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
  • G01N33/48—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
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form

Definitions

• the present invention applies, more particularly but not exclusively, to assay methods and devices which aim to assay immunologically reactive substances of clinical interest, such as the assay of an antigen by an antibody and vice versa.
• the present invention is part of a development and improvement of the method and of the metering device described in patent application FR-A-2604526.
• the prior application FR-A-2604526 describes a method for assaying, in particular for assaying an antigen with an antibody, in which particular phases of agitation and result reading. Synthetic or natural micro-particles or micro-spheres are thus used which, during a stirring phase of determined amplitude and frequency, lead to the immunological and specific agglutination of the antibodies and antigens.
• the microparticles or microspheres chosen have the particularity of offering, for a determined wavelength, visible ultraviolet light, a maximum absorption called ⁇ max.
• US Pat. No. 4,240,751 discloses a method for reading samples having some automation and using a reading device, of the colo ⁇ ' meter type with optical fiber and double beams, provided with a reading head. which is moved manually by the operator above each of the samples to be assayed, which are contained in micro-wells of a micro-plate.
• the method of Reading revealed teaches to use a wide reading band, the upper limits of wavelength of which are less than the average diameter of the microparticles likely to be used. It is therefore not mentioned to have recourse to a specific Reading wavelength, such an approach being even discouraged.
• the dosing method described operates on a principle unrelated to that of FR-A-2604526 and requires numerous manipulations in addition to the manual displacement of the read head leading to a relatively long dosing.
• reading devices are already known which are capable of performing automatic reading, in particular by spectrophotometry, of a large number of samples to be assayed.
• These reading devices are for example used in the field of immunoenzymology and make it possible to read the result of the assay specific to each sample, by means of a micro-plate comprising a series of micro-cups in which the series of samples to be assayed is inserted • and held vertically.
• These may be, for example, micro-plates of the ELISA type, the usual function of which is to allow the reading of a series of metric colo ⁇ ' reactions.
• These micro-plates are usually provided to allow the automatic reading of 96 sample tubes.
• the appropriate reading devices ensure, once the microplate provided with the plurality of tubes to be read is placed in the apparatus, the automatic processing Individualized reading of the sample tubes, by emission of a set of optical beams generally passing vertically through each of the tubes. In most cases, a printer is connected to the reading device to allow immediate printing of the reading results.
• This automatic reading technique is, of course, perfectly suited to the rapid reading of a large series of samples, but its use is currently limited to reading colorimetric reactions. In fact, the optical possibilities of the commonly used automatic reading devices are reduced to a limited number of reading wavelengths characteristic of the reading head or crown of each type of device.
• Another object of the invention is to propose a method and an implementation device allowing the adaptation of the method and the device according to the request FR-A-2604526 to the automatic reading techniques allowed by automatic microplate readers. .
• a secondary object of the invention is to propose a method and its implementation device in which the set of operations for preparing and handling the samples to be assayed and for determining the standard range is reduced and simplified, with a view to reduce the total dosing time.
• the object of the invention is achieved by means of a method for assaying a substance of clinical interest which is immunologically reactive with other substances of the same type, of the type assaying of an antigen by an antibody and vice versa, of the type in which :
• an immunologically active substance acting as a metering agent, is grafted beforehand onto natural or synthetic micro-particles to form a metering reagent
• the dosing reagent is mixed with the substance to be measured diluted in an appropriate manner and in a known manner,
• the sample by comparison with the visible ultra-violet absorption results measured at the same wavelength ⁇ max obtained, under the same conditions, on a standard range, characterized in that, with a view to carrying out the assay a plurality of samples in a single pass in the Reading device:
• mixtures of the dosing reagent are carried out with the plurality of samples to be assayed diluted in a known manner in a series of individual volumes, consisting of a series of micro-wells formed in a micro-plate,
• the plurality of results is read by inserting the micro-plate into a reader spectro-photometric and automatic micro-plate programmed on a Reading wavelength close to the ⁇ max characteristic of the micro-particles chosen.
