibodies against BPA detecting possibly the lowest concentration of BPA (0.1 ng/mL) in urine (4) and contains at least one separate control zone containing bound monoclonal or polyclonal antibodies against antibodies against BPA (5). The termination zone 6 is a sorption membrane holding the migrating sample up.
The entire diaper test is covered with transparent membrane made of material free of BPA and/or its derivatives to protect particular area from damage. Only the absorption area is free of this membrane.
A: diaper test without β-glucuronidase enzyme
Method of test run is based on absorption of biological sample by absorption membrane 1 and flow of sample over subsequent layers 2, 3, 5. Small volume of urine migrates from membrane 1 to membrane 2 that contains color carriers e.g. latex ones covered with antibodies against BPA. In the moment of migration of sample containing given concentration of free BPA over membrane the color complexes carrier-antibody-free BPA are being created in the amount proportional to amount of free BPA in sample. Sample of urine containing color complexes carrier-antibody-free BPA and color carriers with antibodies not bound to BPA migrate in time over the nitrocellulose membrane. Complexes carrier-antibody-free BPA are being caught by antibodies against BPA evenly bound at the detection zone of membrane. Depending on amount of color carrier complexes bound to free BPA from sample of child urine the color area 4 appears of varying color intensity. The more intensive color of this area the more free BPA was present in the sample. The minimal concentration of free BPA in the sample of child urine that could show color reaction was 0.1 ng/mL. Color carriers without BPA bound keep migrating with urine in nitrocellulose membrane and are then being caught by antibodies against antibodies against BPA. It enables appearance of at least one colored control zone 5 confirming proper action of test.
B: diaper test with β-glucuronidase enzyme
Set in this variant is based on using combination of membranes, color carriers covered with antibodies against BPA and monoclonal/polyclonal antibodies against BPA and against antibodies against BPA. Detailed scheme of diaper test is presented in fig. 4. The only difference in construction deals with adding β-glucuronidase enzyme to the absorption 1. Area (place) 1 of putting the biological sample e.g. urine so called sample pad is a membrane that shows high sample absorptivity and will hold up urine of child from diaper. Sample pad membrane is soaked with β-glucuronidase enzyme to initiate hydrolysis of bunds in BPA glucoronate increasing amount of free BPA available. Just below sample pad (overlapping) there is a conjugate pad 2 - membrane at which color carriers are placed (e.g. latex, silicone or made of colloidal gold) covered with monoclonal or polyclonal antibodies against BPA. Layer 3 is a membrane enabling free migration of sample of biological material and carriers covered with antibodies against BPA, also of those bound BPA present in sample. Membrane 3 is evenly covered with monoclonal or polyclonal antibodies against BPA detecting possibly the lowest concentration of BPA (1 ng/mL) in urine 4 and contains at least one separate control zone containing bound monoclonal or polyclonal antibodies against antibodies against BPA 5.
Kit - third variant: cup test
Fig. 13 - scheme of cup test construction, fig. 14-method of cup test action
Set in this variant consists of the reaction vessel covered from bottom till ¾ of height with polyclonal or monoclonal antibodies against BPA (detection zone). Upper part - ¼ of probe from edge is covered with antibodies against antibodies against BPA (control zone). Additionally set contains vessel for urine sample, that contains color carriers covered with polyclonal or monoclonal antibodies against BPA and β-glucuronidase enzyme in the form sprayed on wells of vessel and/or added in the form of solution, powder, tablet. Scheme of cup test construction is presented in fig. 13.
The method of test run is based on binding free BPA to antibodies against BPA adsorbed on carriers. In the reaction vessel for biological sample the β-glucuronidase is placed, in the form of spraying or tablet or pellet or powder or capsule to solution that initiates hydrolysis of bunds in BPA glucoronate increasing amount of free BPA available. Antibodies against BPA are being placed in this vessel and bound to carriers to catch free BPA.
The entire mixture should be poured into reaction vessel covered with antibodies against BPA (detection zone) and antibodies against antibodies against BPA (control zone). At the moment of vessel mixing catching of color complexes carrier-antibody-free BPA takes place in ratio proportional to free BPA content in sample. Depending on content of complexes bound to BPA in sample of patient the test color area appears indicating BPA presence in patient sample. Carriers without BPA bound are being caught by antibodies against antibodies against BPA. It enables appearance of color control zone confirming proper action of test. Figure 5 presents scheme of test run.
Kit: variant fourth: plate test. Fig. 15 - scheme of plate test construction presented, fig. 16 - scheme of plate test action.
Set in this variant constitutes of multi -well reaction plate where each well is a separate detection area (detection zone) covered with polyclonal or monoclonal antibodies against BPA of different concentration. One of wells on plate is covered with antibodies against antibodies against BPA (control zone). Additionally set contains vessel for urine sample, that contains color carrier beads covered with polyclonal or monoclonal antibodies against BPA and β-glucuronidase enzyme in the form sprayed on wells of vessel and/or added in the form of solution, powder, tablet.
The method of test run is based on binding free BPA to antibodies against BPA bound with carriers. In the reaction vessel for biological sample the β-glucuronidase is placed, in the form of spraying or tablet or pellet or powder or capsule to solution that initiates hydrolysis of bunds in BPA glucoronate increasing amount of free BPA available. Antibodies against BPA are being placed in this vessel and bound to carriers to catch free BPA.
Part of mixture was transferred with pipette of proper volume to detection wells of the reaction plate covered with antibodies against BPA (detection zone) and antibodies against antibodies against BPA (control zone). At the moment of plate shaking the reaction of catching color complexes carrier-antibody-free BPA takes place in ratio proportional to amount of free BPA in sample.
