NO860167L - PROCEDURE FOR DETERMINING THE ACTIVITY OF THE BLOOD COMPLEMENT SYSTEM. - Google Patents
PROCEDURE FOR DETERMINING THE ACTIVITY OF THE BLOOD COMPLEMENT SYSTEM.Info
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- NO860167L NO860167L NO860167A NO860167A NO860167L NO 860167 L NO860167 L NO 860167L NO 860167 A NO860167 A NO 860167A NO 860167 A NO860167 A NO 860167A NO 860167 L NO860167 L NO 860167L
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- complement
- activity
- erythrocytes
- plasma
- buffer
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Links
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Electrotherapy Devices (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Measuring Pulse, Heart Rate, Blood Pressure Or Blood Flow (AREA)
Description
Oppfinnelsen vedrører en fremgangsmåte til bestemmelse av aktiviteten av blodets komplementsystem'hos mennesker og dyr. The invention relates to a method for determining the activity of the blood's complement system in humans and animals.
Komplementsystemet utøver i human og dyrisk organisme en rekke funksjoner. Som viktigste gjelder Chemotaxis, opso-nering og lyse av cellene. Det understøtter kroppens immun-system som består av antistoffer og immunkompetente celler. The complement system performs a number of functions in human and animal organisms. The most important concerns Chemotaxis, opsonization and lysis of the cells. It supports the body's immune system, which consists of antibodies and immunocompetent cells.
En hel rekke av berøringspunkter består mellom komplement-A whole range of touch points consists between complement-
og andre reguleringssystemet i blodet, eksempelvis koagulerings- og fibrinolysesystemet. Således kan en ved hjelp av komplement fastslått lyse av leukozytter frigjøre trombo-plastin, hvilket fører til aktivering av det eksogene koa-guleringssystem. På den annen side kan imidlertid også aktiverte koaguleringsfaktorer som brombin aktivere komple-mentsystemets proteiner. Også tilkommer den viktigste koaguleringsinhibitor, Antitrombin III, en regulatorisk funksjon i komplementsystemet. Den ved aktiveringen av koaguleringssystemet samtidig induserte dannelse av plasmin fører også til en aktivering av komplementfaktorene C1R og Cls, ved lengere innvirkning imidlertid til deres desakti-vering. Den ved koagualeringen aktiverte faktor Xlla for-bruker den viktige komplementinhibitor Cl-inaktivator og aktiverer likeledes komplement. and others the regulatory system in the blood, for example the coagulation and fibrinolysis system. Thus, lysis of leukocytes determined by complement can release thromboplastin, which leads to activation of the exogenous coagulation system. On the other hand, however, activated coagulation factors such as brombin can also activate the proteins of the complement system. The most important coagulation inhibitor, Antithrombin III, also has a regulatory function in the complement system. The simultaneous formation of plasmin induced by the activation of the coagulation system also leads to an activation of the complement factors C1R and Cls, with prolonged exposure, however, to their deactivation. The factor Xlla activated by the coagulation consumes the important complement inhibitor Cl-inactivator and likewise activates complement.
En kjent fremgangsmåte til fremstilling av komplementetA known method for producing the complement
er di,-Q-bestemmelsen ifølge Mayer, som vanligvis gjennom-føres i serum. I serum er imidlertid koagulerings- og fibrinolysefaktorene allerede aktivert, også blodplatene har avgitt deres innhold og inhibitorer som AT III er delvis forbrukt, således at den fulle biologiske virkning av komplementsystemet er ødelagt. is the di,-Q determination according to Mayer, which is usually carried out in serum. In serum, however, the coagulation and fibrinolysis factors are already activated, the platelets have also released their contents and inhibitors such as AT III are partially consumed, so that the full biological effect of the complement system is destroyed.
Det er kjent at i visse tilfeller ved anvendelse av serum fåes patologiske verdier ved CHj.^-bestemmelser, enskjønt bestemmelsen av enkelte komplementfaktorer og CHj. Q-aktiviteten i plasma gir normale verdier (oversikter: Th.F. Lint, Laboratory Detection of Complement Activation and Comple ment Activation and Complement Deficiencies, Am. J. Med. technol. (1982) 48, 743 og A.T. Luskin og M.C. Tokin, Alternations of Complement Component in Disease, Am. J. Med. Technol. (1982) 48, 749). Av disse ved innvirkning av koagulasjonssystemet in vitro dannede komplementeffekter synes en bestemmelse i plasma å være mer fordelaktig enn i serum. It is known that in certain cases when serum is used, pathological values are obtained in CHj.^ determinations, despite the determination of certain complement factors and CHj. The Q activity in plasma gives normal values (reviews: Th.F. Lint, Laboratory Detection of Complement Activation and Complement Activation and Complement Deficiencies, Am. J. Med. technol. (1982) 48, 743 and A.T. Luskin and M.C. Tokin , Alternations of Complement Component in Disease, Am. J. Med. Technol. (1982) 48, 749). Of these complement effects formed by the influence of the coagulation system in vitro, a determination in plasma seems to be more advantageous than in serum.
