AU626794B2 - Improvements in diagnostic test strips - Google Patents

Description

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AU-AI-18028/88 WORLD INTELLECTUAL PROPERTY ORGANIZATION Internitional IBurcau JATIONIA1 APP! TCATIONI P1 If! r~i-r~r~ rN3r-~P~ iaw~ PATPNJT COOPERATION TREATY (PCT~

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(51) International Patent Classification 4 er nubraO 8 92 C2Q 1/26, 1/54, 1/60 U~a 9 ll4 mer O88 92 GO IN 33/49, CO IN 33/53 A (3International Publication Date: GOIN 33/66, 33/92 IS December 198$ (15.12,88) (21) International Application Number: PCT/AU88/00171 (81) Designated States: AT (European patent), AU, BE (Europcan patent), BR, CH (European patent), DE (Eu- (22) International Filing Date-, 6 June 1988 (06,06,88) ropean patent), DK, r (uoenptn) B( ropean patent), IT (European patent), JP, LU (Euro.

pean patent), NL (European patent), NO, SE (Euro- (31) Priority Application Number: PI 2329 pean patent), SU, US.

(32) Priority Date: 5 June 1987 (05.06,87) Published (33) Priority Country: AU With jllterflatioflal earch report.

(71) Applicant (for all designated States except US): NA- TIONAL DIAGNOSTIC PRODtICT (AUSTRAL- IA) PTY, LIMITED CAU/AU]h 12 Stellar- Close, Kilta.

ra, NSW 2071 A.V. JP. 9 MAR 1989 (72) Inventor-, and #for U,,S onlyl BRANSOROVE, An.

thony, Brandon 12 Stellar Close, Killat-a, AUSTRAUIAN (74) Agent: WVATERMARK*, Suite 6, Floor 16, T(own Hall 4 JAN 1989 House, 456 Kent Street, Sydney, NSWN 2000 PATEN o 'C~ (5-4)Title:. IMIPROVEM IENTS I N IAGNQSTIC TEST STRIPS (57) Abstract A diagnostic test device r'ot the detection and quantitation of analyles in the cell- and pirtlclc-rrce fractIon or blolog.

lotl flds, such (is blood serum, The device comprises an absorbent carrier matrix impregnated with the N-agents necessary to bring about a graduated response to specific anal> ces contained in the biological fld. The matrix further serves (is a upotfor an overlying porous inembrane cast onto the upper surface of the inatrix. The mcnibrn'i serves as a filter and aout, wipe of( surface. The reagent-containing matrix membrane composite is Ideally attachcd to one end of a conve.

nient length of an inert support base, such as a plastic strip, which can be used to handle the dt-,lme SWO 88/09824 PCT/AU88/00171 1.

IMPROVEMENTS IN DIAGNOSTIC TEST STEIPS TECHNICAL FIELD Tne present invention relates to a diagnostic test strip device for the detection anl quantitation of biochemical compounds (analytes) in the cell and particle free fraction of whole blood, otherw.e known as plasma or serum.

Rapid diagnosis in clinical chemistry by means of test strips carrying dry reagents has assumed an ever increasina degree of importance, and in more recent times has come to be known as "Dry Chemical Pathology". Comparec with conventional methods dry chemistry tests offer the advantages of economy, speed and simplicity.

Test strips are also commonly used for the detection of substances in urine and other body fluids or may be used outside the medical field for monitoring industrial or aoricultural substances.

CURRENT TECHNOLOGY AND BACKGROUND ART Present devices take the form of inert strips (but not exclusively so) to which are attached a pad of absorbent carrier or a water-stable filr, either of which contain all the reagents necessary for the assay. Tho strip with its reagents is then brouaht into contact with the biological fluid being tested and a colour chanqa develops proportional to the co,icontration of the analyte of interest.

In the case of blood this material may be washed or wiped clear to permit viewing of the eolour change or it y may be left at its site of application with the colour change viewed from the opposite side of the strip. In either case the intensity of the colour development can re read by comparison with standards or by the use of a reflectance colourlmeter.

One groat advantage of this method is that It allows the direct application of a small sample of blood, such as obtained fro, a finoeor orick without the

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SWO 88/09824 PCT/AU88/00171 2.

j intermediate need for either measuring or carrying devices.

