AU607698B2 - Antigenic analogues of platelet activating factor (paf) - Google Patents

Description

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PCT

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 87/ 05904 C7F 9/10, G01N 33/92 A l (43) International Publication Date: 8 October 1987 (08.10.87) C07K 15/12 (21) International Application Number: PCT/AU87/00084 (74) Agent: BERRYMAN, Patents Section, ICI Australia Limited, I Nicholson Street, P.O. Box 4311, (22) International Filing Date: 24 March 1987 (24.03.87) Melbourne, VIC 3001 (AU).

(31) Priority Application Number: PH 5175 (81) Designated States: AU, DE (European patent), FR (European patent), GB (European patent), IT (European (32) Priority Date: 24 March 1986 (24.03.86) patent), JP, KR, US.

(33) Priority Country:

AU

Published With international search report.

(71) Applicants (for all designated States except US): THE UNIVERSITY OF SYDNEY [AU/AU]; Parramatta Road, Sydney, NSW 2000 MACQUARIE UNI- VERSITY [AU/AU]; NSW 2109 ROYAL NORTH SHORE HOSPITAL AND AREA HEALTH SERVICE[AU/AU]; St. Leonards, NSW 2065

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(72) Inventors; and Inventors/Applicants (for US only) BALDO, Brian, A Angelo [AU/AU]; 14 Canisius Close, Pymble, NSW 2073 REDMOND, John, William [AU/AU]; 23 Mirool Street, West Ryde, NSW 2114 (AU).

(54) Title: ANTIGENIC ANALOGUES OF PLATELET ACTIVATING FACTOR (PAF) j

I

j d I i i i i i.

j _1 This document contains the amendments made under Section 49 and is correct for printing.

CH

2

-O-R

1 -x 2 1

R

2 COO C H 0~ CH2C -NR R R (57) Abstract Antigens for the production of antibodies to Platelet Activating Factor (PAF). The antigens are PAF analogues of formula wherein X comprises a high molecular weight group, R is a linking group and R 2 to R 5 are selected from Cl to

C

6 alkyl. Other aspects of the invention include PAF-antibodies produced using said antigens, labelled PAF analogues, intermediates for the preparation of PAF analogues and methods and a kit for the immunoassay of PAF.

1- i ii: WO 87/05904 PCT/AU87/00084 ANTIGENIC ANALOGUES OF PLATELET ACTIVATING FACTOR (PAF) Technical Field The present invention relates to novel antigens capable of producing antibodies to Platlet Activating Factor (PAF), novel PAF analogues labelled to enable quantitative assay, intermediates for the production of novel PAF antigens and methods for the preparation of said antigens, and methods of immunoassay of PAF in biological fluid using said labelled analogues and/or labelled PAF-antibodies.

Background Platelet Activating Factor (PAF), 1-0-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine, represents a recently defined example of a class of biologically-active lipids active in the subnanomolar range and possessing a wide spectrum of pathophysiological effects. PAF promotes life -threatening anaphylactic reactions in animals and is suspected of mediating a range of allergic and inflammatory reactions in man. For example, PAF may be important in conditions such as asthma, adult respiratory distress syndrome and shock reactions. However, despite the increasing catalogue of conditions in which PAF maybe involved, greater insights into its role in health and disease are hampered because precise and specific methods 11 Ku.

WO 87/05904 2. PCT/AU87/00084 for its measurement are lacking. The capacity of PAF to" aggregate platelets does not provide a suitable basis for strictly quantitative assay.

It would be desirable to develop an immunoassay for quantitative determination of PAF levels in blood serum. However, it has been found that PAF itself is insufficiently antigenic to produce the necessary PAF-antibodies needed for such an immunoassay.

Novel synthetic PAF analogues have now been found which are sufficiently antigenic to produce PAFantibodies and a method suitable for the immunoassay of PAF levels in biological fluids has been developed.

The Invention Accordingly the invention provides novel compounds of general formula (I) 1 i CH R -X 2 1 R2COO H

O

CH -O-P-O-CH 2

-CH

2 -N R 3

R

4

R

5 0 wherein: R is a C 2 to C25 alkylene or alkenylene linking group substituted by radioactive iodine; X is hydrogen;or WO 87/0504 PCT/AU87/00084 R' is a C 2 to C 25 alkylene, alkenylene or alkynylene linking group optionally substituted by tritium or radioactive iodine; X is selected from: the group consisting of formyl, di(C 1 to C alkoxy)methyl, carboxy, isothiocyanato, N-C 1 to

C

6 alkylamino, N,N-di(C 1 to C alkyl)amino, hydroxy and mercapto; and the group -A-B wherein A is a linking group selected from the groups -NR -COO-, -OCO-,

-CONR

6 -NR6CO-, -NH-CS-NH- and wherein R is selected from hydrogen and C 1 to C 6 alkyl; and B is selected from: monofunctional and polyfunctional protein peptide, carbohydrate and lipid groups and derivatives thereof of molecular weight of at least 2000; and (ii) a label; and

R

2 to R 5 are independently selected from C 1 to C 6 alkyl; and mixtures of the compound of formula and its enantiomer.

In one embodiment the invention provides antigenic PAF analogues of general formula wherein: R is a C 2 to C 25 alkylene or alkenylene linking group; 2 Now' PCT/AU87/00084 I x :I I-a WO 87/05904 X is the group -A-B wherein: A is a linking group selected from -NR -C -OCO-, -CONR 6 -NR6CO- and wherein R 6 is selected from hydrogen and C 1 to C 6 alkyl; and B is selected from monofunctional and polyfunctional protein, peptide, carbohydrate and lipid groups and derivatives thereof of molecular weight of at least 2000 which are capable of eliciting an antigenic response; and

R

2 to R 5 are independently selected from C 1 to C 6 alkyl.

In the antigenic PAF analogues of the invention of general formula -Preferred R 1 include straight chain C 4 to C 16 alkylene.

More preferred R 1 include straight chain C 4 to C 8 alkylene. For convenience R 1 is often chosen from pentylene and hexylene.

-Preferred A include -NR 6 -COO-, -OCO-, -CONR 6 and

R

6 CO- and preferred R 6 include hydrogen and methyl. More preferred A include -NR 6 and -OCO-.

-Preferred B include monofunctional and polyfunctional protein, peptide, carbohydrate and lipid groups of molecular weight at least 5000 and capable of eliciting an antigenic response. More preferred B include monofunctional and polyfunctional groups of molecular weight at least 10,000. Examples of suitable B include Bovine Serum Albumen (BSA), ovalbumen, Porcine Thyroglobulin (PTG), Bovine Thyroglobulin (BTG), keyhole 16 t i i; i6 t i

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WO 87/05904 PCT/A U87/00084 limpet haemocyanin, bacterial cell walls, synthetic polypeptides such as polylysine, poke weed mitagen (PWM), phytohaemoglutinin (PHA), muranyl dipeptidase and lipopolysaccharides.

-Preferred R 2 to R 5 include C 1 to C 3 alkyl, and especially methyl.

