EP0188608A1 - Methode d'essai immunometrique du type a sandwich, par reaction croisee a deux sites avec enzymes marquees - Google Patents

Methode d'essai immunometrique du type a sandwich, par reaction croisee a deux sites avec enzymes marquees

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Publication number
EP0188608A1
EP0188608A1 EP19850903950 EP85903950A EP0188608A1 EP 0188608 A1 EP0188608 A1 EP 0188608A1 EP 19850903950 EP19850903950 EP 19850903950 EP 85903950 A EP85903950 A EP 85903950A EP 0188608 A1 EP0188608 A1 EP 0188608A1
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Prior art keywords
analyte
antibody
interest
solid
reaction
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EP19850903950
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German (de)
English (en)
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Vipin D. Shah
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    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G01N33/54306—Solid-phase reaction mechanisms

Definitions

  • the present invention relates generally to immunological assay techniques for determining the presence of an analyte in serum and more particularly to a two site cross-reaction immunoassay sandwich testing method which has particular application in the qualitative and quantitative determination of the level of the creatine phospho-kinase MB Isoenzyme (CK-MB) in human serum utilizing an enzyme label.
  • CK-MB creatine phospho-kinase MB Isoenzyme
  • sandwich techniques utilize three types of assays.
  • the undesired cross-reaction is reduced based on the observation that the undesirable cross-reactant would not react, at the same time, with antibodies specifically raised in two different animal species .
  • Both of the antibodies are therefore raised against the specific analyte of interest, however they differ in that they are raised in different animal species, such as human beings and guinea pigs.
  • the undesirable effect of the unwanted cross-reactions of the first antibody is reduced through the utilization of the second antibody, whereby an accurate test for the analyte of interest is obtained.
  • This type of sandwich assay technique is used in the detection of hepatitis.
  • the second type of sandwich assay is used when the serum contains, as metabolic by-products , fragments of the analyte to be measured.
  • Two antibodies used for this assay are raised specifically against the analyte of interest. However , one antibody reacts specifically with one portion of the analyte while the second antibody reacts specifically with another portion of the analyte.
  • This prior art immunometric sandwich technique thus util izes antibodies that are specifically raised against the specific analyte to be measured.
  • the unwanted crossreactions of the specific antibody with a fragment of the analyte of interest have caused researchers to develop this two site immunoassay sandwich technique which minimizes the effects of the unwanted crossreactions from a fragment of the analyte.
  • the third sandwich technique uses the same specific antibody twice to detect the analyte of interest.
  • an antibody which is specif ic to the analyte of interest is immobilized on a solid-phase and reacted with the analyte. All other interfering substances are removed before the analyte is reacted once again wi th the same antibody except that the antibody is now tagged for assay purposes.
  • the subject of the present invention is the use of the cross-reactive antibody sandwich technique in which enzyme immunoassay is used rather than radioimmunoassay.
  • the present invention discloses the use of an enzyme label in a matrix aided sandwich technique utilizing cross-reactive antibodies in which CK-MB is specifically and accurately measured at speed and freedom from interference from CK-MM, CK-BB and other isoenzymes of CK to the extent not possible by teachings of U.S. Patent No. 4,353,982. This is achieved by incorporating steps to minimize the effect of unusually large amounts of CK-MM or CK-BB and accelerating the speed at which reactions take place through the use of reaction matrix.
  • the present invention differs from the prior art in that it specifically utilizes the previously avoided crossreactive capabilities of two different- antibodies to create a specific sandwich assay technique wherein an enzymatic label is utilized.
  • the immunoassay technique of the present invention includes the selection of two different antibodies each of which is specific to a different analyte but each of which will cross-react with the analyte of interest.
  • the first such antibody is affixed to a solid-phase and then reacted with the unknown sample. After reaction, the reactants are separated and the solid-phase portion thereof is retained. The second antibody, being tagged, is then reacted with the retained solid-phase portion. After reaction, the reactants are separated and the solidphase portion is tested for the presence of the tag.
  • Calibration tests are conducted concurrently with known amounts of the analyte of interest. The results of the test for the unknown can be compared therewith to provide a qualitative and quantitativedetermination of the presence of the analyte of interest.