1 This inherent simplicity is thus ideally suited for the layman or patients in need of self-monitoring facilities.

J The reaction pad has commonly utilized paper as the absorbent carrier. This material however has jdeficiencies of layer L..ickness, surface regularity and Shomogeneity of composition, all of shich are necessary for precision quantitative tests, and particularly so when using reflectance meters. Thus the cour.e texture of a cellulose or glass fibre matrix will accentuate light scattering I during reflectance measurements.

i Tne introduction of water-stable films according I to DE-1,598,153 overcame many of these problems. These i films are applied as a oolymer solution or dispersion to a substrate. They function primarily as a repository for the I specific reaaents however, they are also homogeneous and I they have a regular and uniform surface which gives minimal Slight scattering. The surface is porous but also sufficiently durable to allow direct wipe off of excess 4 blood.

1 Australian Patent No. 500976 discloser that a j wipe off technique can also be achieved with a paper j absorbent carrier by overlayinq with a polymer stren.thened by cross linkaae. However, this patent does not disclose i any means to overcome the disadvantages of the fibrous I irregular surface.

4 The reaent-containing polymer films or absorbent j pads are selectively permeable to small hydrophylic j molecules absorbino an ultra filtrate (in the manner rf a seml-permeable membrane). When blood is applied proteins, interfering coloured substances, red cells and other material fail to ponotrtte and can be wiped or washed from the surface, These strips are satisfactory for determinations such as* blood glucose, however, they cannot be used for detection o lareoo molecules such as enzymes or proteins and the many hydrophobic substances such an WO 88/09824 PCT/A U88/00171 3 triglycerides or cholesterol which they transport. These molecules do not penetrate, or penetrate only poorly, into the film or pad and so precision assays are not possible.

Various inventions have attempted to address the problem of the failure of Droteins to penetrate water-stable films. Thus in Australian Patent No. 513751, devices known as film openers have been used to increase the porosity of the film.

Although this is satisfactory for serum, it precludes a clean wipe off of whole blood and so interferes with assessment of colour change.

Determents have been proposed in Australian Patent No. 4976R7 as a means of separatina the transported substances such a cholesterol from thec larger carrier proteins. In all cases diffusion of the analyto of interest into the film is slow and imprecise.

In the case of nolymer films, the small pores extend through the full thickness of the film which according to DE-1,598,153 is wet cast with a thickness of 400 microns. This diffusion distance is partly responsible for the slower rate of equilibration of water-stable films and hence the reaction time is longer when compared with the paper absorbent matrix.

DISCLOSURE OP THW INVETIO, The present invention utilizes a much thinner film than heretofore and has thus preserved the regular surface (with its optical advantages) of water-stable films but with enhanced filtering capabilities. It has also transferred the function of reagent carrier to an underlying porous synthetic matrix.

By so separating the filtration function from the reagent repository site finely disoersed soluble material may be incorporated within the substance of te water-stable film at the time of casting, to be later removed by exposure to a suitable aqueous system which otherwise might be detrimental to the reagents. Subseouently the rearents may

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VO 88/09824 PCT/AU88/00171 be applied to the matrix through the membrane.

This improvement of porosity serves the dJal purpose of increasing filtration rate and in turn the reaction speed and allowing access to the matrix by a protein-containing filtrate more representative of blood serum.

Thus according to th.e present invention there is provided a diagnostic test device for the detection and quantitation of analytes ir the cell and particle free fraction of biological fluids.

Such device comp'ising an absorbent carrier matrix impregnated with th. reagents necessary to bring about a graduated response to a specific analyte. The matrix further serves as a s !)porting framework upon which is cast a thin membrane of a water-stable film-forming polymer.

The membrane serves as a filter and a robust wipe off surface. Its porosity is high by virtue of its thinness but this can be further augmented by the inclusion of substances in the casting composition which are soluble under aqueous conditions in which the polymer film is stable.

The reagent-containing matrix membrane composite on its substrate is ideally attached to one end of a convenient length of an inert support base, such as a plastic strip, which can be used to handle the device.

S',MMAPY OF TIHE INVENTION The present invention therefore utilises a wipe off surface which is also a uniquely porous filtration mechanism. This generates a protein-containing filtrate of blood for direct mixing with the reagents. It is thus distinct from other similar test strips which employ an ultra-filtrate. In this manner the bias effect based on molecular site or solubility is minimised. rurthermore, the large pore size causes the filtrate to be imbibed more rapidly and thus the overall reaction speed is faster than N'O 88/09824 PCT/AU88/0017! in other test strips. Finally, because the filter membrane is integral to the whole pad it will not separate and allow red cell penetration and contamination along the edges as occurs with other devices.