In another embodiment the invention provides labelled PAF analogues of general formula wherein:

R

1 is a C 2 to C 25 alkylene or alkenylene linking group substituted by radioactive iodine; X is hydrogen; or

R

1 is a C 2 to C 25 alkylene, alke:hylene or alkynylene linking group; X is a group of formula -A-B wherein: 6 A is a linking group selected from -NR -COOf -OCO-, -CONR 6 -NRCO-, -NH-CS-NH-and -S-Swherein R 6 is selected from hydrogen and C 1 to

C

6 alkyl; B is a label; and

R

2 to R 5 are independently selected from C 1 to C 6 alkyl.

In the labelled PAF analogues of the invention of general formula I wherein X is hydrogen: -Preferred R 1 include straight chain C 4 to C 16 alkylene or alkenylene substituted by radioactive iodine.

-Preferred R 2 to R 5 are methyl.

j i.

i i r 6.

WO 87/05904 PCT/AU87/00084 In the labelled PAF analogues of the invention of general formula I wherein X is a group of formula -A-B: -Preferred R 1 include straight chain C 4 to C 16 alkylene, alkenylene or alkynylene. More preferred R 1 include straight chain C 4 to C 8 alkylene.

-Preferred A include -NR 6 -CO0-, -OCO-, -CONR 6 and -NR6CO- and preferred R 6 include hydrogen and methyl.

More preferred A include -NR 6 and -OCO-.

-In this specification, "label" is used to mean conventional labels used in immunoassay procedures including the radioactive isotope labelled groups based 125 125 125 on 1I-histamine, I-tyramine, I-tyrosine methyl 125 ester and I-Bolton Hunter Reagent; enzymic labels; and photometric labels. Specific examples emzymic labels include horseradish peroxidase, alkaline phosphatase, betagalactosidease and urease. Specific examples of photometric labels include fluorescent groups such as fluorescein and its derivatives, rhodamine .and its derivatives, phycoerythrins, europium, "Texas Red", luminescent labels such as luminol and its derivatives, acridinium esters and umbelliferins.

2 5 -Preferred R to R are C 1 to C 3 alkyl, especially methyl.

In another embodiment the invention provides 1 WO 87/05904 PCT/AU87/00084 compounds of general formula which are useful as intermediates for the preparation of the antigenic PAF analogues of the invention wherein:

R

1 is a C 2 to C25 alkylene, alkenylene or alkynylene linking group; and X is selected from the group consisting of formyl, carboxy, di(C 1 to C 6 alkoxy)methyl, N-C 1 to C 6 alkylamino, N,N-di(C 1 to C 6 alkyl)amino, hydroxy and mercapto.

In the intermediate compounds of the invention of general formula I: -Preferred R 1 include straight chain C 4 to C 16 alkylene, alkenylene and alkynylene. More preferred R 1 include straight chain C 4 to C 8 alkylene.

-Preferred X include formyl, carboxy, dimethoxymethyl and hydroxy.

In another embodiment the invention provides a process for the preparation of compounds of general formula which process comprises: reacting: a compound of general formula (II) i CH -O-R 1

-G

2

R

2 COO H II

CH

2

-H

2 r~ i. e -i7 '7 WO 87/05904 8 PCT/ALU87/00084 wherein RI and R 2 are as hereinbefore defined and G *is selected from di(C 1 to C 6 alkoxy)methyl and groups which may be reacted, using conventional methods, to give a group selected from formyl, di(C 1 to C 6 alkoxy)methyl, carboxy, amino, N-C to C6 alkylamino, N,N-di(C 1 to C 6 alkyl)amino, hydroxy and mercapto; a phosphorylation agent; and an N,N,N-tri(C to C 6 alkyl)ethanolamine derivative to give a compound of general formula (III) 1 CH -O-R -G 2 R 2COO am- C- H 0 1II+ 3 4 CH -0-P-0-CH -CH -N R3R4 R 2 2 2

U_

reacting the product of to convert group G as hereinbefore defined to a group selected from formyl, di(C 1 to C 6 alkoxy)methyl, carboxy, amino, N-C to Cs alkylamino, N,N-di(C 1 to C 6 alkyl)amino, 1 6 6 hydroxy and mercapto and to introduce the desired group X.

In a specific example of the process for the preparation of compounds of general formula ,i1 .WO 87/05P04 PCT/A U87/00084 a compound of general formula wherein G is dimethoxymethyl, R 1 is selected from C 4 to C 16 alkylene, alkenylene and alkynylene and R 2 is methyl, is reacted with phosphorus oxychloride and choline tosylate to give a compound of formula (III), wherein G is dimethoxymethyl, R 1 is selected from C 4 to C 16 alkylene, alkenylene and alkynylene, and R 2 to R 5 are methyl; and the product of is reacted with- acid to give a compound of formula wherein X is formyl and R 1 to R 5 are as hereinbefore defined, which is reacted with a protein or synthetic peptide followed by reduction of the resulting imine to give a compound of general formula wherein R 1 to R 5 are as hereinbefore defined and X is the group -A-B wherein A is the linking group -NR in which R 6 is hydrogen and B is a protein-or synthetic peptide.

It will be recognized by those skilled in the art that in those antigenic PAF analogues of general formula I the group B may be monovalent or polyvalent such that a plurality of residues of general formula typically between 1 and 500 and usually between 2 and 20, are attached to each group B. Therefore, in those antigenic PAF analogues of general formula I in which X is the group if the residue of formula is represented by Z then the invention includes antigenic PAF analogues of formula (Z)nB wherein n is an integer from 1 to 500.

i j

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r r i r 1 i 1

I

r

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i i f: Id-Cr -i

-S.

-i I c WO 87/05904 PCT/AU87/00084 It will also be recognized by those skilled in the art that certain of the PAF analogues of general formula may be non-covalently bonded to or adsorbed onto a solid support. Accordingly in another embodiment the invention provides supported PAF analogues comprising PAF analogues of general formula wherein:

R

1 is a C 2 to C 25 alkylene or alkenylene linking group substituted by radioactive iodine; X is hydrogen; or

R

1 is a C 2 to C 25 alkylene, alkenylene or alkynylene linking group optionally substituted by tritium or radioactive iodine; X is selected from: the group consisting of formyl, di(C 1 to C 6 alkoxy)methyl, carboxy, isothiocyanato, N-C l to

C

6 alkylamino, N,N-di(C 1 to C 6 alkyl) amino, hydroxy and mercapto; and the group -A-B wherein A is a linking group selected from the groups -NR -COO-, 6 -OCO-,-CONR 6 -NRCO-, -NH-CS-NH- and -S-Swherein R 6 is selected from hydrogen and C 1 to

C

6 alkyl; and B is a label; and

R

2 to R 5 are independently selected from C 1 to C 6 alkyl; non-covalently bonded to or adsorbed onto a solid support material.