  • This invention particularly utilizes an enzymatic label.
  • the immunoassay technique of the present invention includes reacting the unknown human serum with CK-BB antibody which is affixed to a solid-phase . After the reaction is complete, the reactants are washed, centrifuged and the liquid portion thereof is aspirated. The solid-phase portion, having been retained, is then reconstituted in solution containing tagged CK-MM antibody and reacted therewith. Upon completion of the reaction, the reactants are washed and centrifuged and the liquid portion is aspirated. The retained solid-phase portion is now tested for the presence of the tag. Calibration tests are also conducted utilizing serum having known amounts of CK- MB to provide a background with which to determine the quantity of CK-MB in the unknown sample.
  • a primary advantage of the present invention is that an antibody that is specific to the analyte of interest need not be created in order to provide an immunolog ical assay test for the analyte. All that is required is that there be found two different antibodies which mutually cross-react only with the analyte.
  • the technique is very useful for those analytes which are unstable and hence cannot be practically used to make antibodies. It is also very useful when it is extremely difficult to purify the analyte, as it is the case with cancer antigens.
  • it is another advantage of the present invention, as applied to the testing for CK-MB that it utilizes the antibodies to CK-MM and CK-BB which are commercially available and inexpensive.
  • test may be performed rapidly and with greater accuracy than test procedures presently available utilizing an enzymatic label.
  • Fig. 1 is a flow chart depicting the two site cross-reaction immunometric sandwich assay method of the present invention
  • Fig. 2 is a schematic diagram of the assay method of the present invention.
  • Fig. 3 is a flow chart depicting the assay method of the present invention as applied to the detection of CK-MB;
  • Fig. 4 is a flow chart depicting an alternative embodiment of the assay method of the present invention.
  • Fig. 5 is a calibration curve for an enzyme labeled test.
  • test procedure of the present invention will permit detection and assay of the mutually cross-reacting analyte of interest. It will be seen that just a situation exists with regard to the immuno-logical assay for creatine phospho-kinase-MB (CK-MB).
  • the general test procedure of the present invention is performed on a serum sample 12 containing three related analytes which are schematically depicted as a diamond shaped analyte 14, hereinafter referred to as AA, a circular shaped analyte 15, hereinafter referred to as CC, and a hybrid analyte 18 of the two other analytes, shown as having both a partial diamond shape and a partial circular shape, hereinafter referred. to as AC.
  • AA diamond shaped analyte 14
  • CC circular shaped analyte 15
  • AC hybrid analyte 18 of the two other analytes
  • anti-AA 19 is affixed to a solid-phase 20 using a standard immunological procedure therefor.
  • antiCC is tagged for later detection. Procedures for tagging antibodies are well known and radioactive, fluorescent, chemluminescent, enzymatic or other types of tags, are appropriate for this test procedure.
  • the serum 12 is combined with the solid-phase anti-AA 20 and reacted 22 therewith for an appropriate time and temperature.
  • the time and temperature will vary with the particular antibodies and analytes involved in the test procedure, as well as the concentrations thereof.
  • the solid phase anti-AA 20 immunochemically binds 23 with the AA 14 and removes all of the AA from the serum as it is specific to that analyte. Additionally, because the anti-AA 19 will cross-react with the AC 18, all of the AC is also removed from the serum.
  • the reactants are separated 24, the solid-phase products 26 being retained and the liquid 28 being discarded.
  • Standard separation procedures such as centrifugation and aspiration, are utilized. It is to be noted that the discarded liquid 28 contains all of the CC analyte.
  • the retained solid-phase 26 is now reacted 32 with the tagged anti-CC 30.
  • the reaction 32 is allowed to continue for an appropriate time at an appropriate temperature. Again, the time and temperature will vary with the particular antibodies and analytes involved in the test procedure, as well as the concentrations thereof.
  • a standard separation step 34 is performed wherein the solid-phase 36 is retained and the liquid 38 is discarded.
  • the tagged anti-CC 30 can only crossreact 39 with the AC which previously cross-reacted with the solid phase anti-AA 26; all of the CC having been discarded in the liquid portion 28 of the first separation step 24.
