Classifications

• • 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
  • G01N33/02—Food
  • G01N33/04—Dairy products
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
  • G01N21/43—Refractivity; Phase-affecting properties, e.g. optical path length by measuring critical angle

Definitions

• the present invention relates to a biosensor based assay method, and, more particularly, to a method for determining the presence and/or amount of an analyte species present in milk.
• milk samples are usually taken by the milk tank lorry driver, both at the milk producing farm and on the tank lorry.
• the sample volumes taken are limited.
• the assay must therefore require only a small sample volume.
• a suspected milk sample can as a rule only be analyzed for a small number of antibiotics due to the limited sample volume.
• Many chromatographic methods, such as HPLC are based on the concentration of a large sample volume for a sufficient sensitivity to be obtained and are therefore usually excluded from the current milk control systems.
• Chromatographic techniques also have the limitations of the requirement of high technical expertise and lengthy and cumbersome sample preparation.
• extraction procedures are critical for the generation of characteristic peaks and to avoid interference from co- eluting material.
• Lengthy and cumbersome sample preparation is also required in the enzyme and radioim unoassay techniques used today for the analyses of analyte species in milk.
• the necessary enzyme or isotope labelling of reagents is, of course, also a disadvantage, both from the viewpoint of sample handling and preparation of enzyme conjugates.
• Microbiological assays like the acidification control used today for detecting antibiotics, are slow (requiring long incubation steps) , and not always reliable. Microbiological assays can, of course, not be used on milk samples to which a preservative, such as e.g. bronopol, has been added in order to prevent growth of microorganisms during storage and transport of the milk sample to the laboratory. Such preservation is used for milk samples collected for a certain type of analysis, e.g. fat, protein and lactose.
• a preservative such as e.g. bronopol
• WO 92/16838 proposes the use of an optical immunosensor based on the surface plasmon resonance (SPR) principle for label-free detection of steroid hormones in various types of samples, including milk.
• SPR surface plasmon resonance
• EP-A2-0389446 discloses an optical measuring device for measuring the protein contents of milk by determining the refractive index of a drop of a milk sample on the surface of a refracting element.
• the present invention aims to provide a method for determining the amount of an analyte species in milk which method requires a minimum sample volume and sample processing to prepare the sample for the assay, while simultaneously being quick and simple to operate and providing for sensitive and accurate measurements.
• the assay may be performed with high sensitivity directly on a raw milk (fresh milk) sample without subjecting the sample to any preparative steps, such as removal of fats etc.
• a raw milk fresh milk
• any preparative steps such as removal of fats etc.
• the present invention provides a method for determining the presence and/or amount of an analyte species in a raw milk sample, which method is characterized by contacting the raw milk sample, without any preparation thereof for removal of milk constituents, with an optical sensor surface and determining the presence and/or amount of the species by measuring a change in refractive index at the surface caused by a specific binding interaction at the surface related to the presence of the species in the raw milk sample.
• the method requires only a small sample volume, no sample preparation and is easy to carry out.
• the method is also apt to automation and may thereby permit screening and/or quantification analyses of a great number of samples with a minimal effort.
• the contacting of the sample with the surface is performed by passing the sample over the surface utilizing a liquid flow system.
• the analyte species or an analyte species analogue is bound to the optical sensor surface.
• the optical surface has an organic polymeric layer, and binding is effected covalently and may take place through a linker molecule.
• the analysis takes the form of an inhibition assay in which an antibody against the analyte species is added to the sample.
• the change in refractive index at the optical sensor surface provides a measure of the amount of the free (i.e. unbound) antibody in the sample from which the concentration of the analyte species can be calculated.
• a receptor such as an anti-analyte species antibody, is bound to the optical sensor surface.
• the amount of analyte species in the sample may then be determined either directly by detecting binding of the analyte species to the receptor, or in a competitive assay by adding to the sample before it is passed over the surface, a substance which also binds to, and competes for, the binding site(s) of the receptor but which produces a greater change in refractive index on binding to the surface.