• the object of the invention is also achieved thanks to a device for implementing the method comprising:
• a dosing reagent comprising micro-particles grafted on an immunologically active substance and capable of exhibiting at least an absorption maximum ( ⁇ max) for at least one reading wavelength authorized by an automatic reading device, of the type spectrophotometer,
• Fig. 1 shows a general schematic and perspective view of a micro-plate used in the method and the device according to the invention.
• FIG. 2 shows a top view of a micro-plate and FIG. 2_a shows a partial view of a specific form of distribution of the standard range.
• Fig. 3 shows a general top view of a box according to the invention.
• the main Reading wavelengths that can be displayed are: 340 nanometers, 405 nanometers, 414 nanometers, 450 nanometers, 492 nanometers, 540 nanometers, 620 nanometers, 690 nanometers.
• patent application FR-A-2604526 has shown that if by visible ultraviolet absorption spectrophotometry by placing itself at a wavelength which corresponds to the absorption max ⁇ of the non-aggregated microspheres , a solution containing a mixture of non-agglutinated microspheres and microparticles and a mixture of agglutinates is read. These, the measurement carried out only concerns, and therefore detects, only the non-agglutinated particles, because the latter are the only ones to absorb at this maximum.
• the maximum absorption ⁇ max is therefore a characteristic of the disaggregated microspheres, and this characteristic is directly related either to the size of the particles, or to their constituent material, or to their color.
• micro-particles or micro-spheres have made it possible to demonstrate that, for commonly used reading wavelengths, the size of the micro-particles or micro-spheres, likely to have a max ⁇ at the Reading wavelengths of micro-plate Reading devices, should be between 0.4 and 0.7 micrometers on the one hand, and between 0 , 8 and 1, 2 micrometers on the other hand, for Reading respectively, for example, in the vicinity of 340 nanometers and 405 nanometers.
• Micro-particles or micro-spheres with a diameter of 1 micrometer having, for example, maximum absorption at a Reading wavelength of exactly 405 nanometers.
• microparticles or microspheres with a diameter of 0.5 to 0.6 micrometers in polystyrene, or 0.6 micrometer in vinyl butadiene are also suitable.
• sizes between 0.1 and 0.5 micrometers are also suitable.
• a diameter of 0.9 to 1.2 micrometers is also suitable.
• the adjustment of the max ⁇ of a given microsphere over a wavelength imposed by the reading device does however have a certain tolerance, insofar as i L has been noted that the microparticles or microspheres of a given size could have a ⁇ max varying by plus or minus 5 nanometers around the displayed reading value without harming significantly to the result.
• micro-spheres or micro-particles on their color characteristic and, in this case, The blue color has been found to give good results in the vicinity of 340 nanometers.
• micro-particles or micro-spheres of blue polystyrene with a diameter of between 0.4 and 0.7 micrometers, allow a reading between 320 and 360 nanometers.
• the microparticles must have a spherical shape, must be synthetic, and chosen from the group consisting of synthetic polymers of vinyl toluene, acrylo-nitri Le, styrene butadiene, acrylo-nitrile derivatives- acrylic acid, acrylic esters, silicones, vinyl butadiene and, preferably, polystyrene.
• synthetic polymers of vinyl toluene, acrylo-nitri Le, styrene butadiene, acrylo-nitrile derivatives- acrylic acid, acrylic esters, silicones, vinyl butadiene and, preferably, polystyrene are preferred.
• an immunologically active substance is grafted, in known manner, playing the role of dosing agent, onto said microparticles or microspheres, in view of constituting a dosing reagent.
• micro-plates 1 are generally in the form of a volume defined in the shape of a rectangular parallelepiped comprising a series of micro-cups 2, preferably of cylindrical shape distributed regularly in the mass of the micro-plate 1.
