METHOD AND DEVICE FOR ANALYSIS OF THE ACTIVITY OF RECEPTORS OF CERTAIN CELLS

Abstract: Method and device for analysis of the activity of receptors in a sample containing certain cells, e.g. blood cells, were the destined reaction occurs at a certain, for example in different individuals varying optimal ratio between the concentrations of the receptor and the agent with which the receptor interact. The method comprises the addition of an agent to a sample containing cells, of which the reaction is to be analysed. The addition of the agent takes place in an extended period of time up to a certain predetermined concentration, such that the cells in one and the same sample are exposed to a raising concentration of agent under stepwise or continuous addition of agent. The cells are exposed to a concentration of agent which is situated so close the concentration of agent, which is optimal for the attainment of an adequate reaction, during such a long time of the lapse of the time that the destined reaction is allowed to take place essentially without purturbation by the addition, which is continued during the extended lapse of time, of the agent. The sample is finally analysed with respect to the result of the reaction. The method may be applied for example in allergy tests.

Description: T 1 i 1t ulee: ; M Meetthhod and device for analysis of the activity of receptors of certain cells Technical field: The present invention relates to a method and a device for analysis of the activity of receptors in a sample containing certain cells , e . g. blood cells , where the desired reaction occurs at a certain indivi¬ dually varying optimal ratial between the concentrations of receptor and the agent with which the receptor interact. Within cell biology knowledge has now been accuired of a multitude of reactions. , released by receptors on cell surfaces . A number of this receptors react according to an all or nothing principle, that is when optimal conditions prevail between the receptor and the agent ^ which effects it, a "total reaction" occurs which can not be imme¬ diately repeated again by the same cell. Often it is so that a certain concentration of agents in relation to the concentration of receptors has to be attained in order that the reaction shall take place. When this "treshold is exceded the reaction takes place rapidly and comple¬ tely. Other receptors have apart from a minimum treshold also a maximum treshold for the amount of agent which can release the reac¬ tion . This concerns e. g. the reactions of mast cells and basophilic leucocytes in allergic individuals , when adequate allergi is exposed to the cells . Another circumstance prevailing in this connection is that large in¬ dividual variation excists what concerns the reaction ability of the receptor, the concentration ratial which is optimal and also how the released reaction then manifests itself in the form of other reactions in the tissue . Background of the invention Testing for allergi ultimately constitutes an analysis of the activity of receptors of basophilic leucocytes and is thus referred to the area of application of the invention . In such testing is used in most OMPI places a practical working schedule with approximately the following appearance : The patient has aquired a picture of his symptoms , which constitutes the base of the anamnesis , which is taken up by the physician. On the basis of his own experiences the patient is often able to specify the allergen (e.g. grass or cat) and with specialist knowledge foun¬ ded on probable allergens in various environment, seasons etc the physician can obtain a fairly good picture of which are the aller¬ gens in question. In order to objectify this there excists tests . These are of two types , partly test on the patient himself (in vivo tests) , partly tests on tissue samples , usually blood samples from the patient (in vitro tests) . These are in brief as follows : In vivo the reaction in question may be achieved by imitating the process which gives the symptom . Thus the provocation may occur by the patient inhaling the allergen , whereby hay-fever and asthma may be dropped into the eyes and release running eyes etc. " The most common in vivo test is however skin tests , of which the most common are so-called spot test, where a drop of the allergen in solution or in powderform is applied to the skin where upon a slender lancet is pricked through the allergen and underlaying skin. After 20-30 minutes the result, which constitutes an itching blush, of which the diameter is a rough measure of the degree of allergi. In a particular drop is usually also added a reference substance allowing better judgement of the reaction (histamine) . The test is usually performed on the arm, this to allow screening of the reaction from the rest of the body if a vigourous reaction results . It is then easier to threat a possible generalisation of the reaction (anaphylactic chock) , which can be fatal. In this way the so-called type 1 reaction (hay-fever, certain forms of asthma and rashes , red eyes and hypersensitivity towards certain drugs , etc) may be objectified. In these tests the allergen is allowed to diffuse to those cells which have receptors on the surface directed towards the allergen . These cells are the so-called mast cells in the tissues . They have ther counterpart in the blood vessel in the so-called basofilic leucocytes , which are present in a low number in comparison to other blood cells (around 0, 5% of all leucocytes or white blood cells) . When the mast cell or the basophilic cell meets with an allergen under certain conditions it empties it content of small granules , granula, from which then histamin is liberated and causes the symptons as it reacts with histamin receptors from other cells . A number of tests may also be executed in vitro in order to objectify an allergy . The most common method in Sweden is PRIST -test (Paper Radio Immun Sorbent Test, Pharmacia) , which means that proportion of IgE -molecules is measured. It is usually higher in allergic persons then in healthy. IgE are those molecules which constitute a part of the receptor on the mast cells and basophilic leucocytes for the allergen . IgE is for¬ med by lymphocytes and particularly when this is stimulated by an allergen . Lymphocytes release allergens in the body fluids and a small portion of these get caught on the mast cells and basofilic leu¬ cocytes (also other cells in certain cases) . Less than 0, 1% of IgE is bounded to the reacting basophilic leucocytes, but probably there is very large variation in this among different individuals . Positive PRIST does thus not mean that an allergical reaction is mea¬ sured but that the fact is made use of that many , but not all, aller¬ gic persons have a higher IgE-proportion in blood then healthy. It is also possible to measure the proportion of specific IgE for a large number of allergens . The test is then called RAST (Pharmacia) . Often there is a good correlation between RAST and a performed Due to a large amount of work this has not been very much spread as a method for allergy test, despite that actually the reaction of the effector cell in aUergy disorder is measured on a sample delive¬ red to a hospital laboratory instead of on the patient as in prick- test. Very recently a couple of publications have described the use of high capacity cell counters in the evaluation of the reaction, but still the blood volume has become large if many allergens are tested, since several portions have been used for each allergen tested. Technic ' problem: The pr titly applicable methods for analyses of the activity of re¬ ceptors _ " ' certain cells, have thus lead to extensive, time consu¬ ming procedures . In particular this has implied a serious problem in allergy tests where is generally required testing of a number of aller¬ gens , in the order of 10-20, for each patient, which thereby, as appeares of the preceding, in their turn have been devided into se¬ veral different samples . For several patients were thus reached a large number of samples , which has meant a complicated handling and requirements of a large amount of blood. The solution: By the present invention is effected a method which permits clinical application of reactions of the type mentioned at the beginning under the heading "technical field" , e.g. for allergy diagnostics , were the required amount of sample, the blood volume, and the amount of work for the execution of the analysis , shall be as small as possible and the method shall also be adapted for automisation . This has been achieved in that the cells in one and the same sample are exposed to a raising concentration of agent under stepwise or continous addition of agent, where this occurs in an extended laps of time up to a certain predetermined concentration, such that the cells are exposed to a concentration of agent, which is optimal for attaining an adequate reaction, during such a long time that the destined reaction is allowed to take place. If the concentration of agent than exceeds the optimal is unimportant since cells already have reacted . The sample is ultimately analysed , after the addition process , with respect to the result of the reaction . Advantages : Through the invention is obtained the advantage that only one sample is required for analyses of the reaction of the cells towards an agent. Despite this is obtained an adequate expressions for the optimal reac¬ tion of the cells without that it is necessary to adapt the addition of the agent to the optimal concentration . This is a great advantage in particular in allergy testing where in general a large number of agent are tried, whereby a plurality of samples for each agent produces a large total amount of samples , which amount thus is reduced considerably through the invention . In addition are obtained those advantages which are achieved when the basophilic leucocytes in blood samples can be utilized in allergy diagnostics , which advantages are : Principally the same initial effector mechanism is studied as the one which releases the symptom of the patient. Blood sample may be sent to laboratory instead of the patient getting of work to occupy a hospital reception , like in pricktest. The risk of anafylactic chock or other reaction during the testing disappear. Certain drugs and other potentially dangerous allergens may be tested without injury to the patient. Only the very allergic reaction is studied, other unspecified reac¬ tions of other type are eliminated and the diagnosis becomes reliable. Theoretical background: The mechanism of the reaction between basophilic