Examples of solid support materials for said supported PAF analogues include proteins, synthetic polypeptides (eg polylysine) carbohydrates and carbohydrate derivatives nitrocellulose, agaroses ;1 ii t g it I iiL WO 87/05904 1 PCT/AU87/00084 such as "Sepharose" (Trade Mark), and lipopolysaccharides] and synthetic polymers such as, for example, polysulphones, polyamides polyacrylamide, nylon 6, nylon 66, nylon 610) and polystyrene in the form of particles, balls or formed articles such as test- -tubes, rods, tubes, fins, wells, beads, disks, slides, plates and micro-titre plates.

Although PAF itself has been found to be insufficiently antigenic to produce the PAF-antibodies required to develop an immunoassay for PAF, surprisingly it has been found that: PAF adsorbed onto or non-covalently bound to a monofunctional or polyfunctional protein, peptide, carbohydrate, lipid or a derivative thereof of molecular weight at least 2000 and capable of eliciting an antigenic response; and the antigenic PAF analogues of general formula stimulate the production of antibodies which are antibodies to PAF. Accordingly in a further embodiment the invention provides antibodies to PAF and methods for their production. Such antibodies, hereinafter referred i to as PAF-antibodies or anti-PAF, may be prepared by those techniques known in the art and conventionally involve introducing an antigenic PAF analogue of general formula into an animal such as a rabbit, mouse, donkey, sheep, etc. to produce antibodies to the antigen and isolating and purifying the antibodies. The 'ii PAF-antibodies of the invention may be labelled with any of the conventional labels used in imunoassay

NT

WO 87/05904 1 2 PCT/AU87/00084 Sj procedures. Such labels include, for example, radioactive labels, enzymic labels and photometric labels such as those hereinbefore described.

The PAF antibodies of the invention include both monoclonal antibodies and polyclonal antibodies and techniques known in the art may be utilized to prepare the required type of antibody. For example, monoclonal antibodies may be produced using the antigenic PAF analogues of general formula of the invention by the techniques taught by G. Kohler and C. Milstein, Nature,256, 495-497 (1975).

The PAF analogues and PAF antibodies of the invention may be used to qualitatively and quantitatively analyse for the presence of PAF in biological fluids.

Accordingly in a further embodiment the invention provides methods for the immunoassay of PAF in biological fluids using the PAF analogues and/or PAF-antibodies of the present invention.

In one method PAF or PAF analogue is immobilised on a solid support and reacted with labelled or unlabelled PAF-antibodies in the presence of known amounts of competing free PAF to generate a graph showing percent inhibition versus PAF concentration. If, unlabelled PAF antibody is used the antibody bound which binds to the first is detected by using a labelled second antibody (goat, donkey, sheep, etc.). Using this graph the amount of free PAF in biological fluids may be quantitatively measured.

In another method, unlabelled anti-PAF bound to a wI^ WO87/05904 WO 87/05904 PCT/AU87/00084 solid support is reacted with a polyvalent antigenic PAF analogue of formula B PAF-polylysine). The resulting complex is then determined using labelled anti- PAF which binds to free PAF residues on the polyvalent antigenic PAF analogue.

In another method, unlabelled anti-PAF bound directly, either covalently or non-covalently, to a solid phase such as magnetized particles, plastic tubes, micro-titre plates,"Sepharose" (Trade Mark) particles, polyaerylamide particles, nylon or polystyrene balls, etc. is mixed in a competition assay with: a known quantity of labelled PAF; and known quantities of unlabelled PAF contained in standard solutions or PAF to be measured in an extract or biological fluid. The concentration of unlabelled PAF in the sample is then determined from a standard curve, for example from a logit/log standard plot.

4 In another method, the procedure above is used except that the anti-PAF is linked to the solid phase by a ligand such as an antibody, protein A, lectin or an enzyme, for example: -solid phase/sheep (or some other species) anti-rabbit(or mouse etc.) immunoglobulin/rabbit (or mouse etc.) anti-PAF; and -solid phase/protein A/rabbit (or mouse etc.) anti-PAF.

In another method, anti-PAF, labelled PAF and PAF to be measured are mixed and the free PAF and antibody-bound PAF are separated using dextran-coated charcoal or some other solid phase adsorbent such as hydroxyapatite etc.

The concentration of unlabelled PAF in the sample being

I

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MONSOON"-

I r"T A /l 1 t

I

-r-I WO 7/ 90 PTA 1U870l--. I WO 87/05904, PCT/AU8700084 measured is then determined from a standard curve.

In another method, anti-PAF/PAF complexes are precipitated with a second antibody or with a protein precipitating reagent such as ammonium sulphate.

Again, concentrations of unlabelled PAF may be determined from a standard curve.

In a further embodiment the invention also provides a kit for the immunoassay of PAF in a biological fluid said kit comprising PAF-antibodies of the present invention.

In practice, it has been found that the PAF present in biological fluids such as blood serum is rapidly degraded by the enzyme PAF-acetylhydrolase which is also normally present in blood serum. Therefore, it is preferable to first deactivate the enzyme. Three methods have been published for the deactivation of the enzyme, namely use of IN hydrochloric acid.,_use of diisopropylfluorophosphate, and use of phenylmethanesulphonyl fluoride, but these methods suffer -the disadvantages of use of drastic conditions and/or toxic substances.

It has now been found that the addition of a detergent to the biological fluid sufficently deactivates the enzyme to enable PAF to be quantitatively determined.

Therefore, in a further embodiment the invention provides a method of immunoassay of PAF in biological fluid which comprises diluting the fluid with an aqueous detergent solution, prior to subjecting the diluted fluid to an immuiassay. Preferably the detergent is a non-ionic detergent, such as those selected from the group

I.

i.

I-

1. j WO 87/05.904 15 PCT/AU87/00084 consisting of: polyalkylene glycols; alcohol, phenol and alkylphenol alkoxylates; castor oil alkoxylates; the partial esters derived from long chain fatty acids and hexitol anhydrides and their alkoxylates; long chain alcohol polyglycol ether acetals; alcohol sugar acetals; and the lecithins. Detergents such as "Nonidet" P40 and "Triton" X100 (Trade Marks) have been found particularly useful.

Industrial Applicability It will be evident to those skilled in the art that the products and methods of the invention find particular use in the medical and veterinary fields for the analysis of PAF.

ii.. WO 87/05904 1 6 PCT/AU87/00084 Preferred Embodiments Embodiments of the present invention will now be described by way of example only.

Example 1 Preparation of 2-O-Acetyl-l-O-(6',6'-dimethoxvhexyl)sn-glyceryl-3-phosphorvlcholine 1. 1,l-Dimethoxycyclohexane.

A mixture of cyclohexanone (52 ml, 0.5 mol), trimethylorthoformate (66 ml, 0.6 mol), methanol (51 ml, 1.26 mol) and concentrated H2SO4 (1 drop) was refluxed for 18 hours. A solution of sodium methoxide in methanol was added until the mixture was neutral, and the mixture was fractionally distilled. 1,1-Dimethoxycyclohexane was i obtained from the fraction b.p. 162-1640C (50.6 g, 2. 1-Methoxvcyclohexene.

1,1-Dimethoxycyclohexane (25 g, 0.174 mol) was heated with p-toluenesulfonic acid (35 mg) at 140°C for 3 hrs.