• the volumes defined by the micro-cups are also preferably constant and equal with respect to each other and have an opening, for example circular, opening at the level of the upper face 3 of the micro-plate 1. It It is obvious that other forms of micro-cups 2 are possible.
• These plates are generally made of a transparent plastic material of the polystyrene type.
• the micro-plates 1 comprise a series of eight rows numbered from A to H, distributed over twelve columns, which leads to micro-plates 1 comprising 96 micro-wells.
• the method according to the invention has the particularity of carrying out the mixing of the dosing reagent, therefore containing grafted synthetic microspheres, with the plurality of samples to be assayed (previously diluted in known manner) directly in each of the individualized volumes defined by the micro-wells 2 of the micro-plate 1.
• Each micro-well 2 is thus capable of containing a mixture of a known amount of dosing reagent, with an also known amount of samples to be assayed, without using tubes of reading.
• the points of the standard range 4 are present at the bottom of the micro-cups 2 which are reserved for them, in freeze-dried form and in quantity also pre-calibrated.
• the The user task CONSI 'ste simply reconstitute the standard range of microplate by simply rehydrating the micro-wells 2 containing the points of the standard range 4.
• the points of standard ranges 4 Lyophilized deposited at the bottom of a micro-cup had the particularity, first of all to be distributed naturally in the form of circular rings (fig.
• micro-cups not occupied by the points of Standard range 4
• pre-calibrated quantities of the dosing reagent under dehydrated or even freeze-dried form distributed in a ring or crescent. The user then only has to deposit at the bottom of said micro-wells the required quantities of each of the samples to be assayed.
• a system for closing the microplate 1 which may, for example, consist of a cover 5 held by any means known to those skilled in the art and, for example, by bonding to the upper face 3 from The micro-plate. By simply detaching this cover 5, the user obtains a ready-to-use micro-plate.
• the microplate 1 is subjected to a periodic movement of frequency between 4 and 40 Hertz, preferably between 5 and 25 Hertz and advantageously close to 16 or 20 Hertz, and of amplitude varying from a few millimeters to a few centimeters , preferably of the order of 4 millimeters.
• the purpose of this periodic agitation movement is to carry out the specific immunological agglutination of said micro-particles or micro-spheres contained in each of the incubation media present in each of the micro-wells.
• recourse is had to conventional agitation apparatus well known to those skilled in the art.
• the blocking of each of the solutions is carried out using a blocking solution which is introduced directly into each of the micro-wells.
• a rest time of the order of, for example, 10 minutes the user can then read directly the plurality of concentration results relating to the plurality of samples by direct introduction of the microplate 1 into a reader. spectro-photomét ⁇ 'and that automatic microplate.
• the user must first select
• the spectrophotometric reading apparatus then ensures, by itself automatically and in a single pass of the microplate 1, the individualized reading of each of the samples by emission of vertical optical rays through each of the micros -cupules 2 of the micro-plate.
• the device for implementing the method according to the invention must therefore consist of at least one microplate 1 wherein is provided a plurality of micro-wells 2, and comprise a reagent doser containing micro-particles or micro-spheres grafted onto an immunologically active substance, able to act as dosing agent and capable of present at least an absorption maximum ( ⁇ max) for at least one reading wavelength authorized by an automatic reading device, of the spectro-photometer type.
• ⁇ max absorption maximum
• the implementation device is in the form of a box 6 (fig. 3) containing at least one and, preferably, three micro-plates 1, each of the micro-plates having a standard range 4 which is originally deposited in lyophilized form and pre-calibrated at the bottom of 27 micro-cups.
• the box 6 also preferably presents bottles of a lyophilized buffer solution 7 for the final dilution of the sample.
• the buffer solution is reconstituted by simple rehydration.
• the box 6 can, in addition, include bottles of a blocking solution 8, as well as tubes of control serum 9, for example two, and, finally, tubes of reagents 10 ready for use for the mixing of samples.
• the points of the standard range 4 are not distributed at the bottom of a series of micro-cups, it is obviously necessary to provide, in the box 6, bottles or tubes containing the points of the range standard.