BEST MODES FOR CARRYING OUT THE INVENTION The invention includes three systems:- The matrix; The reagent system; and The filter membrane.

i) THE MATRIX This is a thin layer of resilient porous material which by a wetting process moves the filtrate away from the site of blood application on the overlying filter.

The trabecula of this layer function as P substrate, upon which is first deposited the chromogen component of the reagent system, preferably as a poorly-soluble colloid, whilst the pores function as the repository for the analyto specific enzymes and other reagents.

Bibulous carriers with a more homogeneous structure than paper are known in the art, The novel advantage accrues when the benefits of this type of matrix are united with those of a water-stable emulsion or solution.

The Matrix is preferably made from a stspension of hydrophylic polymer natural or synthetic such as cellulose or polyamide. It may also include reagents, chromogen, and reflectants such as Barium salts. The suspension is spread directly onto an inert plastic substrate support in a layer preferably between about microns and 250 microns thick.

The unique and novel property of this layer is the nature of its porosity. The pore size is sufficient to allow complete penetration with ease by the particles of the chromogen and the enzyme dispersion or solution. But it will impede penetration by the larger particles of the filter membrane emulsion.

The porosity of the Matrix is controlled by the !A WO 88/09824 PCT/AU88/00171 casting process. The polymer is preferably soluble ;n an organic solvent from which it can be precipitated by a non solvent such as water. Addition of variable amounts of water under conditions of agitation just prior to casting will cause a nucleation process which then determines the pore size of the Matrix.

Several polymer systems have been found to satisfy this requirement and they can be cast by a dipping process, a chromatography spreader, or a Mayer Bar.

ii) THE REAGENT SYSTEM Generally, any reagent system may be employed provided it is able to react specifically with the analyte of interest and bring about a chromogen colour change.

However, a preferred reagent system for the invention is one which provides for a redox reaction in which 02 is the final electron acceptor and H 2

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2 is generated stoichiometrically by specific oxidases acting on the anlayte of interest. Highly sensitive colourimetric assays of H1 2 2 based on the principle first described by Trinder (1969) can then effectively measure the analyto concentration, A preferred system for glucose comprises glucose oxidaso, peroxidase and a redox indicator such as Benzldino and its derivatives or Phonol/4 aminoontipyrine or others known in the art.

Ini cases where the analytes cannot themselves be oxidised they may first be degraded enzymically to oxidase-susceptiblo products. For instance, cholesterol esters to cholesterol, or triglycerides to glycerol, In addition to enzymes and chromogen the reagent system may include other ingredients such an enzyme stabilizers, detergents and buffers, A preferred reagent system of the present invention has some mutually incompatible components, In order to maintain their separation the reagents can be apoliOd to the matrix as two separate solutions, The first WO 88/09824 PCT/A U88/00171 7, containino the chromogen is dried before application of the second containing specific enzymes, buffer and other reagents.

The reagent system may also contain other ingredients, such as hydrophylic polymers, to aid imbibing of the sample fluid, or protective colloids to aid solubility of other reagents or polymers which aid the colour development and stability of the chromogen, iii) THE FILTER OR TOP LAYER This may take many forms but it is essential that it blocks cellular elements and transfers the fluid! component of blood. This requirement is satisfied in the invention by a novel device which incorporates a water-stable polymer film into the surface of the matrix.

This has been done by exposing the matrix to a dilute solution of polymer emulsion with a particle size range between about I and 10 microns.

A novel advantage of the invention has been the incorporation of soluble inclusions in the polymer solution or dispersion which are later dissovled to leave a more porous membrane. These inclusions can take the form of any soluble or particulate matter which will not denature the film-forminq polymer. Thus near saturated solutions of magnesium chloride will not disturb Proplofan but can be washed clear with ease after film formation.

The required porosity of the membrane filter will greatly influence the choice of film-forming polymer. The cold flow characteristics of many polymer films will quickly cause a closure of pores soon after they are formed. It is therefore important for optimal conditions to use a polymer solution or dispersion with a high glassing temperature and a high film-forming temperature. This generally means a high molecular weight.

The following examples are preferred embodiments of the invention which are not to be construed as at all limiting.