  • the remaining solid-phase 36 contains a tagged sandwich of molecules 40 in which the AC (the analyte of interest) is sandwiched between two antibodies, neither of which is specific to that analyte but each of which will cross-react with it.
  • the solid-phase 36 may now be tested 42 for the tag, which test will give an indication of the presence of the analyte of interest.
  • Calibration tests utilizing known amounts of the analyte of interest and the above-described test procedure are conducted simultaneously to create a calibration curve against which to guage the results of the test for the unknown sample. Further tests utilizing known dilutions of the unknown sample and the above-described test procedure may also be performed to obtain test results which fall on sensitive portions of the calibration curve.
  • human serum contains three creatine phospho-kinase isoenzymes; creatine phosphokinase-MM (a skeletal tissue extract, hereinafter referred to as CK-MM) , creatine phospho-k inase-BB (a brain tissue extract, hereinafter referred to as CK-BB) and creatine phospho-kinase-MB (a heart tissue extract, hereinafter referred to as CK-MB) which is a hybrid form of CK-MM and CK-BB.
  • CK-MM a skeletal tissue extract
  • CK-BB a brain tissue extract
  • CK-MB a heart tissue extract
  • CK-MB testing for CK-MB by various methods has proved to be quite difficult as its properties are quite similar to CK-MM and CK-BB, the presence of which can significantly mask small amounts of CK-MB.
  • immunometric assay techniques for the detection of CK-MB these have been made diff icult by the present inabil ity o f researchers to prepare and develop a stable antibody which is specific to the CK-MB (hereinafter referred to as anti-CK-MB) , and no such anti-CK-MB is commercially available at this time.
  • anti-CK-MM and anti-CK-3B will cross-react with CK-MB , in addition to their having specific reactions with CKMM and CK-BB respectively.
  • the test pro.cedure described hereinabove is therefore applicable to the detection of CK-MB in accordance with the following description.
  • anti-CK-BB Prior to the actual assay of serum, anti-CK-BB is affixed to a solid phase as is described hereinbelow. However, as is also described hereinbelow an alternative embodiment of the .above-described test procedure may be performed in which this step is not performed. Additionally, prior to assay the antiCK-MM must be tagged, such as is described hereinbelow by the utilization of radio-active iodine (125 l ).
  • Anti-CK-BB for affixation to a solidphase is commercially available from many sources.
  • the solid-phase selected for linkage with the anti-CKBB was Bio-Rad Immuno-Beads (catalog no. 170-5602) although other solid phases could be utilized such as latex beads or cellulose beads or a membrane strip.
  • Bio-Rad Immuno-Beads are polyacrylamide beads having a diameter of approximately 10 ⁇ (microns) and which are designed for use as a solid phase in immunoassay testing. Following the procedure recommended by the manufacturer, the beads were first covalently linked to goat anti-rabbit Igg.
  • the beads were then suspended in 25 to 50ml of PBS containing 10% calf serum, although other proteins could be used to minimize non-specific binding. Thereafter, the beads were washed with PBS buffer (pH approximately 7.4, 0.02M PO 4 , 0.15M NaCl) . The beads were then slurried in 25 ml PBS containing 1% BSA. Approximately 100 ⁇ l of anti-CK-BB was then reacted with the beads to link the anti-CK-BB to the beads. The antibody coated beads were then washed and separated from unreacted anti-CK-BB with PBS buffer. Trie anti-CK-BB coated beads were then ready for utilization in the test procedure.
  • PBS buffer pH approximately 7.4, 0.02M PO 4 , 0.15M NaCl
  • the anti-CK-MM Prior to testing, the anti-CK-MM is iodinated with radioactive iodine (125 l ) to serve as a tag for the detection of CK-MB.
  • radioactive iodine 125 l
  • Such tagging procedures are well known, see FRAKER, T. and SPECK, J. , Biochemical and Bio-physical Research Commission , volume 80, no. 4, February, 1978.