En ytterligere fremgangsmåte til fastslåelse av komplementaktivitet i serum er omtalt i Z. Naturforschung 20b, 569-574 (1965). A further method for determining complement activity in serum is discussed in Z. Naturforschung 20b, 569-574 (1965).
I denne metode gripes lysen av en saueerytrozytt-suspensjon som ble sensibilisert med kaninantistoffer mot saueerytrozytter ved hjelp av serum og den tid bestemt, hvori sus-pensjonens ekstinksjon er sunket til det halve. Tids-målingen gir dermed en uttalelse over serumets aktivitet. Denne fremgangsmåten er imidlertid omstendelig og fører spesielt ved små komplementaktiviteter til forholdsvis lange måletider. In this method, the light is captured by a sheep erythrocyte suspension which has been sensitized with rabbit antibodies against sheep erythrocytes by means of serum and the time determined in which the suspension's extinction has halved. The time measurement thus gives a statement about the serum's activity. However, this method is laborious and leads to relatively long measurement times, particularly in the case of small complementary activities.
Overraskende ble det funnet at aktiviteten av komplementsystemet av blod kan bestemmes, idet citratplasma blandes med i en kalsium- og magnesiumionholdig puffer suspendert og med antistoffer sensitiverte erytrozytter, tiden til oppnåelse av en bestem£ ekstinétsjonssenking måles og settes i forhold med den tid som måles på en plasmastandard. Surprisingly, it was found that the activity of the complement system of blood can be determined, as citrate plasma is mixed with erythrocytes suspended in a buffer containing calcium and magnesium ions and sensitized with antibodies, the time to achieve a certain decrease in extinction is measured and compared to the time measured on a plasma standard.
Ekstinksjonssenkingen behøver bare å utgjøre en differans på f.eks. 0,1 av den optiske tetthet (O.D.) av suspensjonen direkte etter forening av prøve og reagens, således at det fremkommer en fordelaktig forkortelse av tidsbehovet for en bestemmelse spesielt ved lavere komplementaktiviteter og forenkling sammenlignet med den kjente optiske fremgangsmåte for serum. The extinction reduction only needs to amount to a difference of e.g. 0.1 of the optical density (O.D.) of the suspension directly after the union of sample and reagent, so that there is an advantageous shortening of the time needed for a determination, especially with lower complement activities and simplification compared to the known optical method for serum.
Oppfinnelsens gjenstand er derfor en kinetisk fremgangsmåte til- bestemmelse av aktiviteten av den klassiske vei av komplementsystemet av humant eller pattedyrblod ved hjelp av fotometrisk forfølgelse av lysen av med antistoffer sensitiverte saueerotrozytter i en kalsium- og magnesiumionholdig puffer, idet fremgangsmåten erkarakterisert vedat det anvendes et citratplasma av blodet som prøvematerial. The object of the invention is therefore a kinetic method for determining the activity of the classical pathway of the complement system of human or mammalian blood by means of photometric pursuit of the light of antibody-sensitized sheep erythrocytes in a buffer containing calcium and magnesium ions, the method being characterized by the use of a citrate plasma of the blood as sample material.
En spesielt fordelaktig ny utførelsesform ifølge oppfinnelsen består i at det måles den tid, hvori systemets optiske tetthet ved 578 nm avtar rundt 0,025 til 0,2, fortrinnsvis 0,05 till 0,15. Alternativt kan det også anvendes andre bølgelengder på 540 inntil 800 nm, hvor man kan måle uklar-heten av en erytrozyttsuspensjon. A particularly advantageous new embodiment according to the invention consists in measuring the time in which the optical density of the system at 578 nm decreases by around 0.025 to 0.2, preferably 0.05 to 0.15. Alternatively, other wavelengths of 540 to 800 nm can also be used, where the turbidity of an erythrocyte suspension can be measured.
Det er fordelaktig å bestemme komplementaktiviteten i et prøvematerial, hvori koagulasjonssystemet er best mulig intakt som i citratplasma. Prøven kan i denne form imidlertid også, med de nevnte begrensninger, gjennomføres på serum. It is advantageous to determine the complement activity in a sample material in which the coagulation system is as intact as possible as in citrate plasma. In this form, however, the test can also, with the aforementioned limitations, be carried out on serum.