Ji WVO 88/09824 PCT/AU88/00171 EXAMPLU 1 For the production of a test strip to measure blood glucose.

Matrix Preparation gmi of polyamide M~ poly-caprolactar Aidwich Chemical Co.) were dissolved with the aid of mild heat and stirring, in 100 gm of 90% formic acid. When fully dissolved 14m1 of water was injected rapidly Into the vigorously stirred solution and it was immediately .pplied to a 100 micron thick sheet of polyoarbonate film by a dipping process. The sheet of polycarbonate film was then hung vertically and allowed to harden under conditions of relative humidity, After 24 hours the f i IT was I impregnated with the following solutions I and I1 with drying at 60 0 C after the first solution, V SolUtin' I 3,3 5,5 -tetromethybenzidino 100mg IN UCI I.Oml Water Si 6% hydrolysed rantroz AN 149 (GAF) Hydrolysed Gantroz A'N 149 is a protectvkv colloid with acid functions similar to sodium aleginnte, it has the added advantage of solubility at an acid pH., Solution 1I Glucoso oxidase 116U/mg 10.0mg horseradish pvroxidase 300U2/n j polyvinyl pyrrolidone KO0(GAV) 0ilM Sodium phosphate pH7,0 sodium lauryl sulphate 0.ml Polyvinyl pyrrolidno acto to enhance and A stabilize the chromoqan colour aa does OlrSo sodium lauryl stulphato. After applying the second solution and before it dried solution II-, the polyrmer dinperslon for the filter layor, was applied in a 35 micron thick layer und tubsequontly dried with warm air, So4lution TI WVO 88/09824 PCT/AU88/00171 9, polyvinylpropylene polyvinylchloride (Pr piofan 325D BASF) 10. Om 0.ll Sodium phosphate pH7.0 12.Ornl l01 dioctyl sodium sulphosuccinate O.lml The sheet was then slit into 6mm wide strips which are bonded by double-sided transfer adhesive 3mm from one edge of a 75mm wide plastic base. This base with the glucose sensitive support was slit into 6mm, wide strips.

When tested with glucose-containng blood a well graduated response was evident after 30 seconds of blood contact.

When tested with a reflectance meter and whole blood samples of various glucose content, the followinj results were obtained.

('ucoe rrn's/1l~ Motor readini (Microamps) 303 I 27,5 100 25.0 15.0 19.0 200 151 S 300 Bo0 a0 tXAIPtTri 2 For the production of a tOt strip to measure blood joholestooli The matrix preparation accordin-i to Example I wan moiatenod with distilled water and then layered with a micron thick film of the polymer dipsprsov n from Solution I and then dried with warm air at 500C, Solution I sot pOyvinylpropyleno polyvinylhloride copolymer I(Ppiofan 200D BlASP 1O.Oml Freshly precipittod calcium carbonate 4.0 gm 1 RI Sodium phoophate p117.0 1260 ml dioetyl n0d1i' aulphosuceinato Olml Propiofan 200D, hat a hi(Ih ttolocular wtlnht 4nd a f1im WO 88/09824 PCT/AL188/00171 I0 forming temperature of about Following drying the matrix with its attached membrane was exposed to 0.1 N HC1 for 30 minutes after which it was rinsed for 60 minutes in distilled water and dried. It was next impregnated with Solution I from E.- 1 for 30 minutes and dried. Finally, it was impregnated with Solution II, again for 30 minutes and then dried.

Solution II Cholesterol esterac. 75 microns Cholesterol oxidasL 100 microns Horseradish peroxioase 1800 microns polyvinylpyrrolidone K90 (GAF) 0.1 tI Sodium phosphate pH7.0 sodium lauryl sulphate After application of blood this reactive film was wiped clear o7 red cells and after 60 seconds gave a well graduated response to cholesterol-containing blood.

Although the invention has been described above with reference to examples and to preferred embodiments, it will be appreciated that the invention may bo embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The above description is therefore to be considered as in all respects, illustrative and not restrictive, and all chanqes which come within the meaning and range of equivalency are I intended to be embraced therein.