• a competitive assay by adding to the sample before it is passed over the surface, a substance which also binds to, and competes for, the binding site(s) of the receptor but which produces a greater change in refractive index on binding to the surface.
• the added substance will consist of the analyte species bound to a larger molecule or particle.
• the binding of the analyte species to the optical sensor surface may be detected in a sandwhich assay by a secondary reagent, such as an antibody, which binds to the bound analyte species. If desired, further specificity may be obtained by detecting binding of this secondary reagent by a tertiary reagent which binds to the secondary reagent.
• a secondary reagent such as an antibody
• Binding of the receptor to the surface may be carried out in conventional ways well-known to those skilled in the art. If, for example, the optical surface has a polymeric organic layer at its surface, the receptor may be directly covalently bound to the surface using known linker reagents. Alternatively, an intermediate ligand, such as an antibody, which binds the receptor may first be covalently bound to the surface before this bound intermediate ligand is exposed to the analyte-specific receptor to bind this to the surface. It will be appreciated that covalent and/or affinity bonding may be effective in binding the receptor to the surface. However, when the receptor is bound to the surface, it is important that its ability to bind the analyte species should remain unchanged.
• the term antibody as used herein is to be interpreted broadly.
• the antibody in addition to a whole antibody, may be a fragment thereof, such as an Fab fragment, an Fv fragment, a single chain fragment (scFv) , a single heavy chain or even a peptide (based on the nucleotide sequence of the antibody gene) having binding activity.
• the antibodies which may be used in the invention may be obtained by conventional methods and are many times commercially available. Although polyclonal antibodies may be used in the method of the invention, monoclonal antibodies are preferred for their specificity which enhances the accuracy of the method.
• any analyte species present in milk may be analyzed by the method of the invention.
• exemplary analyte species are drug residues, such as antibiotics and chemotherapeutics, but also hormones, viruses and toxins.
• the measurement of the change in refractive index at the surface may be determined by reflection-optical methods, including both internal and external reflection techniques.
• the measurement is based on evanescent wave sensing, such as surface plasmon resonance (SPR) detection, Brewster angle refractometry, critical angle refractometry, frustrated total reflection (FTR) , evanescent wave ellipso etry, scattered total internal reflection (STIR) , optical wave guide sensors, evanescent wave based imaging, such as critical angle resolved imaging, Brewster angle resolved imaging, SPR angle resolved imaging, etc.
• SPR surface plasmon resonance
• FTR frustrated total reflection
• optical wave guide sensors evanescent wave based imaging, such as critical angle resolved imaging, Brewster angle resolved imaging, SPR angle resolved imaging, etc.
• measurement is based on surface plasmon resonance. This technique is described, inter alia, in EP-A-0305109, EP-A-0267142 and WO-A- 90/05295.
• the optical surface which is used in the measurement based on surface plasmon resonance preferably comprises a gold film and a hydrogel bound to the gold film, as described in WO 90/05303.
• This type of optical surface may easily be regenerated so that a single surface may be used for many analyses. The overall cost per analysis can therefore be reduced considerably.
• Suitable apparatus incorporating such an optical surface is the BIAcore® system available from Pharmacia Biosensor AB, (Uppsala, Sweden) the methods of operation of which are described in the BIAcore® Methods Manual (Pharmacia Biosensor AB) .
• a microfluidic system passes the sample over a sensor chip supporting a gold layer which typically has a thickness of 50 nm.
• a carboxylated dextran is bound to the gold layer via a linker layer. To this dextran layer analyte species or analyte species receptor may be bound depending on the assay format used.
• the method of the invention is conveniently performed directly on a raw milk sample.
• an inhibition type assay for the determination of, for example, an antibiotic in the milk, the milk sample is treated with excess anti-antibiotic antibody and then passed over the optical sensor surface which has the antibiotic or an analogue thereto immobilized thereto.
• Fig. 1 is a diagram showing standard curves (relative response vs concentration) for raw and skim milk analyses of sulfamethazine.
• Fig. 2 is a graph showing typical relative responses for a number of tanker milk samples.
