EP0934525A1 - Dosage immunologique avec double marquage - Google Patents

Dosage immunologique avec double marquage

Info

Publication number
EP0934525A1
EP0934525A1 EP97941088A EP97941088A EP0934525A1 EP 0934525 A1 EP0934525 A1 EP 0934525A1 EP 97941088 A EP97941088 A EP 97941088A EP 97941088 A EP97941088 A EP 97941088A EP 0934525 A1 EP0934525 A1 EP 0934525A1
Authority
EP
European Patent Office
Prior art keywords
component
binding interaction
binding
label
assay
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97941088A
Other languages
German (de)
English (en)
Inventor
David Xenova Limited MACALLAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Terragen Discovery Inc
Original Assignee
Xenova Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Xenova Ltd filed Critical Xenova Ltd
Publication of EP0934525A1 publication Critical patent/EP0934525A1/fr
Withdrawn legal-status Critical Current

Links

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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

Definitions

  • the present invention relates to an improved solid phase assay.
  • a first component of a binding interaction between the first component and a second component is immobilised on a solid support .
  • Any complex formed by the binding interaction is generally isolated on the solid support and the presence of the isolated complex is determined.
  • errors can arise as some of the complex formed by the binding interaction may be lost. Loss of complex may occur if, for some reason, the first component immobilised on the solid support becomes dissociated therefrom. If some of the complex is lost before it can be detected, then subsequent determination of the amount of the complex will give an artificially low result, leading, in some cases, to false assay results.
  • the present invention seeks to provide a solid phase assay which incorporates an internal control to assess whether, or to what extent, the complex formed by the binding interaction is lost before it is determined.
  • the present invention provides a solid phase assay comprising:
  • the assay of the present invention is advantageous as the first label can function as an internal control to assess whether, or to what extent, the complex formed by the binding interaction is lost before it is determined. If complex is lost before it is determined, the signal generated by the first label will be inhibited.
  • the binding interaction can be, for example, (a) an interaction between a ligand and a receptor therefor, for example the binding of a cytokine, hormone, neurotransmitter, growth factor or peptide to a receptor therefor, (b) a protein/protein interaction such as the binding of an antibody to an antigen or a signalling protein interaction, preferably an intracellular signalling protein interaction such as a ras-raf interaction or an interaction between cyclin and cyclin-dependent kinase or (c) an interaction between a DNA sequence and one or more protein which recognises the sequence.
  • the first component and/or the second component may consist of more than one substance.
  • the first component may consist of a DNA sequence and the second component may consist of two proteins which can together bind thereto. Either component of a given binding interaction may be the component immobilised on the solid support .
  • the solid support is an optical quality surface, i.e. a surface that has minimal effect on a signal generation process and on a generated signal.
  • the solid support typically comprises polystyrene beads or the wall of a reaction vessel.
  • the solid support may therefore comprise the walls of a 96 well microtitration plate or the walls of a higher density plate.
  • a higher density plate is a plate of the same footprint size as a 96 well microtitration plate having more than 96 wells. Typically, a higher density plate has about 384 wells.
  • suitable solid supports include optical fibres and glass or quartz surfaces such as quartz cuvettes, flat sheets of quartz or glass and quartz or glass rods.
  • the assay of the invention includes the step of immobilising the first component of the binding interaction on the solid support.
  • the first component can be immobilised on the solid support in any convenient manner such as by a protein capture technique.
  • the first component is covalently or non-covalently bound to a first substance and a second substance, capable of binding to the first substance, is adsorbed onto the solid support. Capture of the first substance by the second substance can then be effected.
  • the first and second substances are, respectively, biotin and streptavidin, an antigen and an antibody therefor, a glutathione-S-transferase fusion protein and glutathione, or a histidine tagged protein and nickel .
  • the first component can be passively adsorbed onto the solid support.
  • the first and second labels are different.
  • the signals generated by the first and second labels can be distinguished.
  • the components labelled with the first and second labels can be distinguished.
  • the first label and/or the second label is preferably fluorometrically detectable. More preferably the first label and/or the second label is fluorescein, calcein, rhodamine or a mutant of green florescent protein. Preferred labels are those which are susceptible of quantitative determination. It is particularly preferred that at least one, more preferably both, of the first and second labels are lanthanide ion labels such as samarium and europium ion labels .
  • a lanthanide ion label is a substantially non- fluorescent lanthanide ion complex covalently bound to the component in question, which is detectable by adding a developer comprising (a) a detergent and (b) a chelating compound with which the lanthanide ion gives fluorescence, thereby dissociating the lanthanide ion from the component and forming a fluorescent lanthanide ion chelate. Further details regarding the developer are given below.
  • the assay of the invention is typically either an assay for an inhibitor of a binding interaction or an assay of a specimen for a predetermined analyte .
  • An assay of the invention for an inhibitor of a binding interaction will now be described in more detail. Such an assay is shown in Figure 1.
  • an assay for an inhibitor of a binding interaction comprises: (I) providing a solid support on which is immobilised a first component of a binding interaction between the first component and a second component, the first component having a first label which is non- radioactive; (II) contacting the said solid support with the second component of the binding interaction and a putative inhibitor of the binding interaction under conditions under which the binding interaction, in the absence of inhibitor, would be expected to occur, the second component having a second label which is non-radioactive and which is distinguishable from the first label; and (III) determining whether the putative inhibitor inhibits the binding interaction.
  • a false positive result is generated when the amount of the complex formed by the binding interaction is reduced for any reason other than inhibition of the binding interaction by the putative inhibitor, for example if the putative inhibitor causes the first component to become dissociated from the solid support. In this event, the amount of complex isolated on the solid support in step (III) will be reduced and both the signal generated by the first label and the signal generated by the second label will be inhibited.