Methanol was distilled off during the reaction.

The residue was fractionally distilled, yielding 1methoxycyclohexene (15.2 g, 80%) b.p. 144-146C.

1 i. -i:

~I;

i:iiii SWO 87/05904 7 PCT/AU87/00084 3. Methyl 6.6-dimethoxyhexanoate.

A solution of 1-methoxycyclohexene (4.5 g, 0.04 mol) in methanol (140 ml) was ozonolysed at 0°C until the uptake of ozone ceased. The solution was degassed and a suspension of reduced Pd/CaCO3 g) catalyst in methanol (30 ml) was added. The mixture was filtered through celite, and the filtrate was evaporated. Trimethylorthoformate (7 ml, 0.06 mol), methanol (5 ml, 0.12 mol) and conc.H2SO4 (1 drop) were added to the residue. After 17 hours, the mixture was neutralized with sodium methoxide solution and then fractionally distilled. Methyl 6,6-dimethoxyhexanoate was collected as the fraction b.p. 80-90 0 C/1.0 mm (4.1 g, 54%).

4. 6,6-Dimethoxyhexan-l-ol To a stirred mixture of lithium aluminium hydride (3.8 g, 0.1 mol) in ether (80 ml) under nitrogen, was added methyl 6,6-dimethoxyhexanoate (15.0 g, 0.079 mol) in ether (50 ml) at a rate to maintain reflux (ca.1.5 hr). The mixture was further refluxed for 1.5 hrs., and then cooled to 0 C. Sodium hydroxide solution (13 ml, 7 M) was added dropwise while cooling in ice. After stirring for 1 hour, the mixture was filtered through a layer of magnesium sulfate. The residue was washed with ether, and the combined filtrates were evaporated. The residue was ":i L, i:i l_ C i-- SWO 87/05904 18 PCT/AU87/00084 subjected to "suction" chromatography.

6,6-Dimethoxyhexan-l-ol was eluted with 25% ethyl acetate in light petroleum (10.3 g, 5. 2-O-Acetyl-3-0-benzl-l-0-(6' ,6'-dimethoxyhexyl) -sn-qlycerol Sodium hydride dispersion (0.377 g, 12.6 mmol, in oil) was washed with dry ether under nitrogen.

The residue was resuspended in dry DMF (30 ml), and 6,6-dimethoxyhexan-l-ol (1.62 g, 10 mmol) was added.

The mixture was heated at 80°C for 1.25 hr., during which time the sodium hydride reacted. (Benzyloxy)-2,3-epoxypropane (1.64 g, 10 mmol) was added and heating was continued for 2 hr. Upon cooling, water (100 ml) was added and the mixture was extracted with ether (100 ml, 2 x 40 ml). The combined extracts were washed with water (2 x 80 ml) and brine (100 ml), dried (MgSO4) and evaporated.

The residual oil (2.8 g) was dissolved in chloroform (36 ml), and cooled to 0 C. Pyridine (3.5 ml, 43 mmol) and freshly distilled acetyl chloride (0.94 ml, 13.2 mol) were added. The mixture was stirred for 0.5 hr. at 25 C, then 2 hr. at room temperature o t I m .m mmm, ;1 SWO 87/05904 PCT/A U87/00084 I WO 87/05,904 WO 87/5.904PCT/A U87/00084 Tce water (100 ml) was added and the layers separated. The aqueous layer was extracted with chloroform (2 x 40 ml), and the combined organic phases were washed with water (100 ml) and brine (100 ml), dried (MgSO 4 and evaporated. The residue was subjected to chromatography and the product was eluted with petroleum ether-ethyl acetate Evaporation of this fraction yielded the product as a colorless oil (1.82 g, 50%) b.p. 170 0C/0.2 mmHg (C 20 'A 3 2 06 requires C, 65.19; H, 8.75%, Found: C, 65.06%; I ~D 1 .98 0 (c 5.06, benzene).

R N.M.R.C 7.36, m,5, ArH; 5.7q1L 5.0 Hz, H2; 4.50,d,2, I 2.5 Hz, benzyl; 4.36, t,1,2 5.7 Hz, -CH(OMe 2 3.62,d,2,2 5.0 Hz, H3; 3.58,d,2,!l 5.2 Hz, 15OC2; 3.8-3.39,m,2,H1; 3.31,s,6,OCH3; 2. 12,s, 3,COCH 3 1.72-l.26,m,8,-CH 2 Mass spectrum: m/e 337, 305, 287, 245, 229, 215, 207, 146, 117, 113, 111, 91, 81, 75, 72.

6. 2.'-0Acetv1-1-0-(6' .6 -dimethoxyhexyl) -sn-cglvcerol 2-0-Acetyl-3-O-benzyl-l-0-(6 6 '-dimethoxyhexyl) -a- -glycerol (369 mg, 1.0 mmol) was hydrogenated in THF ml) over Palladium/carbon (14 mg, 10%) until the uptake of hydrogen ceased (approx. 2.5 The solution was filtered through celite, and t',e filtrate was evaporated to yield a colourless oil (278 mg, 100%) which was used immediately. 1H N.M.R. 6~ 5.04,q,l,l 5.0 Hz,H2; 4.40,t,l,JI 5.7 Hz, -CH(OMe 2 3.S4,d,2,!l 5.0 Hz, H3; 3.65,d,2,!l 5.2 Hz, OCH 2 3.56-3.44,A,2, Hl; 3.35,s,6, OCH 3

K-

'I

I

WO 87/05904 20 PCT/AU87/00084 2.5,s(b),1,OH; 2.14, COCH3; 1.7-1.3,m,8, -CH2-.

7. 2-0-Acetyl-1-0-(6',6'-dimethoxyhexyl)-sn-qlyceryl 3-phospihorvlcholine To a stirred, cold (0 0 C) solution of distilled triethylamine (0.35 ml, 2.5 mmol) in dichloromethane(4 ml) under nitrogen, was added distilled phosphorous oxychloride (0.11 ml, 1.2 mmol) and then 2-0-acetyl-l-0-(6',6'-dimethoxyhexyll-sn-glycerol (278 mg, 1.0 mmol) in dichloromethane (5 ml). The solution was stirred for 1 hr. at RT, and choline tosylate (465 mg, 1.7 mol) in pyridine (10 ml) was added. Stirring was continued for 17 hrs. at RT. Sodium bicarbonate (0.4 g) and water (1 ml) were added and the mixture was evaporated at 30 0 C. The residue was extracted several times with chloroform (total 40 ml) and filtered. The filtrate was evaporated to yield a semi-solid residue (1.3 g).