leucocytes and aller¬ gens may be described as follows . ≥MPI. WiPO On the cell surface are receptors for IgE-molecules, which are pro¬ duced by lymphocytes and released to the body liquids , which in all human beeings contains a small amount of IgE . In allergic persons ® tthhee pprrooppoorrtt:ions is often strongly increased (compare PRIST and RAST® test) There is an equilibrium between the number of free IgE-molecules and the number bound to the mast cell and the cell. Each cell has a large number of IgE-receptor (40-500 000 per cell) . IgE reacts with allergen such that several IgE are bound together. Both free and cellbounded IgE bind allergen , but only the bounded produce continued reaction. The proportion of receptors per cell, the relative number of IgE- molecules bounded, the ratio between free and bounded, the affi- nityl of IgE towards the allergen and the ability for continued reac¬ tion varies from individual to individual. The means that the amount of allergen which has to be added to a sample with cells or through the skin in pricktesting varies considerably with the individual. In addition the requirements of not too much and not too little allergen have to be satisfied. The reaction between allergen and IgE has to be effected in the fol¬ lowing way in order that contined reaction shall be released: Each to the cell surface bounded IgE can react with the allergen but only a small portion of all IgE are needed in order that the reaction may take place, maybe in the order of ten. The releasing factor is the change which occurs around the IgE-molecule in the cell membrane which is produced when an allergen simultaneously binds together two or possibly more IgE-molecules . If there are too few allergens in relation to IgE then two IgE will not be able to simultaneously bind the same allergen whereby nothing further occurs . If there are too many allergen molecules which can bind IgE each IgE will bind the allergen, but the probability of two simultaneously binding the same allergen is reduced. When the ratio between allergen and IgE is optimal, i. e . two or several IgE are bound together by allergen then following occurs : Calcium ions can leak in through a "hole" in the cell membrane, which is held open during a short time, probably during seconds . If enough calcium enters into the cell mechanisms are released which within a couple of minutes cause the granula of the cell to be thrown out of the same . From these are liberated amoung others histamine and the allergic reaction has started. The reaction may be registered in various ways , of which one way is constituted by counting the basophilic leucocytes , where the de- granulated cells not are included. Nowadays there exists automatic devices for counting the basophilic leucocytes , i. e. TECHNICON ® H6000. For research purposes are also employed methods where li¬ berated substances are metered as a measure of the reaction . These methods are however presently laboreus and expensive . The above described reaction occurs very rapidly and critical is that binding together of at least two IgE per allergen must take place . This binding is a very fast reaction while the liberation of histamine subsequently last a few minutes . When the cell once has reacted it is emptied of granula and cannot react further or (as far as is known at least in human beings) not quickly take up granula again . It does not matter then if the concentration of allergen is too high if the reaction already has taken place. This means that the cells may be exposed to a rising concentration of allergen in one and the same blood sample up to a predetermined final concentration under the condition that the time during which the optimal concentration of allergen for the reaction is crossed is sufficiently long, but not too long since then the reaction may pos¬ sibly be inhibited. Thus optimum interval should be crossed during a few minutes whereafter the allergen may rise to very high levels without already produced degradation being influenced. This resemb¬ les the natural process in the body when the allergen defuses to the receptor. OMPI W wiPo Best mode of carrying out the invention: In brief the invention thus implies that the agent, such as an aller¬ gen with which the cell is to react, is added in a slow process . The process shall then cover such a long time that the receptors of the cells attain their reaction level and that the reaction will not deviate on a corresponding level during a period of time which is sufficiently long for the destined reaction without it being disturbed by further substantial addition of the agent in question . This retarded process of addition may principally being carried out in two ways . 1. Stepwise addition to a blood sample with intermediary time inter¬ val in rising concentration of the agent in question. Such a stepwise addition may be carried out manually or in an analysis apparatus by use of pipetts or automatically in a continuous flow system. Both these applications are described under la and lb respectively in the following. la. By use of pipette: From the patient is taken in a Vacutainertube with addition E-DTA, 10 -ml of blood, which is mixed by rocking the tube. To this volume of blood is added 20-25 μl Heparinsolution (Lδvens in ectionliquid Heparin 5000 IE /ml, deluted to 500IE per ml with isotonic sodiumcloride) . The tube is rocked until mixing has been produced. In certain cases up to a week old EDTA-blood may be used if the sample has been stored in refrigerator, possibly after addition of certain nutritive substrates . The blood is devided in portions of 300 μl each in suitable tubes , e.g. polyethylenetubes . 