Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
  • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
  • B29C59/00—Surface shaping of articles, e.g. embossing; Apparatus therefor
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
  • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
  • B29C35/00—Heating, cooling or curing, e.g. crosslinking or vulcanising; Apparatus therefor
  • B29C35/02—Heating or curing, e.g. crosslinking or vulcanizing during moulding, e.g. in a mould
  • B29C35/08—Heating or curing, e.g. crosslinking or vulcanizing during moulding, e.g. in a mould by wave energy or particle radiation
  • B29C35/0805—Heating or curing, e.g. crosslinking or vulcanizing during moulding, e.g. in a mould by wave energy or particle radiation using electromagnetic radiation
  • B29C2035/0838—Heating or curing, e.g. crosslinking or vulcanizing during moulding, e.g. in a mould by wave energy or particle radiation using electromagnetic radiation using laser
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
  • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
  • B29C59/00—Surface shaping of articles, e.g. embossing; Apparatus therefor
  • B29C59/02—Surface shaping of articles, e.g. embossing; Apparatus therefor by mechanical means, e.g. pressing
  • B29C2059/027—Grinding; Polishing
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
  • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
  • B29C59/00—Surface shaping of articles, e.g. embossing; Apparatus therefor
  • B29C59/02—Surface shaping of articles, e.g. embossing; Apparatus therefor by mechanical means, e.g. pressing
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
  • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
  • B29C59/00—Surface shaping of articles, e.g. embossing; Apparatus therefor
  • B29C59/16—Surface shaping of articles, e.g. embossing; Apparatus therefor by wave energy or particle radiation, e.g. infrared heating

Definitions

• the present invention relates to improved assay supports, particularly microtitre plates or trays.
• Enzyme linked immunosorbent assays were introduced in the early 1970's. These assays are now firmly established as precise quantitative methods for the determination of antibodies and antigens.
• An increasing number of commercial diagnostic procedures are based on ELISAs and biological parameters traditionally measured by radioimmunoassays (RIA) are gradually being replaced by ELISAs.
• Applications for ELISAs have been reviewed (1), (2) and include detection of herpes simplex virus, rotavirus, reovirus and virus diseases of trees and plants. Perhaps the main impact of the ELISA has been in the quantitation of antibodies.
• An initial and crucial step in the ELISA protocol is the hydrophobic adsorption of the antigen or antibody to a substrate such as microtitre trays, dipsticks or beads.
• a substrate such as microtitre trays, dipsticks or beads.
• the most commonly adopted protocol for binding involves incubating a solution (1 ⁇ g.ml -1 ) of the protein at pH9.6, 37°C, 16 hours in wells of a polystyrene microtitre tray.
• the present invention results from the discovery that physical abrasion of the internal surface of the walls of the wells can lead to an increased sensitivity of the immunoassay as well as increased reproducibility.
• the invention embodies the improvement of several performance characteristics of substrates when used in ELISAs after abrasion, exposure to light emanating from a laser or exposure to high voltage.
• the invention provides a substrate having improved binding capacity to protein or other organic molecules characterised in that the substrate has been abraded, exposed to light emanating from a laser or to high voltage.
• the improved binding capacity of substrates afforded by the invention finds application in improving the performance of ELISAs carried out in microtitre trays, microtitre wells, dipsticks, beads, cuvettes, test-tubes and the like.
• the substrates find application in other methodologies involving protein binding such as affinity chromotography and medical prostheses. Suitable materials include polystyrene, PVC and other substances to which proteins are known to bind.
• the invention provides a method of increasing the protein or other organic molecules binding capacity of a substrate which method comprises abrading the substrate, exposing the substrate to light emanating from a laser or to high voltage.
• the wavelength of laser light chosen should be a wavelength capable of being absorbed by the substrate. It is most preferred to employ a wavelength which approximates the peak absorbance for the particular substrate.
• the material most frequently used in ELISAs is polystyrene.