  • step (II) above will obviously vary depending on the binding interaction but typically comprise incubation at from 10°C to 40°C, preferably at about room temperature, for from 30 minutes to 2 hours, preferably for about 1 hour .
  • Step (III) is generally carried out by determining whether, or to what extent, the binding interaction has occurred. This can be done by determining the first and second components of the binding interaction. Typically, step (III) comprises (a) rinsing away unbound substances to isolate on the solid support any complex formed by the binding interaction and (b) determining the presence of the labels in the isolated complex. If a substantially non- fluorescent lanthanide ion complex, as described above, is used as the first or second label, step (III b) above typically comprises the detection of the label by:
  • a developer solution comprising (a) a detergent and (b) a chelating compound with which the lanthanide ion gives fluorescence.
  • the lanthanide ion is a samarium or europium ion.
  • the developer solution further comprises a buffer.
  • the detergent is a non-ionic surfactant. More preferably it is a polyoxyethylene ether such as an iso-octylphenoxypolyethoxyethanol in which the polyethoxy chain contains about ten ethoxy units (i.e. Triton X-100 ® ) .
  • a polyoxyethylene ether such as an iso-octylphenoxypolyethoxyethanol in which the polyethoxy chain contains about ten ethoxy units (i.e. Triton X-100 ® ) .
  • the chelating compound is 2- naphthoyltrifluoroacetone, tri-n-octylphosphine oxide or
  • the buffer is capable of maintaining a pH of from 3 to 4 , more preferably about 3.2.
  • the buffer is an acetate-phthalate buffer.
  • Suitable developers are known in the art.
  • An example of a suitable developer is DELFIA ® Enhancement solution available from allac Oy.
  • step (B) is conducted by shaking the solid support with the said complex isolated thereon, in the presence of the developer solution, for from 5 to 20 minutes, for example about 10 minutes.
  • Greater sensitivity may be obtained by exciting the fluorescent chelates obtained in step (B) by means of a light impulse before determining the fluorescent chelate. During the interval between excitation and detection of the fluorescent chelates, the fluorescence from possible sources of interference decays.
  • fluorescent europium chelates are excited from 300 to 500 ⁇ s, preferably about 400 ⁇ s, before they are detected.
  • fluorescent samarium chelates are excited from 40 to 60 ⁇ s, preferably about 50 ⁇ s, before they are detected.
  • fluorescent terbium chelates are excited from 80 to 120 ⁇ s, preferably about lOO ⁇ s, before they are detected.
  • fluorescent dysprosium chelates are detected from 20 to 40 ⁇ s, preferably about 30 ⁇ s, before they are detected.
  • the light impulse is produced by a xenon flash lamp with a flash duration of about 1 ⁇ s .
  • the wavelength of the light impulse is typically from 320-340 nm.
  • the light impulse is generated, and the fluorescence is subsequently detected, in a fluorometer such as the Wallac 1234 ® fluorometer or the Wallac 1420 Victor Multilabel Counter ® .
  • an assay of the present invention which is an assay for an inhibitor of a binding interaction
  • the effect of a putative inhibitor on two or more different bioaffinity reactions can be investigated in the same assay.
  • the effect of a putative inhibitor on one or more binding interactions can be investigated simultaneously.
  • a labelled first component of each binding interaction is provided on the solid support
  • a labelled second component of each binding interaction is contacted with the solid support and the putative inhibitor, the labels for each first component typically being distinguishable from each other and the labels for each second component typically being distinguishable from each other .
  • the second component of each binding interaction has a label which can be distinguished from the label of the first component of each binding interaction.
  • the same label can be used for each second component, in which case any specific inhibition of any of the binding interactions would result in a decrease in the signal generated by the label attached to each second component without a corresponding decrease in any of the signals generated by the labels attached to the first components.
  • the same label can be used for each first component, in which case any non-specific inhibition of any of the binding interactions would result in a decrease in the signal generated by the label attached to each first component .
  • a particularly preferred assay for an inhibitor of a binding interaction is an assay wherein the first component is tumour necrosis factor a (TNFo;) and the second component is the p55 or p75 receptor protein.
  • TNFo; and the p55 or p75 receptor protein are labelled with a substantially non-fluorescent lanthanide ion complex as described above.
  • a complex can be detected by steps (A) to (C) , as described above.
  • the TNF ⁇ is labelled with a samarium complex and the p55 or p75 protein is labelled with a europium complex.
  • TNFo is typically immobilised on a solid phase by biotinylation followed by capture with streptavidin which is adsorbed to the solid phase.
  • biotinylated TNFc is immobilised onto plates which are precoated with steptavidin.
  • TNFo is the first component in the preferred assay described above
  • non-specific binding is significantly lower than in previous TNF ⁇ /receptor protein screening assays.
  • the receptor protein has been immobilised on the solid phase.
  • Non-specific binding consists of all binding between the second component and the solid phase other than binding between the second component and the first component. Non-specific binding can lead to false assay results and a reduction in non specific binding is advantageous.
  • FIG. 1 shows an assay for the detection of inhibitors of the TNFo; and p55 receptor binding interaction.
  • the first component of the binding interaction is tumour necrosis factor cv (TNF ⁇ ) .
  • the TNFo is covalently bound to biotin, which is adsorbed to streptavidin on the solid phase.
  • the second component of the binding interaction is p55 receptor protein.
  • the TNFc is labelled with a samarium ion label and the p55 receptor protein is labelled with a europium ion label.
  • the various symbols have the following meanings:
  • test kit suitable for an assay for an inhibitor of a binding interaction which test kit comprises : (a) a solid support; and (b) labelled first and second components of a binding interaction, as defined above.
  • the first component is immobilised on the solid support.
  • a solid phase assay, conducted to determine whether a specimen contains a predetermined analyte typically comprises :
  • the analyte is typically a substance of diagnostic significance, such as an antibody to a pathogen or an antigen recognised by such an antibody.
  • the first component is typically an antibody or a DNA sequence.