10 ml is enough for about 30 portions and thus principally for 30 allergy tests . To one of the tubes is added 10 μl of 0,06 M CaC and possibly 0,3 M MgC solution. To one of the tubes this addition is not carried out (reference tube) . Allergen, e.g. from DOME (pricktest solution, e. g. 5-grass 4000 PNTJ/ml in 50 % glycerine) is diluted in a number of tubes as follows : OMPI A. Dilution solution Albumine solution 16 g/1 (Kabi , albumine 200 mg/ml diluted with isotonic sodium cloride solution , sterile executed) . Solution 6 : Extract diluted 1+1 with diluent solution . Solution 5 : The above diluted 1+9 with diluent solution . Solution 4: The above (5) diluted 1+9 with diluent solution . Solution 3 : The above (4) diluted 1+9 with diluent solution . Solution 2 : The above (3) diluted 1+9 with diluent solution . Solution 1 : The above (2) diluted 1+9 with diluent solution . Possible further solution may be necessary . The principal is that the concentration in the sample when the solution is added shall be progressively rising, preferably according to a logaritmic curve . The blood samples are placed in water bath at 37°C . 10 μl of these solutions are subsequently added to the two test tubes in the water bath at an interval of a few minutes between each addi¬ tion starting .wit the most diluted solution . When the most concentra¬ ted solution also has reacted for some minutes the reaction is inter- ruped with an excess of EDTA solution , e . g. 50 μl 10 % K g EDTA in water, whereby calcium ions are bounded. The tubes are subsequently placed in a TECHNICON® H6000 appara¬ tus or other detector for measuring the reaction . Positive test implies that basophilic leucocytes of the patient have been reduced in number, often disapeared completely , as the detec¬ tor sees them. (In the reference tube without calcium ions the num¬ ber has not been reduced) . lb . By addition of allergen in a flow system : A continuous -flow equipment is assembled such that blood sample is sucked into the tube at a certain rate (e. g. 500 μl/minute) and then passes "stations" with addition of Ca-Mg solution and allergen in progressively rising (see "Solution 1-6" under la) concentration at certain intervals of time . As the sample appears in other end of the system EDTA addition has also been carried out and the sample may be collected in a tube which subsequently is positioned in the detec¬ tor. Here it is possible to make a direct connection to an automatic de- tectorsystem, e. g. the basophilic channel of a H6000-apparatus . 2. Continuous addition by diffusion , eluation or dissolution of the agent, which may be an allergen, to a blood sample of by chemical reaction with the same. This variant of the invention is described under 2. in the following: 2. On a suitable carrier e.g. a stick of inert plastic material is placed in the one end a device which continuously releases increa¬ singly higher concentration of allergen or other agent for other re¬ ceptor analysis , on the basis of this principal. Also here is thus obtained progressively rising concentration. The liberation in the sample must occur such as the optimum inter- • val is crossed during sufficient but not too long time. A stick sui- table for the purpose may be produced by e. g. GELBOND or dra- wingfilm of which the one side is hydrofilic. To this surface agaros can be b nded. The agaros can then be used to bind allergen solu¬ tion, where the allergen diffuses into the agaros . If additional, pos¬ sibly more concentrated agaros or gelatine is placed on top of the previous agaros , after this has dried, can be diffusion a concentra¬ tion gradient be set up within a very thin layer of agaros , when all has dried after the second diffusion. On top of this may then be placed e.g. gelatine, where a lower concentration of allergen has ben placed, also be dried. If this stick is placed in a tube with blood or suspension of cells in suitable liquid from allergic persons, can, if suitable concentration of agaros has been used, a degranulation reaction be effected. To the sample is also added calcium (or already on the stick in rapidly soluble form) , whereafter the stick is posi¬ tioned in the tube during a certain time. Thereafter it is removed and after possible addition of EDTA the sample is ready for the de¬ tector. This method resembles the diffusion of allergen to the mast cells in pricktest. _ O ΪPI Example on production of stick tested for grass , broadleaf tree , ox- eye daisy, mite, dust, cat and horse on a number of allergic per¬ sons . DOME pricktest allergen as above is used, taken directly from the packing . Var 0, 25 % agaros solution (SEAKEM) in water is poured out onto hydrofobic surface being horisontally positioned as a layer of about 3 mm . After solidification cylinders are punched put with the same diameter as the width of the GELBOND stick and placed immediately adjacent the one end on the hydrofilic side. On this gel cylinder is placed 3 μl