An anion exchange column was prepared from DE-32 celluose (5.5 g) in acetic acid, and washed successively with methanol, methanol/chloroform and chloroform. The mixture (1.3 g) was applied to the column in a small volume of chloroform, and was then eluted with chloroform (100 ml), then methanol in chloroform (100 ml each of 6% The product was contained in the fractions 3-6% methanol in chloroform, as determined by t.l.c (CHCI 3 /MeOH/H 2 0 WO 7/O9O42 1 PCT/AU87/00084 60:35:5). Evaporation of these combined fractions yielded a pale yellow semi-crystalline material (0.21 which was contaminated with a tosylate salt (approx. HN.M.R. 6" 5.13,m,l,H2; z,-H(OMe) 2 4.3-3.2,m,all other protons on COC to 0 or N; 2.06, s,3, COCH 3 104.25, s, -CH(OMe) 2 71.90,d,J 8.0 Hz, C2; 7l.l7,s,-CH 2 O(,r 69.00,s,-CH 2 O(or N); 65.76,s,-CH 0 (or 63.76,d, J 5.1 Hz; -CH OP; 59.03,d,J 4.4 Hz, -CH OP; 53.78,s,-N (CH 53.38,S,QCH 3 32.24,s,-CH 2 ;21.00,s, COCH 3 Example 2 Prepe~ration of 2-0-Acetvl-1-0- (6 '-oxohexv]2 -sn-arlvcery1 -3-phosphorylcholine Crude 2-0-acetyl-1-0- -dimethoxyhexyl) -sn-glyceryl-3 .phosphorylcholine (130 mg) was suspended in ethyl acetate (9 ml) and aqueous trifluoroacetic acid (TFA) (1 7 0,,91, was added. The mixture was allowed to stand at RT for 1.5 hr. and 4 C for 17 hrs., until the deprotection was complete by t.l.q. Toluene (9 ml) was added and the mixture evaporated. The residva -was repeatedly fromntiel from etyluee(0m) The mixture was an 2 WO 87/05904 P CT/A U87/00084 chromacographed on silica gel (70-230 mesn) and the product was eluted with CHC1 3 /MeOH/H 2 0 (40:60:10).

Evaporation of the appropriate fractions yielded a colorless oil (50 mg). 1H N.M.R. 6' 9.78,t,l,j 2.0 Hz, CH=0; 5.l,m,l,H2; 4.4-3.2,m, all other protons on C aC to 0 or N; 2.46, dt,2,J 2.0 7.0 Hz, -CH 2

-CHO;

2.08,s,3,COCH 3 1.7-1.3,m,6,-CH 13 C N.M.R.6 176.05,s, -CHO; 170.79,s, -OCOCH 3 72.07,s,C2; 71.2,L,s,-CH 2 O0 (or 69.27,s, -CH 2 O0 (or 66.12,s,

-CH

2 O0 (or 64.ls, -CH 2 OP; 59.38,s, -CH 2

OP;

54. 20,s, -N +(CH 3 )3 3; 43.78,s, -CH 2

CHO;

29.28,s, -CH 2 25.64 -CH 2 21.80,s, -CH 2 21.26,s,COCH 3 ExamPle 3 Couiplingr 2-0-acetvl-l-0-(6 '-oxohexyl) -sn-crlyceryl-3phosphorylcholine to methylated BSA(PAF-BSA) Methylated bovine serum albumin (250 mg) was dissolved in methanol (90 ml) and 2-0-acetyl-l-0- -oxohexyl) -Arglyceryl-3-phosphorylcholine (25 mg) in methanol (5 ml) was added. The solution was left at RT for 0.5 hr., and then sodium cyanoborohydride (100 mg) was added. The pH of the solution was adjusted to 5 with 1M HCl. After standing for 16 hr. at RT, the mixture was evaporated.

The residue was dispersed in water (90 ml) and dialysed against distilled water (20 The dialysate was 14 14

I

4

I

r- W'O 87/ 05904 23 PCT/AL'87/00084 freeze-dried to yield a fluffy white matefiai (238 mg).

This material was assayed for phosphorous content, which was found to be 100 nanomoles per mg.

ExamTle 4 Couping4re 2-0-ac.etvl-1-0-(6 '-oxohexvli -srl-clceryl -3-phosphorvi-cholile to polylysine (PAF-PL) 2-O--Acetyl-1-0--(6 '-oxohexyl) -sn-glyceryl-3-phosphorylcholine was coupled to the polyvalent synthetic polypeptide polylysine following essentially the same procedure as that described in Example 3.

Examiple Inactivation of PAF-Acetylhydrolase The following experiments demonstrate that PAF-acetylhydrolase can be deactivated by the addition of detergents.

Materials PAF (from bovine heart lecithin) and "Tween" 20 (polyoxyethylene sorbitan monolaurate) were from Sigma (St.

Louis, Mo., USA). Human sera albumin (HSA) was from Commonwealth Serum Laboratories (Melbourne, Australia).

I

q

A

WO 87/05904 PCT/A87/00084 Serum Blood was collected from normal human donors by venipuncture, allowed to clot and the serum collected.

Serum was stored at -20 0 C until used. Similarily, rabbit serum was obtained from the ear veins of normal rabbits.

Platelet-Rich Plasma Whole blood was collected from normal human donors, who had taken no medication for at least 10 days before venipuncture, and mixed with 0.1M trisodium citrate (0.1 vol). Platelet-rich plasma was produced by centrifugation (10 min, 600 and was used within 1 hour.

Dilutionof Sera Sera were diluted 1 in 100 in either PBS or 0.1% "Tween" in PBS Diluted acid-treated sera were prepared by mixing sera (1 vol.) with 0.1M citrate buffer pH (2 vol.), and then 15 minutes later with PBS (98 vol.).

Determination of PAF-acetylhydrolase activity Diluted serum (50/1) was incubated with 3.7 x 10 6

M

PAF (in 2.5% HSA) 50 for 27 hours at 25 0

C.

The solution (501al) was then tested for platelet aggregation activity at 37oC in a Payton dual

J

I__

i LW A~ii SWO 87/05904 25 PCT/AU87/00084 channel aggregometer using human platelet-rich plasma (500,.1).

RESULTS AND DISCUSSION Two human sera and two rabbit sera, each with added PAF, were diluted by the three methods (PBS, "Tween" and acidtreated) and were then tested for acetylhydrolase activity. The results were in the form of light-transmission tracings from the aggregometer. After 27 hours incubation, PAF was destroyed in all sera diluted with PBS whereas the sera diluted in 0.1% "Tween" showed no inactivation of PAF. The "Tween"-diluted sera were tested for platelet aggregating activity, but no aggregation was observed. As a control for the above experiment, PAF was incubated with PBS or 0.1% Tween in PBS. In these experiments platelet aggregation activity was retained.

Disparity between human and rabbit sera was found when the sera were treated with acid. Whereas, rabbit sera no longer destroyed PAF, acid-treated human serum still had acetylhydrolase activity. Human and rabbit sera appear to have the same buffering capacity, so the disparity probably arises from varying acid-sensitivities of the two acetylhydrolases.

These results show that "Tween" 20 inactivates PAF-acetylhydrolase. Dilution in "Tween" is thus a simple and j Ii WO 87/05904 PCT/AU87/00084 mild method of inactivating PAF-acetylhydrolase and this finding will be of great importance in immunoassay procedures used to measure PAF in biological fluids.