Depending in content of complexes bound with BPA in patient sample the color test area appears indicating BPA presence in patients' urine. Carriers without bound BPA are being caught by antibodies against antibodies against BPA. It enables visualization of control zone proving proper action of test.
Kit - fifth variant: capillary test - fig. 17 - detailed scheme of capillary test construction.
Set in this variant is based on using combination of microcapillaries, color carriers covered with antibodies against BPA and polyclonal/monoclonal antibodies against BPA and antibodies against antibodies against BPA. To a glass reaction vessel for urine samples at the bottom of which the bound/sprayed β- glucuronidase is placed (or added in the form of powder, tablets, solution) one adds small volume of biological sample e.g. patient urine. The initiation of reaction of making analyte (free bisphenol, BPA) free takes place to unbound BPA present in glucoronate form in biological material (urine) as a result of β-glucuronidase action. Final samples were obtained with free BPA content reflecting real concentrations of analyte determined.
Flat bottom of reaction vessel is a place where sedimentation of color carriers covered with monoclonal or polyclonal antibodies against BPA would occur.
Place of putting the biological sample e.g. urine is lower open end of glass made (or polymeric but BPA and its derivatives free) capillary set at angle from 60 to 90 degrees to bottom thank to what the migration of sample mixture containing free BPA and color carriers covered with antibodies against BPA constituting complexes with BPA being caught from sample going up in the capillary. Migrating complexes of carriers with BPA are caught during migration up the capillary by antibodies against BPA. In case of presence of free BPA in the biological sample at concentration levels above 1 ng/niL its binding with carriers and binding by antibodies in capillary with result in appearance of color in the detection area. Each capillary has one detection area detecting given BPA concentration by putting proper concentration of antibodies against BPA and one control zone confirming correct action of the test. Appearance of color signal in the control zone is a signal of correct run of test, it results from combining carries, whose antibodies against BPA were bound by antibodies against antibodies against BPA. The test termination zone, marked as absorption zone (Absorbent pad) is a membrane of high absorptivity ratio soaking the migrating sample and color carriers.
Example 5
Diagnostic kit - strip test
Membrane 3 was prepared made of nitrocellulose of 240-280 μιη thickness. Rabbit polyclonal antibodies against BPA were put on the membrane while previously they were put in PBS buffer with addition of 2% BSA (Bovine Serum Albumine). Amount of antibodies put reflected detection of BPA of 2, 3, 10 ng/mL for respective detection areas. The end of membrane goat polyclonal antibodies against rabbit antibodies against BPA were put what constituted the test control zone. At area 2 (conjugate pad) the membrane made of glass fibers was selected and impregnated with PBS with 2% BSA addition. 40 μΐ of 1% solution of latex beads covered with rabbit antibodies against BPA were put at dried via 24 h membrane. Place 1 of putting urine sample (sample pad) constituted membrane made of glass fibers. The set was dried at room temperature for 24 h. The test termination zone 4 (absorbent pad) was a membrane absorbing migrating urine sample and it was made of cotton fibers.
Small volume of patients' urine was added to vessel made of material free of BPA and its derivatives and then with micropipettes 2ml of urine were transferred to glass vial added to test, to reach level marked at site of vessel. As a result of β-glucuronidase (activity of which is >20000 units /g of protein and sprayed at bottom and sides of reaction vessel at concentration of 20 μΐ/ml) action the initiation of reaction of making analyte (free BPA) free took place to unbound BPA present in urine in glucuronate form. Final samples of urine were obtained with free BPA content reflecting real concentration of analyte determined.
100 μΐ of urine sample taken from reaction vessel was put on the place of sample putting on membrane 1. Sample of urine was migrating to the area covered with carrier beads 2, where binding of free BPA in urine took place by antibodies put on color latex beads. Then urine sample, unbound BPA, beads with bound BPA, bead that did not bound BPA were migrating on the nitrocellulose membrane 3. Migration speed of beads was according to membrane parameters 150s/4cm. The amount of antibodies was reflecting detection of BPA in maxima concentration levels respectively 2, 3, 10 ng/mL for given detection area. Value of BPA detected by every detection area where summing up with number of color areas - 1 field for BPA concentration - 2 ng/mL in area (low BPA concentration), - 2 areas for BPA concentration 5ng/mL (medium BPA concentration), - 3 areas for BPA concentration 15 ng/mL (high BPA concentration). Properly acting test (migration of beads properly covered with antibodies) was confirmed by appearance of color of the control zone 6. In fig. 18 the rule of strip test operation is presented and example results of test for different concentration levels of BPA in biological sample.
Photography of test result is presented in fig. 19 where, looking from left there is presented:
1 - negative test results - BPA concentration in patient urine below test detection limit, below 1 ng/mL. Result for chromatographic analyses LC-MS/MS: 0.52 ng/mL.
2 - result showing low BPA concentration in patient urine up to 2 ng/mL. Result for chromatographic analyses LC-MS/MS: 1.09 ng/mL. 3 - result showing medium BPA concentration in patient urine up to 5 ng/mL. Result for chromatographic analyses LC -MS/MS: 4.79 ng/mL.
4 - result showing high BPA concentration in patient urine up to 15 ng/mL. Result for chromatographic analyses LC-MS/MS: 11.28 ng/mL.
Letter and numerical acronyms at test strips show selected type of membrane (made from glass fibers or cellulose). Difference in shape of color indication fields results from hand putting antibodies on carrier membranes.
Example test stripes were also prepared with putting antibodies against BPA to detect lower concentration levels of BPA in urine of children, pregnant women, women and men in reproductive age and test with application of monoclonal antibodies against BPA to confirm their replacement usage in respect to polyclonal antibodies.