Til utvinning av plasma for en undersøkelse av komplement-funksjonen må det være sikret at komplementsystemet ikke inaktiveres ved en antikoagulens, som bevirker fjerning av de komplement- og koaguleringsaktive kalsiumioner og også de for komplementsystemet nødvendige'magnesiumioner. For the extraction of plasma for an examination of the complement function, it must be ensured that the complement system is not inactivated by an anticoagulant, which causes the removal of the complement and coagulation-active calcium ions and also the magnesium ions necessary for the complement system.
I den her omtalte fremgangsmåte kan det anvendes det for koaguleringsundersøkelser vanlige citratplasma, ikke imidlertid EDTA-plasma, som tydeligvis fører til en inaktivering av den klassiske vei av komplementsystemet. In the method described here, the usual citrate plasma for coagulation studies can be used, but not EDTA plasma, which clearly leads to an inactivation of the classical pathway of the complement system.
I motsetning hertil ble det i ovennevnte litteratur også henvist til anvendelse av EDTA-plasma (Lint og Luskin ogTokin). Også heparinplasma kan anvendes i den fotometriske prøve. Det måles fotometrisk i langbølget område av det synlige lys turbiditetsendringen av den ved komplement fastslåtte lyse av suspenderte med antistoffer mot saueerytrozytter av kaniner sensibiliserte saueertrozytter. Erytrozyttene er suspendert i en kalsium- og magnesiumionholdig puffer. Alternativt kan det anvendes en renset immunglobulinfraksjon eller også et antiserum til sensibi-lisering. In contrast, the above-mentioned literature also referred to the use of EDTA plasma (Lint and Luskin and Tokin). Heparin plasma can also be used in the photometric test. The turbidity change of the complement determined lysis of rabbit sensitized sheep erythrocytes suspended with antibodies against sheep erythrocytes is measured photometrically in the long-wave range of the visible light. The erythrocytes are suspended in a buffer containing calcium and magnesium ions. Alternatively, a purified immunoglobulin fraction or an antiserum can be used for sensitization.
Da reaksjonshastigheten øker med økende temperatur, er det fordelaktig å arbeide ved forhøyet temperatur og anvend-elsen av et fortemperert reagens. Det arbeides fortrinnsvis ved 37°C. Den i Z. Naturforschung omtalte metode derimot benytter på is lagret reagens, idet en temperatur-konstant under bestemmelsen ikke er sikret, da det under reaksjonen kommer til en stadig oppvarming av reagenset, således at aktive sera i løpet av kort tid reagerer ved relativt lav temperatur, mindre aktive imidlertid ved høyere temperatur. Overraskende er imidlertid det i eksempel 1 omtalte reagens også stabilt ved 37°C over minst 8 timer og kan derfor godt anvendes ved denne temperatur. As the reaction rate increases with increasing temperature, it is advantageous to work at an elevated temperature and the use of a pretempered reagent. Work is preferably done at 37°C. The method mentioned in Z. Naturforschung, on the other hand, uses a reagent stored on ice, as a temperature constant during the determination is not ensured, as the reagent is constantly heated during the reaction, so that active sera react within a short time at relatively low temperature, less active however at higher temperature. Surprisingly, however, the reagent mentioned in example 1 is also stable at 37°C over at least 8 hours and can therefore be used at this temperature.
Den til suspendering av de sensibiliserte saueerytrozytter anvendte puffer har fortrinnsvis en pH-verdi fra 6-8, fortrinnsvis fra 7-7,5. Som pufferstoffer kan det eksempelvis anvendes Veronal, Hepes, Tris eller Imidazol, fortrinnsvis Hepes . Egnede pufferstoffkonsentrasjoner er 5-100, fortrinnsvis 20 mM. The buffer used for suspending the sensitized sheep erythrocytes preferably has a pH value of from 6 to 8, preferably from 7 to 7.5. For example, Veronal, Hepes, Tris or Imidazole, preferably Hepes, can be used as buffer substances. Suitable buffer substance concentrations are 5-100, preferably 20 mM.
Reaksjonshastigheten avhenger også av saueerytrozyttenes oppladningstetthet. Derfor er en optimal dosering av kaninantistoffer mot saueerytrozytter viktig. En egnet konsentrasjon er angitt i eksempel 1. Selve celletettheten spiller imidlertid for reaksjonstidene bare en underordnet rolle, når man benytter metoden ifølge oppfinnelsen. Derimot er tiden for 50% hemolyse ved høyere celletettheter avhengig av erytrozyttkonsentrasjonen. The reaction rate also depends on the charge density of the sheep erythrocytes. Therefore, an optimal dosage of rabbit antibodies against sheep erythrocytes is important. A suitable concentration is indicated in example 1. However, the cell density itself plays only a subordinate role for the reaction times when using the method according to the invention. In contrast, the time for 50% hemolysis at higher cell densities is dependent on the erythrocyte concentration.