• the far ultra-violet wavelengths have been found most suitable for improving the protein binding capacity of polystyrene. In particular, wavelengths of 193nm, 308nm and especially 248nm have been found satisfactory.
• an electric field strength greater than 1000V.cm -1 is preferred.
• the surface of the support may be treated further with an agent to increase the binding of biologically active molecules thereto, such as glutaraldehyde or other low molecular weight aldehydes and polymers thereof after abrasion or exposure to laser light or high voltage.
• an agent to increase the binding of biologically active molecules thereto such as glutaraldehyde or other low molecular weight aldehydes and polymers thereof after abrasion or exposure to laser light or high voltage.
• Abrasion, in the content of this invention includes roughening, etching and forming a roughened surface by moulding.
• Polystyrene microtitre trays treated according to the invention are capable of binding at least twice as much, and commonly four times as much antibody or antigen as untreated trays. They bind as much antibody or antigen in five minutes as untreated trays do in three hours.
• the range for which the response in the ELISA is linear is extended compared to untreated trays.
• the reproducibility within a tray is improved compared to an untreated tray.
• ELISAs can be performed on samples which could not be performed on untreated trays due to the increased sensitivity of trays treated according to the invention.
• the laser has been employed in two operating modes. Firstly, there is the ablative mode, which causes photochemical change to the surface of the microtitre tray by high energy irridiation of individual wells. The majority of the energy is deposited between about 5ps and about 1ms depending on wavelength. Secondly, the "low fluence mode" is used which involves irradiation of the wells of the ELISA plate with low fluence light i.e. low energy/surface area. Any wavelength of light absorbed by the well surface may be used. The optimum wavelength for this process approximates the peak absorbance of the material. Fluences are usually above about 100mJ.cm -2 in the ablative mode while low fluence usually includes all intensities below about 200mJ.cm -2 .
• the fluence limit depends on wavelength.
• the low fluence light may be applied from a pulsed or continuous source.
• Antibodies and antigens will bind to ablated substrates fn tte presence of detergents. This is not possible with prior-art substrates.
• the high voltage technique generally employs an electric field strength greater than 1000V.cm -1 in the neighbourhood of a well which causes a permanent chemical modification to the well surface.
• a bluish-purple glow occurs near the surface when exposed to the high voltage which concomitantly exposes the surface to ozone and ultra-violet radiation.
• a metal plate under the tray or other substrate during high voltage treatment appears to be beneficial.
• Figures 10a and 10b are a representation of treatment of microtitre trays according to the invention.
• FIGS 11 and 11a illustrate the ablation technique.
• FIGS 12a and 12b illustrate the low fluence technique.
• Figures 13a and 13b illustrate the high voltage technique.
• a microtitre tray was coated with a conjugate of donkey anti-rabbit IgG linked to horseradish peroxidase (Amersham), serially diluted (100 ⁇ l/well) with phosphate buffer (0.1M, pH7). The tray was covered then stored at 37oC for 1.5 hours.
• the conjugate was emptied out of the tray, then it was washed five times with phosphate buffer (0.05M, pH7) containing sodium chloride (0.17M), and Tween (0.05%) (PBS/Tween).
• a microtitre tray was coated with rabbit anti K99 IgG (100 ⁇ l/well) diluted with carbonate buffer (0.1M, pH9.6) for normal, untreated wells, and phosphate buffer (0.1M, pH8) for laser-treated wells.
• K99 is a fimbrial antigen derived from pathogenic E . coli. Typically, a concentration range of 5ng.ml -1 to 10 ⁇ g.ml -1 was used. The tray was covered and allowed to stand overnight at 37°C.
• the IgG solutions were emptied out of the tray, then it was washed five times with PBS/Tween.
• Conjugate (donkey anti-rabbit IgG linked to horseradish peroxidase [Amersham]) was diluted with PBS/Tween (1 in 1000) and then added to the plate 100 ⁇ l/well). The plate was covered and allowed to stand at 37°C for 1.5 hours.