Stripe test to detect BPA in urine of infants and small children - description
The amount of antibodies was reflecting detection of BPA in maximal concentration level respectively 1, 2, 2 ng/mL for given stripe/girdle. Example below illustrates result for morning urine sample of boy, 5 years old. Result for chromatographic analyses LC-MS/MS: 19,9 ± 1,5 ng/mL.
Result of strip test is presented in fig. 20 - colored all three lines proving BPA concentration of total value not lower than 5 ng/mL
Stripe test to detect BPA in urine of female in reproductive age - description
The amount of antibodies was reflecting detection of BPA in maximal concentration level respectively 1, 3, 5 ng/mL for given detection area.
Example below illustrates result for morning urine sample of pregnant woman, 32nd week of pregnancy. Result for chromatographic analyses LC-MS/MS: 9,09 ± 0,33 ng/mL.
Result of strip test is presented in fig. 21 - colored all three lines proving BPA concentration of summary value not lower than 8 ng/mL in sample of urine of pregnant female.
Stripe test to detect BPA in urine of male in reproductive age - description The amount of antibodies was reflecting detection of BPA in maximal concentration level respectively 2, 3, 10 ng/mL for given detection area. Example below illustrates result for morning urine sample, 1981 year of birth. Result for chromatographic analyses LC-MS/MS: 15,88 ± 1,39 ng/mL. Result of strip test is coloring of all three detection areas confirm presence of BPA of summary value not lower than 15 ng/mL in sample of urine of man (1981 year of birth). Result for chromatographic analyses LC-MS/MS: 15,88 ± 1,39 ng/mL. Result of strip test is presented in fig. 22 - colored all three lines proving BPA concentration of summary value not lower than 15 ng/mL in sample of urine of male in reproductive age.
Stripe test with application of monoclonal antibodies to detect BPA - description
Technique of test preparation was different only with fact of using antibodies - instead of rabbit polyclonal antibodies against BPA placed in 3 detection areas and covered on latex beads and goat antibodies against rabbit ones (control) the mice monoclonal antibodies were used (placed in three detection areas and covered on color latex carrier beads) and antibodies against mice antibodies (test control zone).
Amount of antibodies put was reflecting detection of BPA in maximal concentration level respectively 2, 3, 10 ng/mL for given detection area.
Example below illustrates result for morning urine sample, 1981 year of birth. Result for chromatographic analyses LC-MS/MS: 15,88 ± 1,39 ng/mL. Result of strip test is coloring of all three deetction areas confirm presence of BPA of summary value not lower than 15 ng/mL in sample of urine of man in reproductive age. It is identical visual result for the same urine sample as in example where results were given with polyclonal antibodies. Difference in shape of color indication fields results from hand putting antibodies on carrier membranes.
Example 6
Diagnostic kit - diaper set
A: diaper test without addition of β-glucuronidase
Nitrocellulose membrane (sample migration membrane) 3 of 280 μιη thickness. Solution of rabbit polyclonal antibodies against BPA in PBS buffer with addition of 2% BSA (Bovine Serum Albumine) was placed on membrane 3 in detection zone. Amount of antibodies put reflected detection of BPA of 1 ng/mL. Membrane was incubated (dried) for 2 h in room temperature. At the end of membrane 3 in the control zone 5 goat antibodies against rabbit antibodies against BPA were placed (in the form of stripes (as far as it was possible with hand-made application) what was the test control zone. As area 2 (conjugate pad) the glass fibers membrane was selected, and impregnated with PBS with 2% BSA addition. Membrane was incubated for 24h in room temperature. 100 μΐ of 1% solution of latex beads covered with rabbit antibodies against BPA were put at dried membrane. Membrane impregnated with color carrier beads was incubated for 2 h at room temperature and then put on the sample migration membrane (nitrocellulose membrane). Place of putting urine sample 1 (sample pad) was a membrane of high absorptivity of biological samples, in this example these were membranes of glass fibers or of nitrocellulose fibers. The entire set was dried at room temperature for 24 h. The test termination zone 6 (absorbent pad), membrane absorbing the migrating urine sample was membrane made of cotton fibers. In fig. 23 example of negative test is presented - BPA concentration in patient urine sample at level below test limit of detection, below 0.1 ng/mL; result of chromatographic LC-MS/MS analyses< LOD. In fig. 24 - positive test result - BPA concentration in urine. Result of chromatographic LC-MS/MS analyses: 0.52 ng/mL.
B: diaper test with addition of β-glucuronidase
Nitrocellulose membrane (sample migration membrane) 3 of 280 μη thickness. Solution of rabbit polyclonal antibodies against BPA in PBS buffer with addition of 2% BSA (Bovine Serum Albumine) was placed on membrane 3 in detection zone. Amount of antibodies put reflected detection of BPA of 0,5 ng/mL. Membrane was incubated (dried) for 2 h in room temperature. At the end of membrane 3 in the control area 5 goat antibodies against rabbit antibodies against BPA were placed (in the form of stripes (as far as it was possible with hand-made application) what was the test control zone. As area 2 (conjugate pad) the glass fibers membrane was selected, and impregnated with PBS with 2% BSA addition. Membrane was incubated for 24h in room temperature. 100 μΐ of 1% solution of latex beads covered with rabbit antibodies against BPA were put at dried membrane. Membrane impregnated with color carrier beads was incubated for 24 h and then solution of β-glucuronidase (activity of which is >20000 units /g of protein) was added and incubated next 2 h at room temperature. Then samples were put on migration membrane 3. Place of putting urine sample 1 (sample pad) was a membrane of high absorptivity of biological samples, in this example these were membranes of glass fibers or of nitrocellulose fibers. The entire set was dried at room temperature for 24 h. The test termination zone 6 (absorbent pad), membrane absorbing the migrating urine sample was membrane made of cotton fibers. In fig. 25 example of negative test is presented - BPA concentration in patient urine sample at level below test limit of detection, below 0.5 ng/mL; in fig. 26 - positive test result - BPA concentration in urine sample above test limit of detection, above 0.5 ng.mL. In fig. 27 - positive test results - concentration of BPA in patient urine at level above test limit of detection, above 0.5 ng/mL intensive coloring of detection area confirms high BPA content in biological sample.