of current pricktest solution and this is allowed to dif¬ fuse while a heating fan is used to carefully dry the gel or it is positioned such that the gel is hanging up and down over the edge of a table and is dried. When the cylinders have dried to a spot a similar cylinder is placed on top of the spot and the evaporationstep with simultanous backdiffusion of the allergen is repeated. The stick is ready for use . Take e . g. 400 μl of blood with additions or cellsuspension. Add cal¬ cium solution according to previously described manuel method 20 μl 0,6 M Ca and possibly 0,3 M Mg. Place the tube in water bath at 37°C . Place a stick in the tube and shake once in a while the tube or stir with the stick so that the blood is brought in contact with the whole agaros surface at least for shorter periods . Remove the stick after about 30 minutes and add ADTA in order to stop the reaction by binding calcium. The tube is subsequently placed in the detector. Positiv reaction as described above . The stick indicated in the example has been tested by letting radio- actively marked protein having a molecular weight of 22 000 (most allergen are of this size) diffuse out into the bloos as described. After 30 minutes 90 % of the radioactivity was outside the stick, after 5 minutes approximately a third. By modification of binding substance, possible protein binding, of the allergen etc. this can be varied. Continuous addition by diffusion or eluation may also be carried out from substance which itself is being solved in the blood. Simplest is a "tablet" made of e. g. gelatine, which is completely dissolved and disappears as an indication of that the reaction is completed. The allergen may also be bounded to an insolable substance, e. g. a tablet of the type "slow release" , common in connection with drugs. The allergen may also be dissolved from the tube itself or from a cork fitting into the tube, which after corking is turned upside down for reaction and then after "de-corking" is placed in the detector. It is also possible to imagine a cork fitting to the tube and which is designed as a container. The cork is attached a paper strip or a capillary containing an absorbing material, e . g. a thread which has been soaked in allergen solution with suitable additives and dried. The tube with blood is corked with this device . The capillary shall reach down to the blood. Eluation solution is then poured into the cork and is absorbed by the thread whereby the allergen successi¬ vely is liberated in the other end of the capillary to the blood. The allergen may also be added to the sampel chemically bounded to e. g. a protein, from which it is successively liberated to the sample by chemical reaction. In all these cases the allergen thus forms a body, possibly together with a carrier, from which body it is slowly liberated into the sample by diffusion, eluation, dissolution or by a chemical reaction. It is desirable that the analysis may be performed in an analysis apparatus , which is of particular value in allergy tests, where each patient in general must be tested for a large number of allergens . How such an analysis apparatus may function has been described under lb, where stepwise addition is used in a continuous-flow sys¬ tem. However, also the now described method of addition permits continuous addition by liberation into the sample from an allergen body, extra ordinary possibilities to automate the analysis . The ana- OfV.PI lysis apparatus shall then be so designed that it to successively ad¬ ded samples , which preferably are collected groupwise for each pa¬ tient, is added to said body of allergen . Thereafter the respective sample is left in a resting position such that the allergen is liberated into the blood. After an adequate period of time the samples are then fed into a detector, which may be of the previously mentioned kind, for analysis . By a suitable arrangement all sample for one and the same patient maybe analyzed in direct succession after each other, which facilitates the rapport and makes it more reliable . Before the analysis the samples do not have to enter in contact with any flow system, but may pass the system as static amounts of sample, which reduces the need for cleaning of the apparatus and eliminates the risk for inteπnixing of quantities of samples and contamination with not intended allergen . The method of the invention is naturally not limited to the above described embodiments and to the reaction of basophilic leucocytes with allergen. Other fields of application may be the study of neutrophilic leucocytes germicidal ability in patient with defective immundefens e . g. cronic granulating disease, (myeloperoxidedefi- ciency and others) and the reaction of trombocytes on stimulus of the type collagen or of the products of the protease cascade systems . CMPΓ ftϊ WiPO