  • 100 ⁇ g of anti-CK-MM (goat) was dissolved in 100 ⁇ l of water. Sixteen ⁇ l of this solution, to be used for tagging, was combined with 20 ⁇ l (0.25M PO4) PBS buffer (pH, 7.5) in a reaction vial. 480 microcurie of Nal 125 was added to this buffer .
  • test procedure for CK-MB is performed as depicted in Fig. 3.
  • reaction time and reaction temperature being parameters which may be varied depending upon the concentration of the serum and the strength of the anti-CK-BB on the Immuno-Beads. As is demonstrated in Example II hereinbelow, these variables may be adjusted such that good test results are obtainable utilizing room temperature and a reaction time of one hour.
  • the anti-CK-BB coated beads will have reacted with, and therefore removed from solution, all of the CK-BB and CK-MB antigens.
  • the CK-MM antigen does not react with the anti-CK-BB, the CK-MM remains in solution and is discarded with the aspirated waste water 80.
  • the second reaction phase 86 of the test procedure is now initiated by the addition of the r ad io iodinated anti-CK-MM 88 to the precipitate.
  • the reactants are vibrated or vortexed to reconstitute 84 the precipitate into the reaction.
  • the reaction time and reaction temperature are parameters which may be varied in accordance with the concentrations of the reactants. As is demonstrated in Example II good test results may be obtained with the utilization of room temperature and a reaction time of approximately 1 hour.
  • a second separation is performed in the same manner as the prior separation. That is, approximately 2ml of PBS wash buffer 90 containing 0.1% Tween 20 is added to the reactants and the reactants are centrifuged 92 at room temperature for an approximate time of ten minutes at an approximate speed of 2000 rpm to yield a buttonlike precipitate 94 at the bottom of the test vial
  • the liquid 96 in the test vial is then aspirated 98 and discarded in an appropriate manner, in that it contains excess radioactive anti-CK-MM.
  • the buttonlike precipitate 94 is then tested 100 with a scintillation detector to indicate the presence of radiation as a possible indication in the test serum.
  • radioimmunoassay testing that the increase in radioactivity does not vary linearly with increasing amounts of analyte in the test serum.
  • the variation of radiation with concentration of analyte depends upon many variables, such as quantity of antibody, dilution of antibody, antibody-analyte ratio and others.
  • the standard test procedure which is utilized to overcome this testing impediment is to perform a series of tests with varying known dilutions of the unknown test serum. A series of results are then obtained, one or more of which will lie on a sensitive portion of the calibration curve to give an accurate indication of the level of analyte in the sample.
  • the Examples I, II and IV given hereinbelow demonstrate the utilization of this procedure.
  • a variation on the above described test procedure, see Fig. 4, may be conducted in the following manner. Rather than affixing the first antibody 130 to a solid-phase 132, the first antibody 130 is added 136 directly to the unknown serum 134. Thereafter, the solid-phase 132, which has not been coated with the first antibody 130, but which is prepared for coating as described hereinabove, is added 138 to the reactants. The reaction 138 is then permitted to proceed in the manner described hereinabove and the remaining steps of the test procedure are conducted as previously described following reaction 72 of Fig. 3. It is found that sufficient amounts of the solidphase antibody-antigen complex attach to the beads 132 to give adequate test results. Examples IV and V presented hereinbelow are specific examples of the results achieved utilizing this variation of the test procedure.
  • EXAMPLE I This experiment was carried out with anti-CK-BB coated immuno-beads. 100 ⁇ l of sample was mixed with 200 ⁇ l of beads. After overnite incubation at room temperature, 0.5 ml PBS buffer with 10% calf serum was added to the test tube. The beads were separated by centrifugation. They were then reacted for 5 hours at room temperature with radioiodinated anti-CKMM. After incubation, beads were washed with 1 ml PBS buffer with 10% calf serum. The beads were then centrifuged and counted. Results
  • the calibrator samples were made by dissolving a powder containing 6-8% CK-MB in normal human serum at various dilutions.
  • This example shows effect of reducing incubation time and varying the. amount of beads to 200 ⁇ l and 500 ⁇ l.
  • the wash buffer used was 2 ml PBS with 5% calf serum and was added before each centr if ugation.