• the conjugate was emptied out of the tray, then it was washed five times with phosphate buffer (0.05M, pH7) containing sodium chloride (0.17M), and Tween (0.05%) (PBS/Tween).
• a microtitre tray was coated with rabbit anti-K99 IgG (0.5 ⁇ g/ml, 100 ⁇ l/well) in carbonate buffer (0.1M, pH9.6) for untreated wells, and in phosphate buffer (0.1M, pH8) for irradiated wells. The tray was covered, then allowed to stand overnight at 37°C.
• the IgG solutions were emptied out of the tray, then it was washed five times with PBS/Tween.
• the tray was coated with K99 antigen serially diluted with PBS/Tween (2ng/ml -1 to 250ng/ml -1 ; 100 ⁇ /well) then covered and allowed to stand at 37°C for 1.5 hours.
• the antigen solutions were emptied out of the tray, then it was washed five times with PBS/Tween.
• the tray was coated with conjugate (rabbit anti-K99 IgG linked to horseradish peroxidase) diluted with PBS/Tween (1 in 800; 100 ⁇ l/well). The plate was covered then allowed to stand for 1 hour at 37°C.
• conjugate rabbit anti-K99 IgG linked to horseradish peroxidase
• the conjugate was emptied out of the tray, then it was washed five times with phosphate buffer (0.05M, pH7) containing sodium chloride (0-.17M), and Tween (0.05%) (PBS/Tween).
• a microtitre tray was coated with LTB (100 ⁇ l/well) diluted with carbonate buffer (0.1M, pH9.6) for normal, untreated wells, and with phosphate buffer (0.2M, pH8) for irradiated wells.
• LTB is the B-subunit of the heat-labile toxin of an enterotoxigenic E. coli. The tray was covered then stored at 37°C overnight.
• the LTB was emptied out of the tray, then it was washed five times with PBS/Tween.
• the tray was coated with rabbit anti-LTB serum (100 ⁇ l/well) serially diluted with PBS/Tween. The tray was covered then allowed to stand at 37°C for 1.5 hours.
• the serum solutions were emptied out of the tray, then washed five times with PBS/Tween.
• the tray was then coated with conjugate (donkey anti-rabbit IgG linked to horseradish peroxidase [Sigma]) (100 ⁇ l/well) diluted with PBS/Tween (1 in 1000). The plate was covered then stored at 37°C for 1.5 hours.
• conjugate donkey anti-rabbit IgG linked to horseradish peroxidase [Sigma]
• the conjugate was emptied out of the tray, then it was washed five times with phosphate buffer (0.05M, pH7) containing sodium chloride (0.17M), and Tween (0.05%) (PBS/Tween).
• a typical 96 well polystyrene tray was used.
• the internal wall surfaces of each well were mechanically abraded by a wire brush rotated within the well to mechanically abrade the walls.
• the amount of antibody adsorbed to the wells was detected using a peroxidase-labelled antirabbit IgG and 2,2'-azinodi-(3-ethy1benzthiazol inesulfonic acid) as substrate. Absorbance values quoted below are the average of triplicates.
• glutaraldehyde Whilst glutaraldehyde is used to treat the surface of the well, it is likely that the polyglutaraldehyde in the glutaraldehyde solution contributes to the binding capacity of the treated wells.
• Figure 2 shows the effect of pH on the conjugate binding capacity of irradiated and untreated wells. It is clear that the binding to irradiated wells is sensitive to pH and that the optimum pH for binding is 8. Untreated wells are relatively insensitive but show a maximum at pH8 to pH9.6. Most protocols require coating at pH9.6.
• microtitre trays were evaluated according to Method 1 above.
• Figure 3 shows a comparison between the binding of conjugate to an ablated and an untreated well on the same plate.