Example 7 Kit - cup test
The glass vessels 5 of 1ml volume were prepared as well as separate dilutions of rabbit polyclonal antibodies against BPA in PBS solution with 2% BSA (to reach concentrations enabling detection of respectively 10, 5, 3, 2, 1 ng/ ml of BPA). Solution of goat antibodies against antibodies against BPA (concentration of antibodies: 2μg/ml). Standard procedure based on using buffers containing sodium bicarbonate (pH=9.2) and buffers blocking nonspecific binding of antibodies (PBS + 2% BSA) was used to cover vessels walls. The plate was incubated for 24h at room temperature. Correctness of covering with antibodies was checked with using standards containing proper BPA concentrations.
2.5 ml of patient urine was added to the glass vial of 3 cm diameter and 5 cm height, at the bottom of which β-glucuronidase (activity of which is >20000 units /g of protein) was sprayed. As a result of β-glucuronidase action the initiation of reaction of making analyte (free BPA) free took place. 1 ml of 1 % solution of color carrier beads covered with antibodies against BPA was added to the urine sample. 500 μΐ of mixture of urine and color carrier beads that were catching molecules of free BPA, was added to each well and gently shaken, to enable binding of color beads by antibodies against BPA (in the indication area) or antibodies against antibodies (control area). After few minutes the supernatant was gently removed. Coloring of detection area was showing presence of free BPA in given concentration, coloring of control zone was showing correct action of test run.
In fig. 28 the positive result of test run is presented - BPA concentrations in patient urine at the level above the test limit of detection, above 1 ng/mL. Presentation of gradation of coloring of detection areas for biological samples containign different BPA concentrations (increasing from left: 1 ng/mL, 5 ng/mL, 10 ng/mL). Scheme of result of cup test for urine sample for which results of chromatographic analyses were respectively 1 ,09 ng/mL, 4,79 ng/mL, 11 ,28 ng/mL.
Example 8 Kit - plate test
The glass plate was prepared that contained wells of equal 1 ml volume each. Dilutions of rabbit polyclonal antibodies against BPA were prepared in PBS solution with 2% BSA (to reach concentrations enabling detection of respectively 10, 3, 2, 1 ng/ ml of BPA). The last well was covered with goat antibodies against antibodies against BPA (concentration of antibodies: 2μg/ml). Standard procedure based on using buffers containing sodium bicarbonate (pH=9.2) and buffers blocking nonspecific binding of antibodies (PBS + 2% BSA) was used. The plate was incubated for 12h at room temperature.
Correctness of covering with antibodies was checked with using standards containing proper BPA concentrations.
2.5 ml of patient urine was added to the glass vial, at the bottom/walls of which β-glucuronidase (activity of which is >20000 units /g of protein) was sprayed. As a result of β-glucuronidase action the initiation of reaction of making analyte (free BPA) free took place. 1 % solution of color carrier beads covered with antibodies against BPA was added to the urine sample. 500 μΐ of mixture of urine and color carrier beads that were catching molecules of free BPA, was added to each well and gently shaken, to enable binding of color beads by antibodies against BPA (in the detection wells) or antibodies against antibodies (control well). After few minutes the supernatant was gently removed. Coloring of detection wells was showing presence of free BPA in given concentration, coloring of control well was showing correct action of test run.
In fig. 29 the positive result of test run is presented - BPA concentrations in patient urine at the level above the test limit of detection, above 1 ng/mL. Presentation of gradation of coloring of detection areas. Scheme of result of plate test for urine sample for which results of chromatographic analyses was 11.28 ng/mL. in fig. 30 the positive result of test is presentd - BPA concentration in patient urine sample above test detection limit, above 1 ng/mL). Scheme of result of plate test for urine sample the liquid chromatography LC-MS/MS result of which were l,09 ng/mL.
Example 9
Kit - capillary test
1 glass capillary of 5 cm length was prepared at the internal surface of which in the place marked (just above the lower edge) the rabbit polyclonal antibodies buffered with solution of physiological salt with addition of 2 % BSA (Bovine Serum Albumin) were put. The amount of antibodies was appropriate to detect BP A maximal concentrations of 1 ng/mL (1. detection area) in each capillary. At the end of capillary at the internal site the goat polyclonal antibodies against rabbit antibodies against BPA were put (control zone of the test). Just above the control zone the absorption membrane was put being test termination zone. Correctness of putting proper amount of antibodies for given BPA concentration and type of capillary was assessed by using standard solutions containing proper concentration of BPA standard.
2 ml of patient urine was added to the glass vial of 3 cm diameter and 5 cm height, at the bottom of which β-glucuronidase (activity of which is >20000 units /g of protein) was sprayed. As a result of β-glucuronidase action the initiation of reaction of making analyte (free BPA) free took place. 2 % solution of color carrier beads covered with antibodies against BPA was added to the urine sample.