• the kit will not include reagent tubes 10.
• the closure cap 5 it is also possible, if the liquid channel is chosen, to ' distribute the original ready-to-use reagent at the bottom of the micro-wells, the closure cap 5 then ensuring the tightness of the assembly.
• the dosing reagent can finally, of course, be in dehydrated or lyophilized form and be presented in box 6 in tubes or vials.
• the technique used is, moreover, considerably simplified, insofar as the microplate itself is used as a support element for agitation leading to agglutination.
• the automatic reading of the concentration of each of the samples in an automatic reading device allows the user to obtain, almost simultaneously, the concentration results for a large number of samples.
• Example of execution n_ ° _ 1 - The box intended for the simultaneous dosing of a plurality of samples is delivered with three micro-plates, each micro-plate comprising 27 micro-cups reserved for the standard range points distributed over three rows, that is to say 9 points of standard range in triple pre-lyophilized.
• the other 69 micro-wells on each plate, totaling 207 micro-wells, are blank and available for incubation of diluted biological samples. All fluid or liquid distributions can be do this very quickly using multichannel pipettes (8 channels).
• the user simply rehydrates the micro-wells of the standard range, - the user proceeds to the distribution of the biological samples, diluted beforehand, in the virgin micro-wells, at the rate for example of 10 microliters per micro-wells.
• the user distributes, by an eight-channel pipette, the blocking liquid in each of the micro-wells, for example at the rate of 100 microliters.
• the user has, of course, selected the reading filter of the device on the wavelength corresponding to the maximum absorption characteristic of the microparticles of the reagent used.
• Example of realization n ° _2 - Determination of human albumin 1) Distribution of the biological samples: One distributes, in duplicate, in the virgin cups, 10 ⁇ l of the diluted biological samples. For the distribution of 10 ⁇ l of the diluted samples, we use a precision micropipette (capillary pipettes recommended).
• the B2 M SPHEROTEST reagent is vigorously vortexed (1 min).
• the vortexed reagent is placed in a gutter.
• 30 ⁇ l of reagent are distributed, column by column, in all of the columns, except for column 12.
• the microplate is stirred (rotary shaker) immediately after dispensing the reagent:
• the micro-plate is read at a Reading wavelength of 340 nanometers for example.
• NB stability of the micro-plate (crimped with the adhesive film ) : 5 hours (4-8 ° C) a reading made after 5 hours (up to 8 hours) requires rehomogenization of the micro-plate ( stirring 600 rpm - 1 minute).
• n_ ° _3 Determination of human microglobular B2: We proceed exactly as for example n ° 1, making sure to dispense 20 ⁇ l of the reagent (step 3), to incubate for 2 minutes at 1,000 rpm (step 4) and shake the microplate for 2 minutes at 600 rpm (step 5).
• n ° _ 4 Assay of native anti DNA: We proceed as for example n ° 3 taking care, to distribute 20 ⁇ l of biological samples (step 1), and to rehydrate ( step 2) with 20 ⁇ l of water.
• step 4 by distributing 20 ⁇ l of biological samples (step 1) and by rehydrating (step 2) The micro-wells with 20 ⁇ l of water.
• the incubation time (step 4) is exactly 15 seconds at 1,000 rpm, the shaking time of the blocking phase (step 5) being exactly 4 minutes at 600 rpm.
• the invention applies to the assay of antibodies by antigens and vice versa.

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• 1989
  • 1989-08-23 FR FR8911318A patent/FR2651326B1/fr not_active Expired - Fee Related
• 1990
  • 1990-08-23 EP EP90913215A patent/EP0725934A1/de not_active Withdrawn
  • 1990-08-23 WO PCT/FR1990/000623 patent/WO1991002979A1/fr not_active Application Discontinuation
  • 1990-08-23 AU AU63495/90A patent/AU6349590A/en not_active Abandoned

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