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Claims (7)

1. A diagnostic test device for the detection and quantitation of analytes in the cell- and particle-free fraction of biological fluids, comprising an absorbent porous carrier matrix containing predetermined reagent(s) capable of reacting to the presence of specific analyte(s), and a thin filtration membrane integral with said matrix, the membrane being of a porosity such that It is capable of transferring large molecules present In the biological fluids such as proteins or enzymes to the absorbent carrier matrix. 2, A diagnostic test device according to claim 1, wherein the membrane Is formed from a water-stable film.forming polymer cast onto the matrix, 3, A diagnostic test device according to claim 2, wherein the film-forming polymer casting composition Includes compounds which are soluble under aqueous conditions and may subsequently be dissolved out of the mombranu, t 4. A diagnostic test device according to any one of the preceding claims, wherein the matrix comprises a hydrophllic polymer, such as cellulose or polyamido, 5, A diagnostic test device according to claim 4, wheroln the matrix Includes reagents, chromogen, and optionally roflectants, O.t I

6. A diagnostic test device according to any one of the preceding claims, containing #O reagents for the quantitation of glucose and/or cholesterol. S7. A diagnostic test device according to claim 6, containing reagents for the quantitatlon of glucose, cholesterol and other hydrophillt or hydrophobic blood analytos,

8. A diagnostic test device according to claim 5, wherein the chromogen Includes a 3,3'5,S'-totraalkylbenzldone.

9. A diagnostic test device according to any one of the preceding claims, contal0ed on an Inert support base, 11, A diagnostic test device according to any one of the preceding claims, In which Ssample metering is an Intrinsic capability. S12, A diagnostic test device according to any one of the preceding claims, wherein the substrate on which the matrix Is cast is optically clear, and the monitoring of chromogen colour change is possible from the side of the device opposite to that on which the i biloogl"s iuld Is applied.

13. A diagnostic test device according to claim 2, wherein the film-forming polymer j has a glassing temperature such that cold flow will not adversely affect porosity, S14. A diagnostic test devlceo for the dotoetion and quantitatlon of annlytos In the cell. and particle-free fractions of biological fluids, substantially as hereinbefore described vlth reference to the Examples, 1 A method of manufacturing a diagnostic test device the detection and quantltatlon of analytes In the coll* and particlo-froe fraction of biological fluids, comprising forming a porous matrix on an Inert substrate or support, Impregnating the mnatrx with reagents, chromogens and optionally reflectants pnd forming a thin membrane Integral with the matrix from a wator-stable film.forming polymer cast onto !i a surface of the matrix. e 0 S16. A method according to claim 15 wherein the reagents, chromogons and optionally rofloctants are contained In the matrix when It Is formed on the substrate or support,