Previously prepared capillary was immersed into the vial containing sample. Urine sample with color carrier beads was migrating up the capillaries into the site of absorption membrane. Speed of sample migration with color was dependent on capillary angle and diameter. In this case it was 1 cm/s. Amount of antibodies against BPA put in the capillaries was appropriate to detect BPA concentrations in maximal values respectively 1 ng/mL for specific detection area in single capillary. Presence of BPA in urine is confirmed by appearance of color detection area - 1 circle/girdle for BPA concentration of 1 ng.ml in capillary prepared. Properly acting test (migration of carrier beads properly covered with antibodies) was confirmed by appearance of color control zone, just before the absorption membrane.
End of migration occurs when sample of urine and unbound covered beads undergoes absorption at the absorption membrane of high absorptivity (absorbent pad). In fig. 31 set constituting capillary test kit is presented. The reaction vessel containing the samples of patients' urine and β-glucuronidase enzyme. Vial with solution of blue latex beads covered with antibodies against BPA. Glass capillary covered at internal site with rabbit polyclonal antibodies against BPA (detection area) and goat antibodies against rabbit antibodies against BPA (control zone).
In fig. 32 migration of urine sample containing color - blue latex bead covered with antibodies against BPA is presented. Positive result is presented in fig. 33. Coloring of the detection area detecting BPA at 1 ng/mL concentration level. Result of chromatographic analyses LC-MS/MS: 1.09 ng/mL
In another variant of making this set, 1 glass capillary of 5 cm length was prepared at the internal surface of which in the place marked (just above the lower edge) the rabbit polyclonal antibodies buffered with solution of physiological salt with addition of 2 % BSA {Bovine Serum Albumin) were put. The amount of antibodies was appropriate to detect BPA maximal concentrations of 3 ng/mL (1. detection area) in each capillary. At the end of capillary at the internal site the goat polyclonal antibodies against rabbit antibodies against BPA were put (control zone of the test). Just above the control zone the absorption membrane was put being test termination zone.
2 ml of patient urine was added to the glass vial of 3 cm diameter and 5 cm height, at the bottom of which β-glucuronidase (activity of which is >20000 units /g of protein) was sprayed. As a result of β-glucuronidase action the initiation of reaction of making analyte (free BPA) free took place. 2 % solution of color carrier beads covered with antibodies against BPA was added to the urine sample.
Previously prepared capillary was immersed into the vial containing sample. Urine sample with color carrier beads was migrating up the capillaries into the site of absorption membrane. Speed of sample migration with color was dependent on capillary angle and diameter. In this case it was 1 cm/s. Amount of antibodies against BPA put in the capillaries was appropriate to detect BPA concentrations in maximal values respectively 3 ng/mL for specific detection area in single capillary. Presence of BPA in urine is confirmed by appearance of color detection area - 1 circle/girdle for BPA concentration of 3 ng/ml in capillary prepared. Properly acting test (migration of carrier beads properly covered with antibodies) was confirmed by appearance of color control zone, just before the absorption membrane. End of sample migration takes place when sample of urine and unbound covered beads undergoes absorption in the absorption membrane of high absorptivity (absorbent pad)
In another variant of making this set, 1 glass capillary of 5 cm length was prepared at the internal surface of which in the place marked (just above the lower edge) the rabbit polyclonal antibodies buffered with solution of physiological salt with addition of 2 % BSA (Bovine Serum Albumin) were put. The amount of antibodies was appropriate to detect BPA maximal concentrations of 10 ng/mL (1. detection area) in each capillary. At the end of capillary at the internal site the goat polyclonal antibodies against rabbit antibodies against BPA were put (control zone of the test).
2 ml of patient urine was added to the glass vial of 3 cm diameter and 5 cm height, at the bottom of which β-glucuronidase (activity of which is >20000 units /g of protein) was sprayed. As a result of β-glucuronidase action the initiation of reaction of making the analyte (free BPA) free took place. 2 % solution of color carrier beads covered with antibodies against BPA was added to the urine sample.
Previously prepared capillary was immersed into the vial containing sample. Urine sample with color carrier beads was migrating up the capillaries into the site of absorption membrane. Speed of sample migration with color carrier beads was 1 cm/s. Amount of antibodies against BPA put in the capillaries was appropriate to detect BPA concentrations in maximal values respectively 10 ng/mL for specific detection area in single capillary. Presence of BPA in urine is confirmed by appearance of color detection area - 1 circle/girdle. Properly acting test (migration of carrier beads properly covered with antibodies) was confirmed by appearance of color control zone, just before the test termination (absorption membrane, absorbent pad).
Positive result is presented in fig. 34. Coloring of the detection area detecting BPA at 10 ng/mL concentration. Result of chromatographic analyses LC -MS/MS: 11.28 ng/mL
In another variant of making this set, 3 glass capillaries of 5 cm length were prepared at the internal surface of which in the place marked (just above the lower edge) the rabbit polyclonal antibodies buffered with solution of physiological salt with addition of 2 % BSA (Bovine Serum Albumin) were put. The amount of antibodies was appropriate to detect BPA maximal concentrations of 1, 3 and 5 ng/mL (1. detection area) in each capillary. At the end of capillary at the internal site the goat polyclonal antibodies against rabbit antibodies against BPA were put (control zone of the test). Just above the control area the absorption membrane was put being the termination of test. 3 ml of patient urine was added to the glass vial of 3 cm diameter and 5 cm height, at the bottom of which β-glucuronidase (activity of which is >20000 units /g of protein) was sprayed. As a result of β-glucuronidase action the initiation of reaction of making analyte (free BPA) free took place. 2 % solution of color carrier beads covered with antibodies against BPA was added to the urine sample.