Claims: Claims : 1. Method for analysis of the activity of receptors in a sample con¬ taining certain cells , e. g. blod cells , where the intended reactions occurs at a certain, for example for different individuals varying optimal ratio between the concentrations of receptor and the agent with which the receptor interact, where the method comprises the addition of an agent to a sample containing cells , of which the reac¬ tion is to be analyzed, CHARACTERIZED IN, that the addition of the agents takes place in an extended lapse of time up to a predeter¬ mined concentration, such that the cells in the same sample are ex¬ posed to a rising concentration of agent during step wise or conti¬ nuous addition of agent, where the cells are exposed to a concentra¬ tion of agent, which is situated so close to the concentration of agent, which is optimal for achieving an adequate reaction, during such a long time of the time a lapse that the destined reaction is allowed to take place essentially without perturbation by the addi¬ tion of the agent during the extended a lapse of thime and that the sample finally is analyzed with respect to the result of the reaction. 2. Method as claimed in claim 1 , CHARACTERIZED IN, that the addition takes place such that a progressively arising concentration is obtained, preferably arise of concentration according to a logarhyt- mic curve . 3. Method as claimed in claim 1, where the receptors are constitu¬ ted by IgE-molecules to the cell surface of basophilic leucocytes and the agent of allergin particles or molecules and where at positive result, at a certain optimal ratio between IgE-molecules and the aller- gin takes place a, by certain ions, e.g. Ca 2+ , initiated basophilic, degranulation of the cells with accompanying liberation of hystamin, CHARACTERIZED IN, that to the sample, which is a blood sample or other tissue sample or cell suspension in suitable medium, is ad¬ ded stepwise or continuously allergin such that the concentration of this is successively increased in the sample to the predetermined concentration, and that said increase in concentration is carried out by means of said extended a lapse of time during such a long time, that the destined reaction is allowed to take place at essentially op¬ timal concentration of allergin, whereafter completed addition analyzis of the sample is carried out by classification of basophilic leucocytes with respect to their granulation or alternatively by measurement of histamin or other substances which are liberated during the degra- nulation , or their effect on other measurable processes . 4. Method as claimed in claim 1 , 2 or 3, CHARACTERIZED IN , that the agent is added to the sample stepwise in rising concentration at certain intevals of time. 5. Method as claimed in claim 1 , 2 or 3, CHARACTERIZED IN, that the agent is added to the sample banded as a body from which it is successively liberated for example by diffusion , eluation , dissolution or chemical reaction . 6. Device for analyzis of the activity of receptors in the sample containing certain cells , e. g. blood cells, where the destined reac¬ tion takes place at a certain individually, for example for different individuals varying optimal ration between the concetrations of re¬ ceptor and the agent with which the receptor interacts , where the method comprises the addition of an agent to a sample containing cells , of which the reaction is to be analyzed, and according to the method as claimed in any of the claims 1-5, CHARACTERIZED IN , that it comprises means , which are arranged to add the agent in an extended a lapse of time up to a certain predetermined consentration , whereby the cells are exposed to a concentration of agent, which is situated so close to the concentration of agent, which is optimal for achieving an adequate reaction, during such a long time of the lapse of time that the destined reaction is allowed to take place essentially without perturbation by the addition of the agent, continued during the extended lapse of time. 7. Device as claimed in claim 6, CHARACTERIZED IN , that it in addition to said means comprises a detector device for analyzis of the sample with respect to the result of the reaction. 8. Device as claimed in claim 7, CHARACTERIZED IN, that it comp¬ rises a device for displacement of sample being included in a series of samples and being contained in a sample container or constituting a separated sample volume in a continuous flow system relative to a number of stations arranged for the addition of agent to the sample in a successively rising concentration , the displacement velocity re¬ lative to the station being so adaped to said extended lapse of time that the optimal concentration is attained before the sample reaches the detector for analyzis. 9. Device as claimed in claim 6, CHARACTERIZED IN, that the agent is arranged to form a body, in which it is banded in such a way that the agent upon contact with the sample is successively libe¬ rated from the body by diffusion, eluation, dissolution or chemical reaction . 10. Device as claimed in claim 9, CHARACTERIZED IN, that it comp¬ rises a storage area for the sample and for a series of samples , which are feaded to the device free from the agent, at least one in the area arranged means by which the agent is added in form of said body to the samples, arrested position, in which the samples may remain during such a long period of time that the optimal reaction has time to take place during collection in the sample of said body, and nearby the resting position the detector device for reception an analyzis of .

Inventors: NILSSON TOMAS, NILSSON, TOMAS
Applicants: BIO INSTRUCTA LABKONSULT, BIO-INSTRUCTA LABKONSULT
Application year: 1984
Main classification: C12Q1
IPC codes: C12Q1, G01N33