Example 6 Preparation of PAF-antibodies 2-O-acetyl-lQ--(6'-oxohexyl)-sn-glyceryl-3-phosphorylcholine coupled to methylated bovine serum albumin prepared as described in Example 3 (PAF-BSA) was used as an antigen in rabbits and the immunoglobulin fraction was isolated from the rabbit anti-PAF serum produced by affinity chromatography on "Sepharose"/protein A.

The presence of PAF-antibodies in the isolated immunoglobin fraction was determined by a direct :.ding .assay showing binding to tritium labelled PAF H-PAF) as described below.

A sample of the immunoglobulin fraction (Ig) was mixed in an assay tube with a mixture (3-5mg) of "Sepharose" (solid support) and protein A (a ligand to link the antibody to solid support) and 3 H-PAF in a total volume of 50 to 100 /1 and incubated at room temperature overnight. The resulting mixture was centrifuged, washed twice with phosphate buffered saline containing 0.1% "Tween" 20, centrifuged and the sediment transferred in 4 IWO 87/ 05904 P CT/A U87/00084 water (200/dl) to the liquid scintillant "Aquasol" (3mi) and counted in a liquid scintillation counter.

The results, tabjulated below, indicate significant uptake of 3 H-PAF by the immunoglobulin isolated from rabbits treated with the PAF-BSA antigen in comparison to "normal" iminunoglobulin isolated from control rabbits.

Rabbit Ig 3H-PAF Assay Count 3H-PAF No Lc) (cpDM' Uptake(% 2 2 2 Control Control Control None 28, 123 28, 123 28,123 28,123 28, 123 28, 123 28,123 28,123 28, 123 .28, 123 5,046 5,124 3,967 4,449 3,001 2 ,189 326 492 281 140 17 .9 18 .2 14.*1 15. 8 10.7 7.8 1.2 1.7 *"Sepharose", "Tween" and "Aquasol" are Trade Marks.

p ii J WO 87/05904 28 PCT/AU87/00084 Example 7 The following experiments demonstrate the use of PAF-antibodies of the present invention in a competition or inhibition assay with a known quantity of labelled PAF and known quantities of unlabelled PAF or PAF analogues of the invention which can be used to establish standard plots from which the quantity of PAF in sample can be determined. They also demonstrate the binding of the PAF-antibodies of the invention to PAF and the PAF analogues of the invention PAF-PL of Example 4) in comparison to lyso-PAF, lecithin and lyso-lecithin.

A standard quantity of immunoglobulin containing PAF-antibodies (Ig) prepared as described in Example 6 was mixed in an assay tube with a mixture (3-5mg) of "Sepharose" and protein 4, H-PAF (22,676 cpm), and a Ssample of a "test" substance for competitive binding to PAF-antibodies in a total volume of 100 to 200/d1 and the mixture was incubated at room temperature overnight. The resulting mixture was centrifuged, washed twice with phosphate buffered saline containing 0.1% "Tween" centrifuged and the sediment transferred in water (20091) to the liquid scintillant "Aquasol" (1ml) and counted in a liquid scintillation counter.

WO. 87/U5904 29 PCT/AU87/00084 The results, tabulated below, indicate: PAF-antibodies of the present invention may be used in a competition assay with known amounts of radiolabelled PAF and PAF to develop a standard plot for the quantitative determination of PAF by competition assay; and (ii) the specific binding of the PAF-antibodies of the invention to PAF and the PAF-analogues of the invention PAF-PL) TEST SUBSTANCE ASSAY COUNT ASSAY/CONTROL Name ng cpm PAF 5,000 228 4.1 PAF 500 598 18.6 PAF 50 1,602 57.8 PAF 5 2,316 85.7 PAF 0.5 2,561 95.2 PAF-PL 27,000 435 12.2 PAF-PL 2,700 662 21.1 PAF-PL 270 429 11.9 PAF-PL 27 1,329 47.1 PAF-PL 2.7 2,338 86.5 i.; WO 87/05904 P CT/AU187/00084 lyso-PAF lyso-PAF lecithin lecithin 5,800 580 5, 000 500 2,746 2,744 2,994 2,545 2,658 2,668 2,683 lyso-lecithin 5, 000 lyso-lecithin 500 99.6 99.0 99.4 100. 0 Control No Ig 123 Assay Count Assay Count No Icr Assay Count Control Assay Count No Ig x 100 i~e.(Assay Count 123) x 100 2560 1i~ a d

Claims (23)

1. Compounds of general formula CH -0-R -X 2 R 2 COOCCH S i R4R5 CH -0-P-O-CH -CH -NR R R 2 2 2 wherein: R 1 is a C 2 to C25 alkylene or alkenylene linking group substituted by radioactive iodine; X is hydrogen; or R 1 is a C 2 to C 25 alkylent, alkenylene or alkynlene linking group optionally substituted by tritium or radioactive iodine; X is selected from: the group consisting of formyl, di(C 1 to C 6 alkoxy)methyl, carboxy, isothiocyanato, N-C 1 to C 6 alkylamino, N,N-di(C 1 to C 6 alkyl)amino, hydroxy and mercapto; and the group -A-B wherein A is a linking group selected from the groups -NR -CO0-, -OCO-, -CONR 6 -NR6CO-, -NH-CS-NH- and wherein R 6 is selected from hydrogen and C1 to C6 alkyl; and B is selected from: monofunctional and polyfunctional protein, peptide, carbohydrate and lipid groups and derivatives thereof of I, :I I I r w I~ I <I WO 87/05904 PCT/AU87/00084 molecular weight of at least 2000; and (ii) a label; and R 2 to R 5 are independently selected from C 1 to C 6 alkyl; and mixtures of the compound of formula I and its enantiomer.

2. Antigenic PAF analogues of general formula (I) CH -0-R 1 -X 2 12 R C00amCH I CH -0-P-0-CH -CH -NR R R I 0o wherein: R is a C 2 to C 25 alkylene or alkynylene linking group; X is the group -A-B wherein: A is a linking group selected from -NR -COO-, -OCO-, -CONR 6 -NR6CO- and -S-S wherein R 6 is selected from hydrogen and C 1 to C 6 alkyl; and B is selected from monofunctional and polyfunctional protein, peptide, carbohydrate and lipid groups and derivatives thereof of molecular weight of at least 2000 which are capable of eliciting an antigenic response; and R 2 to R 5 are independently selected from C 1 to C 6 alkyl.

3. Antigenic PAF analogues according to claim 2 wherein: R is selected from straight chain C 4 to C 16 alkylene; X is a group -A-B wherein: *I '1k I r .r i N I WO 87/05904 PCT/AU87/00084 A is selected from -NR 6 -COO-, -OCO-, -CONR 6 and -NR6CO- wherein R 6 is hydrogen or methyl; and B is selected from monofunctional and polyfunctional protein, peptide, carbohydrate and lipid groups and derivatives thereof of molecular weight at least 5000 which are capable of eliciting an antigenic response; and R 2 to R 5 are independently selected from C 1 to C 3 alkyl.