Previously prepared capillaries are immersed into the vial containing sample. Urine sample with color carrier beads was migrating up the capillaries into the absorption membrane.. Amount of antibodies against BPA put in the capillaries was appropriate to detect BPA concentrations in maximal values respectively 1, 3, 5 ng mL for specific detection area in single capillary Presence of BPA in urine was confirmed by appearance of colored detection areas - 1 circle/girdle for BPA concentration - 1 ng/mL in the first capillary, 2 circle/girdle for concentration 3ng/mL in the second capillary, 3 circle/girdle in for concentration 5 ng/mL in the third capillary. Detected amounts are summing up. Properly acting test (migration of beads properly covered with antibodies) was confirmed by appearance of color of the control zone, just before to the absorption membrane.
Termination of sample migration occurs when sample of urine and unbound covered beads undergoes absorption in the absorption membrane of high absorptivity (absorbent pad). In Fig. 35 simultaneous use of capillaries to measure BPA in biological sample is presented.
Example 10
Confirmation of results of diagnostic sets action
Results of instrumental analyses refer to all examples dealing with diagnostic sets: example 4-9.
Correctness of test action and BPA content in urine samples used were verified with liquid chromatography coupled to tandem mass spectrometer (LC-MS/MS) (Shimadzu LCMS-8050, Poland). Each measurement of BPA content in sample was performed in triplicate. Final results of analyses of BPA content in urine samples are given as mean value and its uncertainty.
Chromatographic conditions
During the studies the Lichrospher CI 8 (250 mm χ 4 mm; 5 μιη) column was used. Chromatographic separation was performed under isocratic elution conditions and mobile phase contained 50% water and 50% of acetonitrile. Volumetric flow of mobile phase was equal to lcm3/ml and temperature of column thermostat was set to be 45 °C.
Method of determining free BPA in urine samples
Extraction of free BPA was performed with Fabric phase sorptive extraction method. 500 μΐ of urine was added to the probe, together with 100 μΐ of internal standard, and 4400 μΐ of buffer consisting of 0.1 M ammonia formate of pH = 4.5. The content was vortexed. The probe was transferred to water bath and hydrolysis was performed in 37 °C for 2 h. Then the material with sorption material being previously conditioned in the methanol/acetonitrile mixture the sample was placed in the probe and shaken for 30 min. after isolation of BPA this substance was eluted from the fabric surface with 1 cm3 of acetonitrile/isopropanol mixture. The extract was centrifuged and dosed to HPLC-MS/MS system.
Method of total BPA determination in urine samples
500 μΐ of urine was added to the probe, together with 100 μΐ of internal standard, 20 μΐ of β- glucuronidase and 4380 μΐ of buffer consisting of 0.1 M ammonia formate of pH = 4.5. The content was vortexed. The probe was transferred to water bath and hydrolysis was performed in 37 °C for 2 h. Then the material with sorption material being previously conditioned in the methanol/acetonitrile mixture the sample was placed in the probe and shaken for 30 min. after isolation of BPA this substance was eluted from the fabric surface with 1 cm3 of acetonitrile/isopropanol mixture. The extract was centrifuged and dosed to HPLC-MS/MS system - fig.36 and fig.37.
In table results of chromatographic determination of free BPA in samples of urine of healthy women (21-24 years old) prior to and after adding the β-glucuronidase (incubation 20 μ1/500 μΐ of urine) is given. Examples illustrating action of proposed variants of test are performed based in these urine samples. Table. Results of chromatographic analyses of determining free BPA in urine samples collected from patient and after incubation of given sample with β-glucuronidase. sample without β-glucuronidase (BPA with β-glucuronidase (BPA ng/mL) ng/mL)
1. 1.15±0.13 10.22±1.5
2. 0.19±0.02 1.89±0.09
3. 0.81±0.07 1.35±0.08
4. <LOD 1.01±0.08
5. 2.46±0.29 4.79±0.36

Claims

Claims
1. Method for determining bisphenol A in biological material that relies on binding BPA from biological material with antibodies against bisphenol A and subsequent quantitative determining of BPA content, characterized in that, a migration membrane is being prepared to determine BPA from the migrating biological material so that the membrane is divided into at least a zone of binding with a carrier, a detection zone and a control zone while color medium covered with monoclonal or polyclonal antibodies against BPA is placed at the zone of binding with the carrier and the detection zone is divided into at least two detection areas while at the first detection area a selected concentration of antibodies against BPA is adsorbed so that BPA can be detected in the range of 0.5 ng/mL to 50 ng/mL and on the second detection area selected concentration of antibodies against BPA is adsorbed so that BPA can be detected in the 0.5 ng/mL to 50 ng/mL and on the control zone monoclonal or polyclonal antibodies against monoclonal or polyclonal antibodies against BPA are applied and subsequently analysed biological material is being applied at the zone of binding with carrier wherein in case of BPA presence the BPA is being bound with color medium and thus the created complexes of BPA- color medium are directed into the detection zone in which the complexes of BP A-color medium are bound to the antibodies against BPA while the color medium not bound with BPA are directed to the control zone where the color medium is bound with the antibodies against antibodies against BPA while the BPA presence is determined by color indication in at least one detection area of the detection zone and by determination of color at the control zone.
2. Method according to the claim 1, wherein the zone of binding with carrier is an additional carrier membrane and/or reaction vessel free of BPA, on which the color medium covered with the monoclonal or polyclonal antibodies against BPA is placed.
3. Method according to the claims 1 or 2, wherein on the migration membrane an additional membrane is placed to separate blood morphological elements and the additional membrane is made of woven fibers characterized with high water absorptivity higher than 70 mg/cm3.