  • the incubation time was one hour for each of the reactions at room temperature.
  • Other test conditions were identical to those of Example I. Results
  • Example II The method described in Example II was used v/ith 200 ⁇ l of beads and an incubation time of 1.5 hours for each reaction.
  • the tracer used in this experiment had higher non-specific binding.
  • Several serial samples of a patient just admitted to a hospital for a myocardial infarct were tested. Sample Count
  • the rising levels of CK-MB shown in this Example demonstrate the well known increase in CK-MB levels in the patient following a myocardial infarct or similar incident.
  • polyacryl amide beads were used as a precipitating antibody.
  • diluted (1:30) anti-CK-BB was added to the sample.
  • Bio-Rad Immuno-beads were suspended in 50 ml PBS buffer with 1% BSA, 500 ⁇ l of these beads being comparable in number of beads to 250 ⁇ l of beads used in previous examples. These beads were not precoated with anti-CK-BB.
  • the reactants were centrifuged and aspirated.
  • a second incubation of one hour was carried out in the normal manner. Therafter, 2 ml of PBS buffer with 1% BSA and 1% Tween was added and reactants were centrifuged, aspirated and the solid-phase counted. The results were as follows: Sample Count NHS 2076
  • the above sample contained roughly 5 mlU/ml and 10 mlU/ml CK-MB which is roughly the limit of normal range.
  • the use of a membrane as a solid-phase is demonstrated by using anti-CK-BB immobilized on Whatman no. 1 filter paper as described hereinabove.
  • the antibody coated membrane (3/8" x 1") was p l a c e d i n a m i x t u r e o f s a m p l e a n d 2ml PBS 5% BSA buffer. After a first incubation of 1 hour, the liquid was aspirated and the membrane was thoroughly washed. Radioiodinated anti-CK-MM was added. After a second incubation of one hour the liquid was aspirated. The membrane was washed thoroughly and transferred to another tube. The results were as follows:
  • the present invention derives its uniqueness from the utilization of the cross-reactive capabilities of the antibodies that are selected for use. This is in contradistinction to standard immunological assay techniques in which the cross-reactive capabilities of the antibodies are minimized or avoided through the use of multiple antibodies which are each specific to the analyte of interest. Utilization of the instant test technique for the detection of CK-MB in human serum provides a rapid and effective means for the detection and identification of myocardial infarcts and similar heart disturbances in patients immediately following their occurance.
  • CK-BB and CK-MM isoenzymes utilized to create appropriate antibodies as well as the anti-CK-BB and anti-CK-MM were produced according to methods described in the article IMPROVED RAD IOIMMUNOAS S A Y FOR CREATINE KINASE ISOENZYMES IN PLASMA, authored by Ritter, Mumm and Roberts, Clinical Chemistry, Volume 27, No. 11, 1981, Page 1878, et seq.
  • the macrobeads Prior to conducting the test, the macrobeads are coated with anti-CK-BB.
  • the procedure to accomplish the coating of the macrobeads utilizes goat anti-rabbit antibody which is diluted 1:50 in Bicarbonate-Carbonate buffer, pH 9.6.
  • the diluted antibody was added to the macrobeads (approximately 250 ul of diluted antibody per bead) and allowed to stand at room temperature overnight. The beads were than removed from the antibody solution.
  • Rabbit-antiCK-BB was prepared by adding the antibody to TrisAcetic acid buffer containing 0.1% BSA plus 3% PEG; the dilution of the antibody depends upon the titer of the antibody and such dilution is performed so that approximately 250 ul of the antibody solution provides more than enough rabbit antibody to fully bind all of the goat anti-rabbit antibody previously bound to the macrobeads. The macrobead solution was then incubated for 24 hours at 2-6 degrees Centrigrade.
  • HRP Horse Radish Peroxidase
  • the enzyme labeled test procedure is conducted utilizing the coated macrobeads ajnd the conjugate.
  • 200 ul of sample to be tested is added to a test tube with a coated macrobead and incubated for 30 minutes. Thereafter, 2 ml of wash buffer is added and then aspirated. Thereafter, 200 ul of conjugate is added to the bead and incubated for 30 minutes. Three wash steps are then conducted wherein 2 ml of wash buffer are added and then aspirated. This washing is important in obtaining consistant and accurate results.