  • the conditions in step (II) generally comprise incubation at from 18°C to 40°C, preferably at about 37°C. The incubation period will depend on the particular binding interaction but is typically from 10 to 60 minutes, for example about 30 minutes.
  • the specimen is a serum, plasma, urine, saliva, or cerebrospinal fluid specimen, more preferably a serum or plasma specimen.
  • Step (III) is generally carried out by determining whether, or to what extent, the binding interaction has occurred. This can be done by determining the first component of the binding interaction and the complex formed by the binding interaction.
  • step (III) the complex formed by the binding interaction is typically determined by (a) rinsing away unbound specimen components to isolate on the solid support any complex formed by the binding interaction, (b) adding a further reactant which recognises the said complex and which is labelled and (c) determining the label of the further reactant .
  • the label of the further reactant is distinguishable from the first label. It can be detected by conventional methods. It may be any conventional label such as an enzyme label, a fluorescent label or a radioactive label.
  • the further reactant can be anti-human IgG conjugated with the enzyme horseradish peroxidase (hrp) .
  • the hrp conjugated anti- human IgG binds to complex formed by the binding interaction. Unbound anti-human IgG may be rinsed away and the bound anti-human IgG may be detected by adding a chromogenic substance containing tetramethyl benzidine (TMB) . This reacts with the hrp conjugated to the anti- human IgG to form a blue coloured product .
  • TMB tetramethyl benzidine
  • the first label is preferably a substantially non- fluorescent lanthanide ion complex, as described above. It can be detected by steps (A) to (C) , as described above. If the first component should be become dissociated from the solid support, the signal generated by the first label will be inhibited. Thus, one source of false negative results can be detected by means of the present invention.
  • the present invention also provides a test kit suitable for an assay of a specimen for a predetermined analyte, which test kit comprises a solid support upon which is immobilised a labelled first component of a binding interaction as defined above.
  • the present invention provides a solid phase assay in which (i) a solid support is provided, upon which a first component of a binding interaction between the first component and a second component is immobilised, (ii) a step is carried out in which the second component may bind to the first component and (iii) whether, or to what extent, the binding interaction has occurred is determined, wherein the first component is labelled with a non- radioactive label.
  • Lyophilised protein carrier free TNF ⁇ was reconstituted in sodium carbonate buffer at pH 9.6. It was then simultaneously labelled with biotin and samarium (Sm) chelate by incubation with biotin- e-aminocaproic acid-N- hydroxysuccinimide ester and samarium chelated with N 1 - (p- isothiocyanatobenzyl) -diethylenetriamine-N 1 , N 2 , N 3 , N 4 - tetraacetic acid for 18 hours at 4°C.
  • the labelled protein was purified by gel filtration chromatography, with an 80% yield. The concentration of the purified labelled protein was estimated to be 80 ⁇ g/ml. The labelling ratio was calculated to be 0.5:0.7:1 Sm : biotin: TNFo; .
  • the labelled purified protein was stabilised with 1 mg/ml Bovine Serum Albumin (BSA) and stored at -70°C.
  • BSA
  • Tris-HCl buffered NaCl assay buffer solution pH 7.8 containing 0.05% NaN 3 , BSA, Bovine gamma globulins, Tween 40, diethylenetriaminepentaacetic acid and an inert red dye (available from Wallac Oy as DELFIA ® assay buffer) was used for diluting the labelled protein.
  • Streptavidin coated 96 well polystyrene plates of good optical quality were used to capture the Sm-TNF ⁇ -biotin. Increasing concentrations of Sm-TNF ⁇ -biotin were added to streptavidin coated wells (100 ⁇ l/well) and incubated for 1 hour at room temperature with shaking.
  • Binding was observed to increase linearly with increasing concentrations of Sm-TNFce-biotin.
  • Lyophilised protein carrier free recombinant p55 receptor protein was reconstituted in sodium carbonate buffer at pH 9.6. It was then labelled with europium (Eu) chelate by incubation with europium chelated with N 1 - (p- isothiocyanatobenzyl) -diethylenetriamine N 1 , N 2 , N 3 , N 4 - tetraacetic acid for 18 hours at 4°C.
  • the labelled protein was purified by gel filtration chromatography, with an 80% yield. The concentration of the purified labelled protein was estimated to be 80 ⁇ g/ml. The labelling ratio was calculated to be 2.1:1 Eu:p55.
  • the labelled purified protein was stabilised with lmg/ml BSA and stored at -70°C.
  • the control samarium signal was as expected in the assay at around 15,000 samarium counts.
  • Example 1 a 2 hour incubation period was used for binding of the p55-Eu to the Sm-TNFc-biotin.
  • a time course of binding of p55-Eu to Sm-TNF ⁇ -biotin at a fixed final concentration of 7nM was performed with 15 minute time points.
  • the assay was conducted in the same way as in Reference Example 2 except that, as explained above, the concentration of p55-Eu was fixed at 7nM and the incubation time was varied. The results are shown in Table 4.
  • a Sm-TNFc-biotin complex was prepared as described in Reference Example 1.
  • the labelled protein was diluted with the Tris-HCl buffered NaCl assay buffer described in Reference Example 1.
  • 384 well white polystyrene plates of good optical quality were coated with 40 ⁇ g/ml streptavidin overnight at 4°C. These streptavidin coated plates were used to capture the Sm-TNF ⁇ -biotin.
  • the plates were first blocked using 2% BSA and then increasing concentrations of Sm-TNF ⁇ -biotin were added to the streptavidin coated wells (total volume 50 ⁇ l/well) . Incubation then took place for 1 hour with shaking.
  • the samarium ions were determined as described in Reference Example 1. The results are shown in Table 5.
  • Purified p.55 receptor protein labelled with europium chelate was prepared as described in Reference Example 2. Streptavidin coated 384 well plates to which Sm-TNF ⁇ -biotin was bound were prepared as in Reference Example 5 using 50 ⁇ l of 120 ng/ml Sm-TNF ⁇ -biotin. The plates were then washed three times with a wash buffer as described in Reference Example 1. The purified p.55-Eu was then added at various concentrations (total volume 50 ⁇ l/well) . Incubation took place at room temperature with shaking for 2 hours .
  • Table 7 show 50% inhibition at a p55 concentration of 4.12nM.
  • the Cheng-Prusoff equation was used to determine the p55 Ki and hence p55-Eu Kd from the p55 IC 50 , as shown in Example 1.
  • Tables 9, 10 and 11 show the data for the 96 well plates.
  • Tables 12, 13 and 14 show the data for the 384 well plates.
  • the TOTAL values are those obtained adding both Sm- TNF ⁇ f-biotin and p55-Eu in the assay.
  • the NSB non-specific binding refer to those values obtained using p55-Eu alone.
  • the concentrations used were 120ng/ml SM-TNF ⁇ -biotin and 7.7nM p55-Eu.
  • the TOTAL values are those obtained adding both Sm- TNF ⁇ -biotin and p55-Eu in the assay.
  • the NSB non-specific binding refer to those values obtained using p55-Eu alone.
  • concentrations used were 120ng/ml SM-TNFc ⁇ -biotin and