• the sensor surface was activated for 18 min with 25 ⁇ l N-hydroxysuccinimide (NHS) /N-ethyl-N'- (dimethyla inopropyl)carbodiimide (EDC) 1:1, and sulfamethazine was then immobilized to the surface by amine coupling by contacting 30 ⁇ l of sulfamethazine solution with the activated surface for 2.5 h.
• NHS N-hydroxysuccinimide
• EDC dimethyla inopropyl
• any remaining active carboxyl groups were deactivated by placing 25 ⁇ l of ethanolamine in contact with the surface for 18 min, followed by a final wash step.
• the prepared surface was then conditioned for 1 min with 25 ⁇ l 100 M NaOH + 20% DMF and then for 1 min with 25 ⁇ l 100 mM HC1 + 20% DMF.
• the surface was washed three times with 30 ⁇ l HBS buffer between each step.
• the sensor chip was put on a wet paper towel and covered by a box to prevent the chip from drying. Contacting of the sensor surface with the above-mentioned solutions "was performed by carefully pipetting the respective solutions onto the surface from a corner thereof and were gently thrown off after incubation.
• Analyses were performed on samples of (i) non-treated, full fat raw milk (i.e. milk taken directly from the milk tank) and (ii) skim milk. To remove the milk fat from raw milk to obtain skim milk, raw milk was centrifuged at 1000 g for 10 min and the fat on top was discarded.
• Polyclonal antibodies prepared from serum from sulfamethazine immunized rabbit by ammonium sulphate saturation and dialysis against PBS with 0.15 M NaCl, were diluted in HBS to a concentration of 4.5 nM.
• 35 ⁇ l samples of milk/antibody from the above prepared raw and skim milk samples were analysed in the BIAcore® system, using a flow rate of 5 ⁇ l/min. Regeneration of the surface between the different analyses was performed with 15 ⁇ l of 50 mM NaOH, pH 12.2, and 15 ⁇ l of 75 mM HC1.
• standard solutions of sulfamethazine were used to prepare known concentrations of sulfametahzine in milk and HBS buffer. 10 mg of sulfamethazine were dissolved in 10 ml of 50 mM borate buffer, pH 8.5.
• the relative response for milk from a negative milk sample (no sulfamethazine) with 2.25 nm antibodies varied between 800 and 1100 resonance units (RU) , probably depending on the amount of immobilized sulfamethazine and the condition of the sensor chip.
• Standard curves of sulfamethazine (SMZ) in skim milk and full fat raw milk are shown in Fig. 1.
• "A” is negative control (skim milk)
• "B” is full fat raw milk
• "C” is skim milk.
• the differences between raw milk and skim milk were insignificant, indicating that removal of milk fat has no influence on the results, or , in other words, that milk fat did not interfere in the assay.
• Sulfamethazine in milk from a cow treated after morning milk on the 27th of April 1994 with 33 g of the drug sulfamethazine (SMZ) was tested for residues of the drug by HPLC for a number of days after the treatment.
• samples of the milk were frozen and tested later by BIAcore® analysis as described above after thawing and defatting. The results are presented in Table 1 below.
• Tanker milk samples were randomly collected from a screening survey for antimicrobial residues within an integrated control system in Schleswig-Holstein, Germany. All samples were negative in the microbial inhibitor assays (brilliant-black reduction test, a modified Blue Star test and Delvotest SP Special) . The samples were frozen and then analysed for sulfamethazine (SMZ) by the BIAcore® analytical procedure described in Example 1 above.
• SZ sulfamethazine
• Typical responses in the above analysis of tanker milk samples are shown in Fig. 2. These tanker milk samples are all negative and designated A in the graph. Included in the graph are also two positive standard milk samples (1.4 ppb SMZ), designated D; two negative standard milk samples, designated C; as well as one incurred tanker milk sample, designated B.

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• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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• 1994
  • 1994-09-15 SE SE9403078A patent/SE9403078D0/xx unknown
• 1995
  • 1995-09-15 JP JP8510123A patent/JPH10505899A/ja active Pending
  • 1995-09-15 EP EP95932289A patent/EP0781412A1/de not_active Withdrawn
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