4. Antigenic PAF analogues according to claim 2 or claim 3 wherein: R 1 selected from straight chain C 4 to C 8 alkylene; X is a group -A-B wherein: A is selected from -NH- and -COO- and B is selected from monofunctional and polyfunctional protein and peptide groups of molecular weight at least 10,000 which are capable of eliciting an antigenic response; and R 2 to R 5 are each methyl. Antigenic PAF analogues according to any one of claims 2 to 4 inclusive wherein: R 1 is hexylene; X is a group -A-B wherein: A is and B is selected from a protein residue derived from bovine serum albumen and a peptide residue derived from polylysine; and t s ii 1 i t-1:u WO 87/05904 PCT/AU87/00CS4 R 2 to R 5 are methyl.

6. Labelled PAF analogues of general formula (I) CH -o-R 1 -X 22 R2COO-C-MH (I) O 1 CH -O-P-O-CH -CH -NR3 R R I- 0 wherein R 1 is a C 2 to C 25 alkylene or alkenylene linking group substituted by radioactive iodine; X is hydrogen; or R is a C 2 to C 25 alkylene, alkenylene, or alkynylene linking group; X is a group of formula -A-B wherein: A is a linking group selected from NR -COO-, -OCO-, -OCO-, -CONR 6 -NR6CO-, -NH-CS-NH- and wherein R 6 is selected from hydrogen and C 1 to C 6 alkyl; B is a label; and R 2 to R 5 are independently selected from C 1 to C 6 alkyl.

7. Labelled PAF analogues according to claim 6 wherein: R 1 is selected from straight chain C 4 to C 16 alkylene; X is a group of formula -A-B wherein: A is selected from -NR 6 -C0O-, -OCO-, -CONR 6 and -NR 6 CO- wherein R6 is hydrogen or methyl; and I i I! ;i q Ii r 3 1: ii l^j b SWO 87/05904 PCT/A U87/00084 B is labelled group selected from: 125 radiolabelled groups based on I-histamine, 125 I-tyramine, 125 I-tyrosine methyl ester and 12 -Bolton Hunter Reagent; enzymic labels; and photometric labels; and 2 5 R to R are independently selected from C 1 to C 3 alkyl.

8. Compounds of general formula which are intermediates for the preparation of PAF analogues CH -0-R -X 2 12 R2COO-C-WH 0 S+ 34 CH -0-P-O-CH -CH -NRR4R 2 2 2 0 wherein: R 1 is a C to C25 alkylene, aikenylene or alkynylene linking group; and X is selected from the group consisting of formyl, carboxy, di(C 1 to C 6 alkoxy)methyl, N-C 1 to C6 alkyl)amino, hydroxy and mercapto.

9. Compounds according to claim 8 wherein: R 1 is selected from straight chain C 4 to C 16 and X is selected from formyl, carboxy, dimethoxymethyl and hydroxy. J S. i l i WO 87/05904 36 PCT/AUi87/00084 A process for the preparation of compounds of general formula which process comprises: reacting: a compound of general (II) CH 2-0-R 1-G 12 2 R COO m-C -s CH -OH 2 wherein R1and R 2herein before defined and G is selected from di(C to C 6 alkoxy)- 1 6 methyl and groups which may be reacted, to give a group selected from formyl, di(C to C 6 1 6 alkoxy)methyl, carboxy, amino, N-C 1 to C 6 alkylamino, N,N-di(C to C 6 alkyl)amino, hydroxy an N,N,N-tri(C 1 to C 6 alkyl) ethanolamine deriatie t gie acomoun ofgeneral formula 2 CH P--CH-CH -NR R R I -l r i r I LI W 87/05904 PCT/AU87/00084 reacting the product of to convert group G as hereinbefore defined to a group selected from formyl, di(C 1 to C 6 alkoxy)methyl, carboxy, amino, N-C 1 to C 6 alkylamine, N,N-di(C 1 to Cg alkyl)amino, hydroxy and mercapto and to introduce the desired group X.

11. Supported PAF analogues comprising: PAF analogues of general formula (I) CH -O-R -X R2COOM -C H 0 4 CH -0--0-CH CH -NR RR 2 26 wherein: R 1 is a C 2 to C25 alkylene or alkenylene linking group substituted by radioactive iodine; X is hydrogen; or, R 1 is a C 2 to C 25 alkylene, alkenylene or alkynylene linking group optionally substituted by tritium or radioactive iodine; X is selected from: the group consisting of formyl, di(C 1 to C 6 alkoxy)methyl, carboxy isothiocyanato, N-C 1 to C 6 alkylamino, N,N-di(C 1 to C 6 alkyl)amino, hydroxy and mercapto; and 1~ t~ I WO 87/05904 38 PCT/AU87/00084 the group A-B wherein A is a linking group selected from the groups -NR-, -COO-, -OCO-, =CONR 6 ,-NR 6 CO-, -NH-CS-NH- and wherein R 6 is selected from hydrogen and C1 to C 6 alkyl; and B is a label; and 2 3 R to R are independently selected from C 1 to C 6 alkyl; and a solid support material upon which said PAF analogues are covalently bound.

12. PAF antibodies prepared using as antigen: PAF adsorbed onto or non-covalently bound to a monofunctional or polyfunctional protein, peptide, carbohydrate, lipid or a derivative thereof of molecular weight at least 2000 and capable of eliciting an antigenic response; or the antigenic PAF analogues of general formula as defined according to any one of claims 2 to 5 inclusive.

13. PAF or antibodies prepared using as antigen an j antigenic PAF analogue of general formula as defined according to claim 4 or claim

14. A method for the preparation of PAF antibodies which method comprises: introducing an antigen selected from: PAF adsorbed onto or non-covalently bound to a monofunctional or polyfunctional protein, SWO 87/05904 9 PCT/AU87/00084 peptide, carbohydrate, lipid or a derivative thereof of molecular weight at least 2000 and or capable of eliciting an antigenic response; 4 ad the antigenic PAF analogues of general formula as defined according to any one of claims 2 to 5 inclusive; into an animal; and isolating the antibodies produced in response to said antigen. A method for the preparation of PAF antibodies which method comprises: introducing an antigen selected from the antigenic PAF analogues of general formula as defined according to claim 4 or claim 5 into an animal; and isolating the antibodies produced in response to said antigen.

16. PAF antibodies as defined according to claim 12 or claim 13 which have been labelled with a radioactive, enzymic or photometric label.

17. PAF antibodies as defined according to claim 12 or claim 13 which are polyclonal.

18. PAF antibodies as defined according to claim 12 or claim 13 which are monoclonal.

19. A method for the immunoassay of PAF in biological fluids which method comprises using a PAF antibody as defined according to any one of claims 12, 13 and 16 to 18 inclusive. A 4 1 NY i& T 4 4 0 PCT/AU87/00084 A method for the immunoassay of PAFAin biological fluid wherein said biological fluid is diluted with a detergent before subjecting said biological fluid to immunoassay.

21. A method according to claim 20 wherein said detergent is a non-ionic detergent.

22. A method according to claim 20 or 21 wherein said detergent is selected from the group consisting of: polyalkylene glycols; alcohol, phenol and alkylphenol alkoxylates; castor oil alkoxylates; the partial esters derived from long chain fatty acids and hexitol anhydrides and their alkoxylates; long chain alcohol polyglycol ether acetals; alcohol sugar acetals; and the lecithins.