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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)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un dosage en phase solide consistant à: (a) réaliser un premier support solide sur lequel est immobilisé un premier composant d'une interaction de liaison entre un premier composant et un second composant, le premier composant portant un premier marqueur qui est non radioactif; (b) mettre en contact ledit support solide avec (i) un inhibiteur présumé de l'interaction de liaison et le second composant de l'interaction de liaison, le second composant ayant un second marqueur qui est non radioactif et qui peut être distingué du premier marqueur, dans des conditions dans lesquelles on s'attend à ce qu'en absence d'inhibiteur, l'interaction de liaison ait lieu ou (ii) avec un spécimen à analyer pour un analyte prédéterminé, qui est le second composant de l'interaction de liaison, dans des conditions dans lesquelles on s'attend à ce que l'interaction de liaison se produise lorsque l'analyte est présent dans l'échantillon; et (c) à déterminer si, et dans quelle mesure, l'interaction de liaison s'est produite.
EP97941088A 1996-10-11 1997-09-22 Dosage immunologique avec double marquage Withdrawn EP0934525A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB9621256 1996-10-11
GBGB9621256.8A GB9621256D0 (en) 1996-10-11 1996-10-11 Assay
PCT/GB1997/002552 WO1998016833A1 (fr) 1996-10-11 1997-09-22 Dosage immunologique avec double marquage