4. Method according to one of the claims 1-3, wherein the detection zone is divided into three detection areas while at the first detection area selected concentration of antibodies against BPA is adsorbed so that BPA can be detected in the range of 0.5 ng/mL to 50 ng/mL and on the second detection area selected concentration of antibodies against BPA is adsorbed so that BPA can be detected in the range of 0.5 ng/mL to 50 ng/mL while on the third detection area selected concentration of antibodies against BPA is applied so that BPA can be detected in the range of 0.5 ng/mL to 50 ng/mL.
5. Method according to one of the claims 1-4, wherein the detection zone is divided into detection areas so that at every detection area the different maximal amount of antibodies specific against BPA is applied.
6. Method according to one of the claims 1-5 wherein as the migration membrane the membrane made of nitrocellulose and/or cotton and/or glass fibers and/or polyester fibers and/or their composites is used.
7. Method according to one of the claims 1-6 wherein as the color medium color spatial structures made of materials free of BPA, especially latex, rubber, silicone, polystyrene , gold with diameter of single structure >0.1 nm is used.
8. Method according to the claim 2, wherein as the carrier membrane, membrane made of nitrocellulose and/or cotton and/or glass fibers and/or polyester fibers and/or their composites is used.
9. Diagnostic device for detection bisphenol A BPA in biological material characterized in that, the device comprises a migration membrane to determine BPA from the biological material migrating on the migration membrane while the membrane comprises a zone of binding to carrier, a detection zone and a control zone while the zone of binding with carrier contains color medium covered with monoclonal or polyclonal antibodies against BPA and the detection zone contains at least two detection areas so that at the first detection area selected concentration of antibodies against BPA is applied in such an amount that it is able to detect BPA in biological material in the range from 0.5 to 50 ng/mL while at the second detection area selected concentration of antibodies against BPA is adsorbed in such an amount that it is able to detect BPA in biological material in the range from 0.5 to 50 ng/mL while the control zone contains monoclonal or polyclonal antibodies against monoclonal or polyclonal antibodies against BPA.
10. Device according to claim 9, wherein the zone of binding with carrier is an additional carrier membrane and/or reaction vessel free of BPA at which color medium covered with monoclonal or polyclonal antibodies against BPA is placed.
1 1. Device according to the claim 9 or 10, wherein on the migration membrane an additional membrane is placed to separate blood morphological elements and the additional membrane is made of woven fibers characterized with high water absorptivity higher than 70 mg/cm3.
12. Device according to one of the claims 9-10, wherein the detection zone is divided into three detection areas so that at the first detection area selected concentration of antibodies against BPA is adsorbed in such an amount that it is able to detect BPA in biological material in the range from 0.5 to 50 ng/mL while at the second detection area selected concentration of antibodies against BPA is applied in such an amount that it is able to detect BPA in biological material in the range from 0.5 to 50 ng/mL while at the third area selected concentration of antibodies against BPA is added in such an amount that it is able to detect BPA in biological material in the range from 0.5 to 50 ng/mL.
13. Device according to one of the claims 9-1 1, wherein the detection zone is divided into detection areas so that at each detection area different maximal amount of antibodies specific against BPA is adsorbed.
14. Device according to one of the claims 9-12, wherein as the migration membrane, membrane made of nitrocellulose and/or cotton and/or glass fibers and/or polyester fibers and/or their composites is used.
15. Device according to one of the claims 9-13, wherein as the color medium color spatial structures made of materials free of BPA, especially latex, rubber, silicone, polystyrene , gold with diameter of single structure >0.1 nm is used.
16. Device according to claim 10, wherein as the carrier membrane, membrane made of nitrocellulose and/or cotton and/or glass fibers and/or polyester fibers and/or their composites is used.
17. Diagnostic kit for detection of bisphenol A BPA in biological material characterized in thet, the kit comprises: carrier enabling absorption and/or migration of biological material such as membrane and/or reaction vessel and/or multi-well plate and/or capillary,
- monoclonal and/or polyclonal antibodies against BPA, monoclonal and/or polyclonal antibodies against antibodies against BPA,
color carriers covered with monoclonal or polyclonal antibodies against BPA,
β-glucuronidase.
18. Kit according to the claim 17, wherein the carrier contains at least one separate detection zone and at least one separate control zone and preferably the termination zone for test in a form such as strip, capillary, diaper.
19. Kit according to the claim 18, wherein the detection zone enables detection and determination of BPA content in the biological material by bound in the zone monoclonal or polyclonal antibodies against BPA being applied in the concentration to detect BPA.
20. Kit according to the claim 18, wherein the control zone contains monoclonal or polyclonal antibodies against antibodies against BPA.
21. Kit according to the claim 18, wherein the absorption of migrating biological material takes place at a termination zone.
22. Kit according to the claim 17, wherein the color carriers are covered with monoclonal or polyclonal antibodies binding BPA in the biological material in the free form.
23. Kit according to the claim 17, wherein the β-glucuronidase is sprayed at the bottom and/or walls of reaction vessel or added to the membrane and/or added to the biological material in the form of tablets or capsule or powder or solution to transform bound BPA into free form.
24. Kit according to the claim 17, wherein as the carrier it is used a membrane with at least one separate detection zone that contains such an amount of antibodies that enables detection and determination of BPA content in the minimal range of concentrations from 1 to 15 ng/mL.
25. Kit according to the claim 17, wherein as the carrier it is used a membrane with at least one separate detection zone that contains such an amount of antibodies that enables detection and determination of BPA content at the minimal concentration level of 0.5 ng/mL in case of adding β-glucuronidase to the biological material.
26. Kit according to the claim 17, wherein as the carrier at least one separate detection zone that contains such an amount of antibodies that enables detection and determination of BPA content at the minimal concentration level of 0.1 ng/mL is used.
27. Kit according to the claim 17, wherein as the carrier it is used a reaction vessel with at least one separate detection zone that contains such an amount of antibodies that enables detection and determination of BPA content in the minimal concentration level of 1 ng/mL.