• the conjugate was serially diluted froa 1 in 500 to 1 in 100000. At a dilution of 1 in 500 the ablated plate binds 400X more conjugate than the untreated wells.
• Microtitre trays were irradiated by the same method as Example 3. Columns 9, 10, 11 and 12 were then evaluated as follows.
• Irradiated columns 1 to 5 appeared slightly yellow. The total energy delivered to each well was 4.69J. Columns 7 to 12 of microtitre trays were untreated. Columns 6 and 7 were evaluated according to Method 1 above.
• Figure 8 shows a comparison between a low fluence half plate and the untreated half for a full, double sandwich ELISA as in method 4 above.
• antigen not antibody
• the results in Figure 8 show that the treated plate showed a much higher binding capacity for the antigen at both concentrations and that the irradiated plate reflected the titration of the anti serum. The untreated plate was ineffective.
• Figure 9 shows the comparison between a low fluence half plate and an untreated plate for a half sandwich according to method 2 above. Rabbit anti K99 IgG was adsorbed to the plate and then conjugate was added. Figure 8 shows that the laser treated wells exhibit a higher sensitivity.
• FIGS 10a and 10b schematically illustrate a processing plant for manufacture of microtitre trays having the improved characteristics of the invention.
• Microtitre trays 1 are supplied from a feed system, 2, to a conveyor belt, 3, travelling in the direction of arrows, A.
• the conveyor belt, 3, moves the microtitre trays, 1, passed a treatment zone, 4, and onto a stack, 5.
• a sensor, 6, detects the presence of microtitre trays, 1, at the approprite position and enables the conveyor belt, 3, to be stopped whilst a tray, 1, is aligned in a treatment area, 7, for treatment by laser or high voltage generator, 8.
• the laser ablation technique is illustrated schematically in Figures 11a and 11b.
• the ablation technique requires a tightly focussed beam in each well of the microtitre tray.
• a broad beam, 9, of laser light, preferably of wavelength 248nm, is split by miirors, 10, to narrow beams, 11, which are focussed into the wells, 12, of a microtitre tray, 1, by cylindrical lens, 11.
• the conveyor belt, 3, is stopped by sensor, 6, (shown in Figure 10b) to permit exposure of successive rows of wells, 12.
• the low fluence technique illustrated in Figures 12a and 12b requires an expanded beam to cover an entire microtitre tray.
• a beam of laser light, 9, is reflected by convex mirror 14, to form a diverging beam, 15, which intercepts the whole surface area of microtitre tray 1 on conveyor belt 3.
• the sensor (6 in Figure 10b) enables the conveyor belt 3 to be stopped at the appropriate position such that divergent beam 15 is able to irradiate the whole surface of the microtitre tray 1.
• Lambda Physik EMG 150 ETS This laser was employed at a repetition rate of 25Hz, average power of 5W and a pulse energy of 0.20J. It is a rare gas halide exciter laser. The gases employed were krypton and fluorine.
• Figure 13a shows a high voltage probe 16 placed in proximity to a well 12 of a microtitre tray 1 which is supported on a metal plate 17.
• Discharge 18 eminates from the probe 16 and modifies the well 12 so as to improve its protein binding capacity.
• Figure 13b illustrates a Tesla coil which is a preferred method of generating the high voltage suitable for treating microtitre trays as illustrated in Figure 12a.
• a high voltage 19, usually in the order of 10 to 30kV is applied across a condenser 20.
• the circuit includes a spark gap 21 and transformer 22 having a very short coil 23 wound with a long coil 24 associated with high voltage output 25.
• the substrates of the invention find use in any application where proteins or other organic molecules need to be bound to a solid surface.

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• 1986
  • 1986-10-31 WO PCT/AU1986/000329 patent/WO1987002619A1/en unknown
  • 1986-10-31 NZ NZ21812886A patent/NZ218128A/en unknown
  • 1986-10-31 EP EP19860906245 patent/EP0245310A1/de not_active Withdrawn

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