17. A method according to claim 15 or claim 16 wherein the film-forming polymer includes compounds which are soluble under aqueous conditions and which may subsequently be dissolved out of the membrane, iI 13 1 8. A method of manufacturing a diagnostic test device for the detection and quantitation of analytes In the cell- and particle-free fraction of biological fluids, subs~tantially as hereinbefore described with reference to the Examples. Dated this 20th day of May, 1992. NATIONAL DIAGNOSTIC PRODUCTS (AUSTRALIA) PTY. LIMITED WATERMARK PATENT TRADEMARK ATTORNEYS FLOOR 4, AMORY GARDENS, 2 CA VI LL AVENUE, ASHFIELD, NEW SOUTH WALES P131, AUSTRALIA, j *1 a *1 a II I 4 4 S S S I S *5 a S S a a. a S A AUI 002888.WPO DOC016 INTERNATIONAL SEARCH REPORT IntgrftelIeftai Atiolicatio. No PCT/AU 88/00171 1, CLA s sic A Tio of ZUMJECT MA TTIA 1 9, $fe4-1 :1611CVo lvgmoo I loilt Act:49 fll Acco,.g aom 41 IAlafm&aloanol 10agent Ctassa9li:a10 cIrCI Of 10 0099M NOItiOA, C181616941110A Ind Ipc Int.C14 C12Q 1/26, 1/54, 1/60l GOIN 33/49, 33/53, 33/66, 33/92 11 F1111,01 11ARCHIO Clallb?ll9, $Ft I CIA1ias9~cilon $?Most$ i 4 C12Q 1/26,1/54,1/60,GOlN 33/52,3/66,33l/92,33/49,31/22 IC WPIL GOIN 33/49, 31/22, C12Q 1/54, 1/26 KEYWORDS:BLOOD,GLUCOSE, Chemical Abstracts: KEYWORD GLUCOSE, BLOOD, *REOOX oacumnon~sIlon 3@?04io 0mf them ?i~nmiiun oticurnmlanSA to Ithe (Il nt 111SCft OQCI4 l'ltitI are Inm9viodd f 160 hori l Soilthti 5a Icj III. OOCUMINT$ MKNIII111 TO E1 AIt.(VANTI C111corl C111Alio f @9 o ea 1 -Ilpv lir i llaim. -ht# .o0tovir~ax, of ate polovapt Iamamg It Alqffn 10 Cie.- 110 11 XP AU-A-76758/87 (LIFESCAN INC.) 18 February 1988, 1-16 (18 .02.88) X AU-B-28564/84 (MILES LABORATORIES INC.) 1-16 6 December 1984 (06.12.84) XY AU-B-58425/86 (MILES LABORATORkIES INC.) 1-16 January 1987 (15.01.87) Y AU-B1-87557/75 (BOEHRINGER MANNHEIM Gmbh) 1-16 23 June 1977 (23,06.77) Y AU-A1-59651/86 (MUREX CORPORATION) 4 Dccember 1986 1-16 (04.12.86) 36(i C6164114 01g~la 116 dh i *tahftsAll. It *T li9So KIfn lt kwspeI' s h~o t t e 1999,9 A iOt!I 041f A ~Si~eA fl qAOS9 ~09 **.ofiil *49* 66 t COA~@A,91 .i9IN fthe 1%.1~9~I sfA'*, 11: NZ 09f~~ the ilt..94 s,,tae at9 th0 am-lf o.oil'#IA40041gft0 trh. 01006160 Of triO*1p y~AdPpAf the Alingwe CAif* 01 top-1144164 A4141 61 t19AOI We 0111166161 to 4 011vinomf ht h 9.ft fir tho9aw 004010O isfito clalmt) of li,*Et**i afsAvaA9,.4 *i#0 .AftA1 tS iqlod It **Ia koIft the 0ii~lCI i f 61 B SA41hf 9 4 **t1fo*Al It 'tVIt 190itt, the tI@,n',g ifist49.6ft

161.6 A a~ ti It.9aIa$A too *Eatifs@0 9jAft0 t9ioge 4# 00itoi-0 OAft *91Alloo silo wRO#A IC d0 i vsl~eA 4460i@ftog to 0909 40s4t9C1i,. 419*. W1100116A Op 4O(Ig4Yilfu9 WI oold E$ .A 0 11A 4 it l tP S S4.ftofO it *9Ag OI 'lfto Fiil. W J I I OM610111160 IVAg 461P0410 Of 0 *St$GA 06.14 WOe MIAR inf, 0ollif 0090 4l~ffl*# -t *ttvIRmfti oviombo*I th time saw litft AM flit CI~1lTIPCATOP* Coto S1 ike' AtiIva Combloia of 10. lAA6lfi*hk1 Sii Oslo a9 W' 4 14. fI IA ifta S14 tw Asian 29 August 1988 (29,08.88) aa -x IAI#'A.9ol looa!(At Autlitt, IIA60 $1 liholifl Ofii.0o AUSTRALIAN PATENT OFFICE IW0HN ASHiMAN ~Ti ANNEX THE IERNATIONAL SEARCH REPORT ON INTERNATIONAL APPLICATION NO. PCT/AU 88/00171 This Annex lists the knci-n publication level patent family members relating to the patent documents cited in the above-mentioned intermational search report. The Australian Patent Office is in no way liable for these particulars which are rarely given for the purpose of information. Patent Dcument Cited in Search Patent Family Members Report AU 76758/87 DK 4191/87 EP 256806 F1 873356 JP 63101757 AU 28564/84 CA 1223183 DK 2706/84 EP 130335 ES 532954 ES 8605102 FI 842189 IL 71704 JP 59228166 NO 842055 US 4543338 ZA 8403494 AU 58425/86 EP 207360 JP 62005182 AU 87557/75 AR 206739 AT 9700/75 BE 836787 CA 1060906 CH 619048 DE 2460903 FR 2295425 GB 1464359 GB 1464360 JP 51089491 JP 54036237 AU 59651/86 BR 8606624 FI 870379 NO 870300 WO 8606978 CN 86103715 EP 206561 ES 555520 ES 8801725 ES 557760 GB 8529067 GB 8528938 ,7D OF ANNEX A tr 23/15o/&/2 L