Oppfinnelsen skal forklares nærmere ved hjelp av noen eksempler. The invention will be explained in more detail with the help of some examples.
Eksemgel_lEczema gel_l
200 |il av en suspensjon av vaskede saueerytrozytter ble blandet med 200 ul av en oppløsning av kaninantistoffer mot saueerytrozytter (Ambozeptor 6000, (Behringwerke AG, Marburg, BRD, fortynning 1.50, eller også en renset immunglobulinfraksjon av dette antiserum) og oppfylt til 10 ml med en puffer som inneholdt 0,15 mmol/1 CaCl2, 0,5 mmol/1 MgCl2, 20 mmol/1 Hepes, pH 7,3, 150 mmol/1 NaCl og 1 g/l gelatin. Erytrozytt-suspensjonens celletetthet utgjorde 2 x 10^ celler/ml. 200 µl of a suspension of washed sheep erythrocytes was mixed with 200 µl of a solution of rabbit antibodies against sheep erythrocytes (Ambozeptor 6000, (Behringwerke AG, Marburg, BRD, dilution 1.50, or also a purified immunoglobulin fraction of this antiserum) and made up to 10 ml with a buffer containing 0.15 mmol/1 CaCl2, 0.5 mmol/1 MgCl2, 20 mmol/1 Hepes, pH 7.3, 150 mmol/1 NaCl and 1 g/l gelatin. The cell density of the erythrocyte suspension was 2 x 10^ cells/ml.
Etter ca. 10 minutter er reagenset bruksferdig. Før anvendelse i den fotometriske prøve ble suspensjonen oppvarmet til 37°C og eventuelt sedimentert erytrozytter forsiktig opp-rystet. After approx. After 10 minutes, the reagent is ready for use. Before use in the photometric test, the suspension was heated to 37°C and any sedimented erythrocytes were gently shaken.
Under en bestemmelse kan det imidlertid sees bort fra cellenes sedimentasjon. During a determination, however, the sedimentation of the cells can be disregarded.
Eksem<p>e<l>_<2>Eczema<p>e<l>_<2>
MålefremgangsmåteMeasurement procedure
50 pl human-citratplasma ble pipettert i en til 37°C foroppvarmet kyvette og tilsatt 500 \ il av det ovenfor frem-stilte reagens, som var blitt holdt på 37°C. Etter sammen-blanding av komponentene ble ekstinksjonen bestemt ved 578 nm. Som måleparameter bestemmes tiden for oppnåelse av en ekstinksjonsdifferans på -0,1 O.D., som frembringes ved lyse av erytrozytter ved komplement. Denne tid er selvsagt kortere enn den for en lyse av halvparten av erytrozyttene. Spesielt ved patologiske plasmaer benyttes nedsatt komplementaktivitet viser tidsgevinsten seg ved anvendelse av fremgangsmåten ifølge oppfinnelsen sammenlignet til en bestemmelse av den for den halvmaksimale lyse nødvendige tid (fig. 1). 50 µl of human citrate plasma was pipetted into a cuvette preheated to 37°C and 500 µl of the reagent prepared above, which had been kept at 37°C, was added. After mixing the components together, the extinction was determined at 578 nm. As a measurement parameter, the time to achieve an extinction difference of -0.1 O.D., which is produced by lysis of erythrocytes by complement, is determined. This time is of course shorter than that for a lysis of half of the erythrocytes. Especially in the case of pathological plasmas, reduced complement activity is used, the time gain is shown when using the method according to the invention compared to a determination of the time required for the half-maximal light (Fig. 1).
Eksemgel_3Eczema gel_3
1 ml citratplasma ble blandet med 200 |il av en oppløsning av Fab'-fragmenter av kaninantistoffer (Behringwerke AG, Marburg, BRD) mot komplementfaktorene Clq, Cls, C3, C4 eller C5 av den klassiske vei eller faktor B av den alternative vei. For fortynning av oppløsningen av Fab'-fragment ble den blandet med fysiologisk koksaltoppløsning. 1 ml of citrate plasma was mixed with 200 µl of a solution of Fab' fragments of rabbit antibodies (Behringwerke AG, Marburg, BRD) against the complement factors Clq, Cls, C3, C4 or C5 of the classical pathway or factor B of the alternative pathway. For dilution of the Fab' fragment solution, it was mixed with physiological saline solution.