23. A kit for the immunoassay of PAF in biological fluid said kit comprising PAF antibodies as defined according to any one of claims 12, 13 and 16 to 18 inclusive.

24. Compounds of general formula according to any one of claims 1 to 9 inclusive substantially as herein described with reference to Examples 1 to 4. Method according to claim 10 for the preparation of compounds of general formula I substantially as herein described with reference to Examples 1 to 4.

26. PAF antibodies according to any one of claims 12, 13 and 16 to 18 inclusive substantially as herein described with reference to Example 6. 1 i- WO 87/05904 PCT/A U87/00084 IFa II i I -4 ih

27. Method according to claim 14 or claim 15 for the preparation of PAF-antibodies substantially as herein described with reference to Example 6.

28. Method for the immunoassay of PAF in biological fluids according to any one of claims 19 to 22 inclusive substantially as herein described with reference to Example 6 or Example 7. a ii B jit 11 i j i V ii 7] INTERNATIONAL SEARCH REPORT Interriational Apat~cat~on No POT/AU 87/00084 I. CLASSIFICATION Of SUM JtCr MATTER I 069a ds.' cilonm %moo I sooaf, ro..:fc0e1t Accodin to tnmsnatou,.IPatent Ctaaamficamgorm (IPC) or 10 calm National ClteioricAtion and IPC Int. C1. 4 C07F 9/10, G01N 33/92, C07K 15/12 11, FitLDS SEARCHED tMiinijum Documentation Starchied. Clam mn'sc allaihS5,11in Clams'ila or S n oll IPC C07F 9/10, GOIN 33/92, G01N 33/16, C07K 15/12, C07G 7/00 Documenrtation Searched otier thaim Miinum, Oocuim.niation to the Extent that such Oocurnents are Included in the Fields Searched I AU IPC as above Ill. DOCUMENTS CONSIOEMEO TO 01 RKLEVANT' Category Citation of Oocurnt, -1 -itt, indication. wherv aoarocriate, ofthttiarelevant casacema 1 I2F'eirtnt to Clamn,No i X US,A, 4370311 (ILEKIS) 25 January 1983 (25.01.83) (20-22) See column 2 lines 11-18 A Journal of Immunology Vol. 134 No.2 (1985) M. Odo et al, "Molecular Species of Platelet-Activating Factor Generated by Human Neutrophils challenged with Ionophore A23187" pages 1090-3 A US,A, 3708558 (KNY) 2 January 1973 (02.01.73) A CA,A, 1169433 (GOVERNMENT OF THE UNITED STATES OF AMERICA) 19 June 1984 (19.06.84) A Patent Abstracts of Japan, C-9, page 117, JP 55-28955 (TOYAMA KOGAKU KOGYO 29 February 1980 (29.02.80) A US,A, 4329302 (HANAHAN) 11 May 1982 (11.05.82) A Chemical Abstracts, Volume 104, No.5 issued 1986, Lakin K. et al, "Activation of Rabbit Platelets induced by 1-0-al kyl -2-0-acetyl -sn-glycerophosphochol ine"I see page 388, abstract No. 32325s, Byull. Eksp. Biol. Med. 1985 100(10)410-12 (Russ). Spacial categories of cited documents: ImeIT" ltedocument Published tat rthe Inormation~al Citig date A" documentl del'.rin the geonsie a te of the art which Its ot11or priority dame end not in conflict with the application Out citad to unditstand the grmnesoleor theory underlying the considered to be at aticut role-onace imnonf Searlier document but puablshed on oatoiler the imternattonal X. documnernt Ot oorliCUli oerlovrmce-e:laicmed nerliorr fling date cannot be Conmidered novel of cannam be can s.bered to LIdocument whch maly throw doubte on oriort, clrimfaf or involve anmnentiveasto which is Cited to eaablimn te putI.cation date I1,1aoteher document lOf peijiculir rloeteace: the claitmed Inventionm C, fallon or oitier special lesson Itee eaecir-fa cannr~i be conaideted to inolve an Inrete e lowhen the, ~Qdocument referrng to an oral disclosure. use. *ishiitiorn or doctvAienl to comoined with or* at maote other ouCh dOCu' other means malls. oucn combination oemrmg obvious to a person skilled F. documnentDu bihed armor to the internattonel fiing dote but tn the ,rm. lter them the priority dote Ctaimed '4 documenat member ot thes Wme Patent tamity IV. CERTIFICATION Osto of the Actual Complton of the Ilnenaln Seafcn Dale of Melting of thins International Seorch Report 18 June 1987 (18.06.874 ;z 7Vt-/ t 7 tnteirnatimnol Searching Aujthority Signature ot Authormsd Officer Australian Patent Office 0 J.G. HANSON form ICTttSAIZIO $second shei January145 tUOJ b A I I International Anolicaion No. POT/AU 87/00084 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET V[]OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHA13LE'I This International search report h~as not been established In respect of certain clims under Articie 17(2) for the following reasons: IQE Claim numbers because they relate to subject matter not required to be searched by this Authority, namely: Claim numbers because they relate to paris of the interrratio~nai application that do not comply with the prescribed require- ments to such an extent that no meaningfl international search can be carried out, specifically: 3]Claim numbers.......because thiey are depenoent claims and are not drafted In accordance Vpith tis second and third sentelneSS Of PCT Rule 5.4(a). VIfZ O3SERVATIONSWIERK UNITY OF INVENTION IS LACKING. a This International Searching Authority found multiple Invention& In this International application as follows: Claims 1 to 11, 12(b),13,14(b),15 to 19,and 23 to 28 Claims 12(a) and 14(a) Claim 20 to 22 IM As all required additional search tees were timely paid by the applicant, this International search report covers all searchable claims of the International application. 2.M As only some of the required additional sech fees were timely paid by the applicant, this International search report covers only thoseii claims of the International application for which fsk's were paid, specifically claims: 3M No required additional search lees were timely paid by the applicant. Consequently, this International search report Is restricted to the Invention first mentioned In the claims; ItIe covered by claim numbers: 47 As ali searchable claims could be searched without effort justifying an additional lee, the International Searching Authority did not invite payment of any additional fee. Remark on Protest M The additional search fees were accompanied by applicant's protast. I~No protest accompanied the payment of additional search lees. Form PCTIiSAi2O laupplemeiltal shoet 121) Jianuary 195 11 fI I 11 *I ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL APPLICATION NO. PCT/AU 87/00084 This Annex lists the known publication level patent family members relating to the patent documents cited in the above-mentioned international search report. The Australian Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent Document Cited in Search Patent Family Members Report US 3708558 BE 763578 CH 542247 DE 2009341 ES 388446 GB 1280788 IL 36299 NL 7102495 YU 343/71 ZA 7101241 US 4329302 US 4504474 US 4551446 END OF ANNEX J 8,23'90/4 r 1 r -r l r