  • 200 ul of OPD Buffer are added and incubated for 30 minutes. The incubation is then haulted by adding 1 ml IN H 2 SO 4 . The enyzme reading is then taken at 490 NM.
  • several calibrators with known differing concentrations of CK-MB are simultaneously tested such that a calibration curve against which the unknown sample test results can be compared is created.
  • Table I presents results utilizing the abovedescribed enzyme labeled test procedure for "Sample A” and 5 calibrators (A, B, C, D, E) and a control. The test was run twice and an average reading calculated. Figure 5 presents a calibration curve of the results. EXAMPLE VIII.

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Abstract

Une méthode d'essai immunométrique à sandwich, par réaction croisée à deux sites pour la détection et la mesure d'un analyte, comme la créatine phospho-kinase-MB, dans un sérum, comprenant la sélection de deux anticorps différents, chacun desquels est spécifique à un analyte différent mais produira une réaction croisée avec l'analyte concerné. L'on fait réagir le premier anticorps avec l'échantillon inconnu à l'aide d'une phase solide afin de lier le premier anticorps. Les parties solide et liquide de la première réaction sont séparées, et l'on fait réagir la partie solide avec le deuxième anticorps qui est marqué. Les parties solide et liquide de la deuxième réaction sont séparées et la partie solide est soumise à un essai pour permettre de déterminer le marquage, comme indication de la présence dudit analyte. Pour ce qui concerne en particulier les essais de détection de créatine phospho-kinase -MB dans le sérum humain, on utilise comme anticorps de réaction croisée un anticorps vis-à-vis de la créatine phospho-kinase-BB et de la créatine phospho-kinase-MM, ainsi qu'un marqueur d'enzyme.
EP19850903950 1984-07-30 1985-07-29 Methode d'essai immunometrique du type a sandwich, par reaction croisee a deux sites avec enzymes marquees Withdrawn EP0188608A1 (fr)

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Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5382515A (en) * 1987-07-21 1995-01-17 International Immunoassay Laboratories, Inc. Creative kinase-MB immunoassay for myocardial infarction and reagents
US4900662A (en) * 1987-07-21 1990-02-13 International Immunoassay Laboratories, Inc. CK-MM myocardial infarction immunoassay
US5382522A (en) * 1987-07-21 1995-01-17 International Immunoassay Laboratories, Inc. Immunoassay for creatine kinase-MB and creatine kinase-BB isoforms and reagents
DE3838900A1 (de) * 1988-11-17 1990-05-23 Scheefers Borchel Ursula Pyruvatkinase-isoenzym typ m2 (tumor m2-pk) spezifische antikoerper/verfahren zu deren herstellung und deren verwendung
US5972628A (en) * 1989-11-17 1999-10-26 Schebo Tech Medizinisch-Biologische Forschungsgesellschaft M.B.H. Pyruvatekinase-iosenzyme typ-M2 (Tumor-M2-PK)-specific antibody/process for the preparation and use thereof
CA2431201A1 (fr) * 2000-12-13 2002-08-01 Bio-Rad Laboratories, Inc. Standard diluent for multiplex assays
ES2708781T3 (es) * 2014-04-30 2019-04-11 Fujifilm Wako Pure Chemical Corp Método para ensayar la isozima MB de la creatina quinasa y kit para usarse en el mismo

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Publication number Priority date Publication date Assignee Title
US4237219A (en) * 1977-10-27 1980-12-02 Washington University Radioimmunoassay assay and reagents for MB isoenzyme of CK
DE2836046A1 (de) * 1978-08-17 1980-02-28 Behringwerke Ag Immunologisches bestimmungsverfahren
US4244940A (en) * 1978-09-05 1981-01-13 Bio-Rad Laboratories, Inc. Single-incubation two-site immunoassay
US4353982A (en) * 1980-04-10 1982-10-12 Hoffmann-La Roche Inc. Immunochemical assay for creatine kinase-MB isoenzyme

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Title
See references of WO8600992A1 *

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