Tabell_lTable_l
Innvirkning av antistoff-fragment Fab' mot Clq,Cls, C3, C4, C5 og faktor B på reaksjonstiden. Effect of antibody fragment Fab' against Clq, Cls, C3, C4, C5 and factor B on the reaction time.
Blanding: 60 ja 1 plasmaMixture: 60 ja 1 plasma
30 |il Fab'- resp. NaCl-oppløsning30 |il Fab'- resp. NaCl solution
600 fil suspensjon av sensitivert erytrocytter 600 fil suspension of sensitized erythrocytes
(37°C) (37°C)
I tabell I ser man hvorledes stigende Fab'-konsentrasjoner mot Clq, Cls, C3, C4 og C5 forlenger reaksjonstiden for kontrollene (Fab'-konsentrasjon = 0, bare NaCl-oppløsning). Antistoffer mot faktor B viser derimot ingen innvirkning. Dette resultat viser at reagenset anviser følsomt alle undersøkte faktorer av den klassiske vei, imidlertid ikke en mangel på faktor B fra den alternative vei av komplementet. Table I shows how increasing Fab' concentrations against Clq, Cls, C3, C4 and C5 prolong the reaction time for the controls (Fab' concentration = 0, only NaCl solution). Antibodies against factor B, on the other hand, show no effect. This result shows that the reagent sensitively indicates all investigated factors of the classical pathway, however not a deficiency of factor B from the alternative pathway of complement.
Eksemp_el_4Example_el_4
Bestemmelse_av_en_enkeltf aktor_.(_C4]__Determination_of_a_single_proctor_.(_C4]__
C4-mangelserum ble oppnådd fra marsvin med genetisk betinget C4-mangel. Til bestemmelse av aktiviteten av faktor C4 C4-deficient serum was obtained from guinea pigs with genetic C4 deficiency. For determining the activity of factor C4
ble humant citratplasma utfortynnet med NaCl.human citrated plasma was diluted with NaCl.
20 ul plasma ble blandet med et overstående av 100 ul mangelserum og reaksjonen startet ved tilsetning av sensitiverte erytrozytter. Ved dobbelt logaritmisk oppføring ble det funnet et lineært forhold mellom C4-konsentrasjoner og reaksjonstid. 20 ul of plasma was mixed with a supernatant of 100 ul of deficient serum and the reaction started by the addition of sensitized erythrocytes. By double logarithmic entry, a linear relationship was found between C4 concentrations and reaction time.
En sammenlignbar blanding lar seg gjennomføre med et C2-mangelserum for C2. A comparable mixture can be made with a C2-deficient serum for C2.
Eksemp_el_5_ Example_el_5_
Jh2Yi£lS!3i22_5Y_££2£2§ser _§Y_IS2É2yi§liD2§§Yt§m§t_2i_komgle-mentaktiviteten Jh2Yi£lS!3i22_5Y_££2£2§ser _§Y_IS2É2yi§liD2§§Yt§m§t_2i_comgle ment activity
Innvirkningen av trombin, kallikrein og plasmin, som akti-veres under koagulering på komplementaktiviteten av plasma viser tabell 2. Av disse resultater lar det seg avlede at koaguleringssystemet kan påvirke komplementaktiviteten. Således fåes ved trombin- og kallikreininnvirkning på plasma lengere måletider. Table 2 shows the effect of thrombin, kallikrein and plasmin, which are activated during coagulation, on the complement activity of plasma. From these results, it can be deduced that the coagulation system can influence the complement activity. Thus, the effect of thrombin and kallikrein on plasma results in longer measurement times.
Blanding:Mixture:
20 ul enzymoppløsning og 60 ul citratplasma ble inkubert 60 sekunder ved 37°C. Deretter ble det tilsatt 600 ul raegens ifølge eksempel 1. Det bestemmes tiden for Delta-E = 0,1. 20 µl of enzyme solution and 60 µl of citrate plasma were incubated for 60 seconds at 37°C. Then 600 ul of reagent according to example 1 was added. The time for Delta-E = 0.1 is determined.
Claims (7)
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DE (1) | DE3501496A1 (en) |
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US7411006B2 (en) | 2000-10-23 | 2008-08-12 | Shanbrom Technologies, Llc | Enhanced production of blood clotting factors and fibrin fabric |
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US4492761A (en) * | 1982-04-05 | 1985-01-08 | Duke University | Complement assay method |
EP0132537A1 (en) * | 1983-05-31 | 1985-02-13 | Denka Seiken Co., Ltd. | Immunoassay |
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