28. Kit according to the claim 17, wherein as the carrier it is used a multi-well plate with at least one separate detection zone that enables detection and determination of BPA content in the minimal range of concentrations from 1 to 15 ng/mL.
29. Kit according to the claim 17, wherein as the carrier it is used a microcapillary or set of microcapillaries with at least one separate detection zone that enables detection and determination of BPA content in the minimal range of concentrations from 1 to 15 ng/mL.
30. Kit according to the claim 17 that comprises:
- set of membranes enable absorption and migration of biological material and color carriers covered by monoclonal or polyclonal antibodies,
- at least one separate detection zone that enables detection and determination of BPA concentrations by binding in this areas area/areas proper concentrations of monoclonal or polyclonal antibodies against BPA, while the reaction plate with at least one detection zone contains such an amount of antibodies that enables detection and determination of BPA concentration in minimal concentration range from 1 to 15 ng/mL,
- at least one control zone containing bound monoclonal or polyclonal antibodies against antibodies against BPA,
- termination zone where absorption of biological material and unbound beads covered with antibodies takes place,
- β-glucuronidase sprayed at the bottom and/or walls of reaction vessel or added to the biological material in the form of tablets or capsule or powder or solution and color carriers covered with monoclonal or polyclonal antibodies against BPA.
31. Kit according to the claim 17 that comprises:
- set of membranes enabling absorption and migration of biological material and of color carriers covered with monoclonal or polyclonal antibodies against BPA,
- at least one detection zone that enables detection and determination of given BPA concentrations by binding in this area/areas proper concentrations of monoclonal or polyclonal antibodies against BPA, while the membrane with at least one detection zone contains such an amount of antibodies that enables detection and determination of BPA concentration at minimal level of 0.5 ng/mL,
- at least one control zone containing bound monoclonal or polyclonal antibodies against antibodies against BPA,
- termination zone where absorption of biological material and unbound beads covered with antibodies takes place.
- β-glucuronidase enzyme added to the adsorption membrane.
32. Kit according to the claim 17 that comprises:
- set of membranes enabling absorption and migration of biological material and of color carriers covered with monoclonal or polyclonal antibodies against BPA,
- at least one detection zone that enables detection and determination of given BPA concentrations by binding in this area/areas proper concentrations of monoclonal or polyclonal antibodies against BPA, while the membrane with at least one detection zone contains such an amount of antibodies that enables detection and determination of BPA concentration at minimal level of 0.1 ng/mL,
- at least one control zone containing bound monoclonal or polyclonal antibodies against antibodies against BPA,
- termination zone where absorption of biological material and unbound beads covered with antibodies takes place.
33. Kit according to the claim 17 that comprises:
- reaction vessel as the carrier for detection and control zone,
- at least one detection zone that enables detection and determination of given BPA concentrations by binding in this area areas proper concentrations of monoclonal or polyclonal antibodies against BPA, while the reaction vessel with at least one detection zone contains such an amount of antibodies that enables detection and determination of BPA concentration at minimal level of 1 ng/mL,
- at least one control zone containing bound monoclonal or polyclonal antibodies against antibodies against BPA,
- β-glucuronidase sprayed at the bottom and/or walls of reaction vessel or added to the biological material in the form of tablets or capsule or powder or solution and color carriers covered with monoclonal or polyclonal antibodies against BPA.
34. Kit according to the claim 17 that comprises:
- reaction plate with wells constituting detection zone,
- at least one separate detection zone that enables detection and determination of given BPA concentrations by binding in this area/areas proper concentrations of monoclonal or polyclonal antibodies against BPA, while the reaction plate with at least one detection zone contains such an amount of antibodies that enables detection and determination of BPA concentration in minimal concentration range from 1 to 15 ng/mL,
- at least one control zone containing bound monoclonal or polyclonal antibodies against antibodies against BPA,
- β-glucuronidase sprayed at the bottom and/or walls of reaction vessel or added to the biological material in the form of tablets or capsule or powder or solution and color carriers covered with monoclonal or polyclonal antibodies against BPA.
35. Kit according to the claim 17 that comprises: microcapillars as the carrier, enabling migration of biological material,
at least one detection zone that enables detection and determination of BPA content by binding in this area proper concentrations of monoclonal and polyclonal antibodies against BPA, where microcapiUaries and/or set of microcapiUaries contains at least one detection area with such amount of antibodies that enables detection and determination of BPA concentration in the minimum concentration range of BPA from 1 to 15 ng/mL,
at least one control zone containing bound monoclonal or polyclonal antibodies against antibodies against BPA,
β-glucuronidase sprayed at the bottom and/or walls of reaction vessel or added to the biological material in the form of tablets or capsule or powder or solution and color carriers covered with monoclonal or polyclonal antibodies against BPA.
EP18770405.1A 2017-03-21 2018-03-20 Method for determining bisphenol a in biological material, diagnostic device for detection of bisphenol a in biological material, diagnostic kit for detection of bisphenol a in biological material Pending EP3602057A4 (en)

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PL420935A PL235548B1 (en) 2017-03-21 2017-03-21 Diagnostic kit for detection of bisphenol A (BPA) in biological material
PL424955A PL242100B1 (en) 2018-03-19 2018-03-19 Method for determination of bisphenol A in biological material and the diagnostic device for detection of bisphenol A in biological material, preferably in blood
PCT/PL2018/000030 WO2018174733A1 (en) 2017-03-21 2018-03-20 Method for determining bisphenol a in biological material, diagnostic device for detection of bisphenol a in biological material, diagnostic kit for detection of bisphenol a in biological material

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