CA1172958A - Two site cross-reaction immunometric sandwich assay method - Google Patents

Abstract

TWO SITE CROSS-REACTION IMMUNOMETRIC
SANDWICH ASSAY METHOD

ABSTRACT
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 serum. Conventional immunometric techniques depend upon the immunochemical reaction between a tagged antibody which is raised to specifically react with the particular analyte of interest. However, such antibodies may not be available, or if available, then unstable.
The disclosed two site-reaction immunometric sandwich assay method provides for 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 antibody is reacted with the unknown sample of utilizing a solid-phase to bind the first antibody. Separation of the solid and liquid por-tions of the first reaction is accomplished and the solid portion thereof is reacted with the second antibody which is tagged. The solid portion and liquid portion of the second reaction are separ-ated and the solid portion is tested for the tag as an indication of the presence of said analyte. With particular reference to test-ing for creatine phospho-kinase-MB In human serum, the cross-reacting antibodies utilized are antibody to creatine phospho-kinase-BB and creatine phospho-kinase-MM.

Description

117~5~
TWO SITE CROSS-REACTION IMMUNOMETRIC
SANDWICH ASSAY METHOD
___________________________________ TECHNICAL FtELD
The present Inventton relates generaliy to Immuno-loglcal assay technlques for determlnlng the presence of an analyte tn serum and more partlcularly to a two slte cross-reactton Immunoassay sandwlch testlng method whlch has partlcular appllcatlon In the qualltattve and quantltatlve determlnatlon of the level of the creatlne phospho-klnase MB
Isoenzyme (CK-M8) In human serum.
BACK~ROUND OF PRIOR ART
Conventlonal Immunometrlc technlques depend upon the Immunochemlcal reactton between a tagged antlbody and the analyte to be assayed. Such anttbodles are raised to spectflcally react wlth the partlcular analyte and may be tagged In a radloactlve, fluorescent, chemlumlnacent, enzymatlc or other manner. However, many antlbodles have cross-reactlons wlth materlals other than the speclflc analyte to be assayed whereupon the results of stmple anttbody-analyte tests become unrellable. To overcome thts problem, sandwlch type assay techntques hDve been developed whlch utlllze two antlbodles to sandwtch the analyte therebetween.
Conventtonal sandwich techntques uttltze three types of assays. In the flrst type, the undestred cross-reactton ts reduced based on the observatton that the undeslr-able cross-reactant would not react, at the same tlme, wlth antlbodles speclflcally ralsed In two dlfferent anlmal specles. 8Oth of the antlbodtes are therefore ratsed agalnst the speclflc analyte of Intersst, however, they dlffer In that they are ralsed tn dtfferent antmal specles, such as human belngs and gulnea plgs. Utlllzlng thls sandwlch technlque, the undeslrable effect of the unwanted cross-reacttons of the flrst antlbody Is reduced through the utll-tzatlon of the second antlbody, whereby an accurate test forthe analyte of Interest Is obtalned. This type of sandwlch assay technlque Is used In the detectlon of hepatltts.
The second type of sandwlch assay ts used when the serum contains, as metabollc by-products, fragments of the analyte to be measured. Two antlbodtes used for thls t,~ " 7 ~ ~ ~ S ~

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 utilizes antibodies that are specifically raised against the specific analyte to be measured. The unwanted cross-reactions 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 cross-sections from a fragment of the analyte.
The third sandwich technique uses the same specific antibody twice to detect the analyte of interest. In this tech-nique, an antibody which is specific 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 with the same antibody excent that the anti-body is now tagged for assay purposes.
The present invention provides for an improvement in a two site immunometric sandwich immunoassay method for the detection and measurement of an analyte of interest in serum where the analyte of interest has at least two immunochemically differinq binding sites, and is present along with other molecules which have one immunochemically identical binding site to the analyte of interest, the improvement in combination therewith comprising the utilization in said immunoassay method of two antibodies, neither of which antibodies is specifically produced against said analyte of interest and each of which said antibodies will cross-react with only one of said differing sites of said analyte of interest, wherein said analyte of interest becomes immuno-chemically sandwiched between said two antibodies, and wherein one of said two antibodies is tagged for detection as an indication of the presence of said analyte of interest.
As will be seen from the description of the preferred embodiment hereinbelow, the present invention differs from the prior art in that it specifically utilizes the previously avoided cross-reactive capabilities of two different antibodies to create a spec-ific sandwich assay technique.

