Patent Application: US-98123998-A

Abstract:
the invention relates to a process for producing ferrofluid - forming magnetic particles substituted by dmsa that can be covalently coupled , directly or through a difunctional reactant , to an effector , which process comprises combining dmsa with ferrofluid - forming magnetic particles to form ffss particles having disulfide bonds , and reducing the disulfide bonds of the ffss at basic ph to form ffsh . the invention includes a composition comprising such ffsh magnetic particles . the invention also relates to a method in which ffsh magnetic particles are incubated with an effector comprising at least one functional group which forms an s -- s , c -- s , c -- c , or c -- n bond with dmsa , so that a covalent effector - ffsh complex is formed . the invention further includes a method for separating and / or distinguishing between molecules or cells having a ligand that forms an affinity complex with an effector - ffsh particle of the invention , and those molecules or cells that do not have such a ligand .

Description:
the starting ferrofluid is a nitric ferrofluid synthesized according to the method of french patent 2 662 539 . it consists of nanoparticles of maghemite γfe 2 o 3 of a diameter of between 3 and 30 nm ( mean diameter : 9 nm ) charged positively at the surface in acidic medium ( ph & lt ; 6 ) or negatively in basic medium ( ph & gt ; 10 ). the preparation of a sol which is stable at about ph 7 - 8 for producing a coupling in a homogenous liquid phase in conditions which are not denaturing for the proteins ( in this case annexine ) or other biological molecules is done by complexing of the surface irons using dmsa . the free thiol groups permit the formation of s -- s bond with the spdp bound beforehand to the protein via a peptide bond . 1st stage : 5 cm 3 of nitric ferrofluid ([ fe ] = 1 mol 1 - 1 ) are added to 250 cm 3 of a 10 - 3 mol 1 - 1 solution of dmsa in degassed distilled water . the [ dmsa ]/[ fe ] ratio is 0 . 05 . flocculation takes place and after 30 minutes the ph is near 4 . 5 . 2nd stage : the excess dmsa is removed by centrifuging : 10 minutes at 3000 rev / min . 3rd stage : the flocculate is peptized with a 0 . 1 mol 1 - 1 sodium hydroxide solution to ph 11 . a sol stable between ph 3 and 11 is formed . the volume is adjusted to 50 cm 3 and the ph to 7 with a dilute hcl solution . this dmsa - ferrofluid ([ fe ] = 10 - 1 mol 1 - 1 ) is stable in time and serves as master solution for its subsequent applications . 4th stage : before coupling to the protein - spdp , 1 cm 3 of the above ferrofluid is treated by the addition of 1 cm 3 of an aqueous solution of dtt at a concentration of 10 - 2 mol 1 - 1 at about ph 9 . it is left to incubate for 1 hour and the mixture is then flocculated by the addition of 0 . 1 mol 1 - 1 hcl to ph = 2 . after centrifuging for 10 minutes at 5000 rev / min the flocculate is peptized with a pbs solution ( 0 . 1 mol 1 - 1 ) and the ph is adjusted to 7 . 4 . this precipitation - peptization sequence is recommenced twice in order to remove completely the excess dtt and the dmsa possibly released during the cutting of the disulfide bridges . during the last peptization the volume of pbs is adapted to the desired concentration for the ferrofluid before its coupling by direct incubation with the protein - spdp . 1 ) the ferrofluid is treated with dimercaptosuccinic acid ( dmsa ) and reduced with dithiothreitol at ph 9 . 2 and at ordinary temperature ( ffsh ). after flocculation of ffsh at ph 2 and centrifuging , the supernatant containing the excess dtt is removed and the flocculate retained by a magnet is washed and then dispersed in a phosphate buffer ( pbs ). the thiol groups of the ffsh are determined using ellman &# 39 ; s reagent ( 5 , 5 &# 39 ;- dithiobis ( 2 - nitrobenzoic acid ) ( 31 ), with minor modifications to remove the interference by the ferrofluid particles in the spectrophotometer reading . 2 ) an annexine , in this case recombinant annexine v , is reacted with spdp in pbs medium , the spdp being in excess in relation to the annexine ( 2 . 5 moles of spdp per mole of annexine ). the spdp is dissolved in an anhydrous solvent , 1 - methyl - 2 - pyrrolidone , and then added to the annexine in an aqueous medium ; the mixture is left to incubate at ambient temperature . the annexine - spdp is obtained and the excess spdp is hydrolyzed spontaneously . 3 ) various quantities of annexine spdp are incubated with the ffsh for 2 hours at ambient temperature , the optimum ratio of the two components being determined experimentally , for example by measuring the change in the electrophoretic mobility of the particles in solution by employing the doppler effect . in this case the electrophoretic mobility of the anx ff complex is compared with that of the ffsh ; another method is the determination of the enzymatic activity or of another functional property of the effector , such as its affinity for an antibody , or the ability to form an affinity complex with antibodies of a peroxidase / protein a complex immobilized on the particles , and the determination of the peroxidase activity . an optimum activity is obtained by the binding of 1 . 5 to 2 μg of annexine on 1 ml of ffsh , as the use of the above - mentioned methods has shown . 4 ) the dmsa which has not reacted with the effector and other potentially reactive sites on the ferrofluid particles are masked by saturation with 1 % of bsa ( for bovine serum albumin ) in 50 mm of nahco 3 buffer and ph 9 . 6 to give the anx ff . in some cases other proteins such as human serum albumin may be more appropriate . a particular attention is paid to avoid a flocculation resulting from an excess of saturation . one method of producing the coupling of anx ff to erythrocytes is to mix : the reaction mixture is incubated for 30 min at 37 ° c . and then diluted with 6 ml of tris buffer to which has been added 0 . 