Publications (1)

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EP0934525A1 true EP0934525A1 (fr) 1999-08-11

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EP (1) EP0934525A1 (fr)
AU (1) AU4311497A (fr)
GB (1) GB9621256D0 (fr)
WO (1) WO1998016833A1 (fr)
ZA (1) ZA978504B (fr)

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EP1216417A1 (fr) * 1999-09-28 2002-06-26 Evotec OAI AG Analyse quantitative et de classement par types des particules infracellulaires
DE10109779A1 (de) * 2001-03-01 2002-09-19 Infineon Technologies Ag Vorrichtung und Verfahren zum Erfassen von makromolekularen Biopolymeren mittels mindestens einer Einheit zum Immobilisieren von makromolekularen Biopolymeren

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Publication number Priority date Publication date Assignee Title
IL68506A (en) * 1982-05-04 1988-06-30 Syva Co Simultaneous calibration heterogeneous immunoassay method and device and kit for use therein
GB8619206D0 (en) * 1986-08-06 1986-09-17 Ekins Roger Philip Fluorometric determination of analyte concentration
DE69008178T2 (de) * 1989-06-26 1994-10-06 Tritech Partners Analyse von vitamin b12.
JP3185215B2 (ja) * 1990-07-13 2001-07-09 三菱化学株式会社 免疫測定法

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Title
See references of WO9816833A1 *

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GB9621256D0 (en) 1996-11-27
AU4311497A (en) 1998-05-11
WO1998016833A1 (fr) 1998-04-23
ZA978504B (en) 1998-04-01

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