1 % bsa and 5 mm of dtt . example 4 : separation of the cells retained by anx ff from the unretained cells this separation is carried out by an automatic apparatus , an example of which , given in fig1 is the following : a tygon tube is placed in a magnetic field and connected to various storage containers via solenoid valves . the electromagnetic field is brought into action and the reaction mixture described in example 3 above is , after an incubation at 37 ° c ., introduced into the tube by actuating the appropriate valve and passes through the magnetic field at a rate of 120 ml per hour , and the effluent is collected . a valve controlling a washing storage container is actuated and 15 ml of buffer pass through the tube . the washing buffer is harvested at the same level as the effluent consisting of cells not retained by the anx ff . the cells retained by the anx ff are eluted from the tube by switching off the magnetic field and by harvesting the cells retained in 8 ml of buffer . the cells retained and not retained are concentrated by centrifuging at 3000 g for 7 min and at 4 ° c . and then counted before any eventual subsequent treatment or characterization . a quantification of the receptors with annexine in a cell population can be done by employing annexine tagged with iodine 125 . if reference is made to fig1 during the first stage of washing with physiological water the valves 1 and 4 are open and the valves 2 , 3 , 5 and 6 are closed ; the pump is at its maximum for 2 to 10 minutes and then stopped . the buffer is next introduced through the same tube ( valves 2 and 4 open ). the sample of the concentrate is introduced with a flow rate of 160 ml / hour , valves 3 and 5 being open ( 1 , 2 , 4 , 6 closed ). the pump operates between 3 and 60 seconds and then stopped . the fraction not retained in the presence of cacl 2 buffer is next collected by passing the buffer at a rate of 160 ml per hour , valves 2 and 5 being open ( 1 , 3 , 4 , 6 closed ). the pump is in action for 2 to 10 minutes and then turned off . the fraction retained is obtained after washing with physiological water , valves 1 and 6 being open ( 2 , 3 , 4 , 5 closed ), the electromagnet being switched off and the pump brought into action for 2 to 10 minutes . a last washing with physiological water is performed by opening valves 1 and 4 ( 2 , 3 , 5 , 6 closed ) for 2 to 10 minutes . fig2 shows the percentage of cells retained on the anx ff particles as a function of the presence or absence of cacl 2 . it is seen that 44 % of the cells are retained in the presence of calcium ions , whereas only 8 % are retained in the absence of calcium ions . fig3 shows the percentage of erythrocytes bonded to anx ff as a function of the time of in vitro storage of the red corpuscles . it is clear that this percentage increases logarithmically as a function of the storage time , indicating an increasing deterioration of the membranes of the said red corpuscles . one week of storage results in 22 % of coupling , one month in 30 % and three months in 42 %. in the mouse a storage of 24 hours already entails a coupling of 13 %, whereas no bonding is observed with freshly collected blood . c ) effect of the erythrocyte concentration on the separation of the erythrocyte - anx ff complexes . this effect is illustrated in fig4 which shows that the result obtained is independent of the concentration of red corpuscles in the solution : in the conditions employed , between 20 and 24 % of the cells are retained , whatever the concentration employed . fig5 shows the percentage of red corpuscles retained on the anx ff in the presence of 20 μl or 30 μl of annexine - ffsh . it is seen that 20 μl of anx ff are sufficient to obtain a maximum retention starting with a young erythrocyte population , whereas 30 μl are needed for aged erythrocytes . f ) relationship between the anx ff coupling of the erythrocytes and the physiological or pathological state of these same red corpuscles . the table below shows the results obtained individually on control samplings performed by the french blood agency . ______________________________________fba normal sickle cell other control sr anemia high sr pathologie s______________________________________19 . 60 21 . 73 50 . 00 36 . 94 49 . 76 0 . 00 16 . 75 49 . 00 36 . 37 63 . 00 14 . 80 19 . 56 64 . 00 51 . 26 63 . 00 12 . 00 27 . 59 35 . 00 54 . 49 11 . 90 32 . 00 49 . 00 49 . 85 18 . 00 53 . 20 46 . 80 0 . 00 65 . 70 12 . 60 71 . 00 14 . 00 5 . 90 12 . 00 10 . 00 8 . 70 . sup . ( *. sup .) 10 . 7 ± 5 . 9 23 . 5 ± 6 . 2 50 ± 9 . 3 51 . 5 ± 12 . 3 58 . 6 ± 7 . 6______________________________________ . sup .(*. sup .) mean the mean retention of the red corpuscles on the anx ff is 10 . 7 plus or minus 5 . 9 . the results given in the last four columns relate to blood samples of subjects who have attended medical analysis laboratories . the samples were assigned to us , the anonymity of the subjects being preserved . only the sedimentation rate ( sr ) was known to us . results given in the second column show a moderately raised degree of retention in subjects with normal sr whose pathology has remained unknown to us . the results given in the fifth column relate to subjects also with normal sr but exhibiting a markedly higher degree - of retention , in whose cases it was possible to identify the pathology after investigation : one case of carcinoma ( the subject being under chemotherapy ), one case of deforming rheumatism and one case of onset of rhinopharingitis . the results given in the third and fourth columns relate to blood samples with a high sr from subjects presenting a known ( third column ) or unknown pathology ( fourth column ). these results clearly indicate that an abnormally high percentage of retention on the anx ff is the reflection of a physiological or pathological state which must cause the transfuser to reject the batch of red corpuscles for transfusion purposes . 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