:~7~58 - 2a -B~IEF SUMMARY OF INVENTION
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 solid-phase 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 quan-titative determination of the presence of the analyte of interest.
With particular regard to the detection of CK-MB the immuno-assay technique of the present invention includes reacting the unknown humar; serum with CK-BB antibody which is affixed to a solid-phase.

., .

After the reactlon ts complete, the reactants are washed, centrl-fuged and the llquid portlon thereof Is asplrated. The soJid-phase portlon, havlng been retalned, Is then reconstltuted In solutlon contalnlng tagged CK-MM antlbody and reacted there-wlth. Upon comptetlon of the reactlon, the reactants arewashed and centrlfuged and the llquid portlon Is asplrated.
The retalned solld-phase portlon Is now tested for the presence of the tag. Callbratlon tests are also conducted uttllzlng serum havlng known amounts of CK-MB to provlde a background wlth whlch to determlne the quantlty of CK-MB In the unknown sample.
A prlmary advantzge of the present Inventlon Is that an antlbody that Is speclflc to the analyte of Interest need not be created In order to provlde an l~munologlcal assay test for the analyte. All that Is requlred Is that there be found two dlfferent antlbodles whlch mutually cross-react only wlth the analyte. Thls technlque Is very useful for those analytes whlch are unstable and hence cannot be practlcally used to make antlbodles. It Is also very useful when It Is extremely dlfflcult to purlfy the analyte, as It Is the case wlth cancer antlgens.
It Is another advantage of the present Inventlon, as applted to the testlng for CK-MB, that It utlllzes the antlbodles to CK-MM and CK-B8 whlch are commerclally avallable and lnexpenslve.
It Is a further advantage of the present Inventlon, as applled to the testlng for CK-MB, that the test may be performed rapldly and wlth greater accuracy than test procedures presently avallable.
These and other obJects and advantages of the present Inventlon wlll no doubt become apparent to those of ordlnary sklll In the art after havlng read the followlng detalled descrlptlon of the preferred embodlments whlch are Illustrated In the several flgures of the drawlng.
BRIEF DESCRIPTION OF DRAWINGS
FIG. I Is a flow chart deplctlng the two slte cross-reactlon Immunometrlc sandwlch assay method of the present Inven~tton;
FIG. 2 Is a schematlc dlagram of the assay method of the present Inventlon;
FIG. 3 Is a flow chart deplctlng the assay method of the '7;~5~

present Inventlon as applled to the detectlon of CK-MB;
FIG. 4 Is a flow chart deplctTng an alternatlve embodl-ment of the assay method of the present Inventlon.
DETAILE~ DESCRIPTION OF THE INVENT_ON
In standard sandwlch assay technlques, two antlbodles are utlllzed, each of whlch Is ralsed agalnst the speclflc analyte of Interest. The utlllzatlon of the two speclflc antlbodles glves a very hlgh probablllty of detectlon of the ana;yte and a very low probablllty of error due to unwanted cross-reactants. However, such sandwlch techn7ques cannot be utlllzed where an antlbody that Is speclflc to the analyte of Interest has not been created or Isolated. The present lnventlon has applIcatlon In Just such a clrc~mstance.
Where It has proven dlfflcult to create and/or Isolate a speclflc antlbody for the detectlon of an analyte of Interest, It may well be the case that known antlbodles exlst whlch, whlle speclflc to other analytes, wlll mutually cross-react only wtth the analyte of lnterest. Where two such antlbodles exlst, each belng speclflc to a dlfferent analyte but each belng capable of cross-reactlng wlth the analyte of Interest and there belng no other analyte to whlch they both wlll cross-react, the test procedure of the present Inventlon wlll permlt detectlon and assay of the mutually cross-reactlng analyte of Interest. It wlll be seen that Just a sltuatlon exlsts wlth regard to the Immuno-loglcal assay for creatlne phospho-klnase-M~ (CK-MB).
As deplcted In FIGS. I and 2, the general test procedure of the present Inventlon Is performed on a serum sample 12 con-talning three related analytes whlch are schematlcally deplcted as a dlamond shaped analyte 14, heretnafter referred to as AA, a clrcular shaped analyte 16, herelnafter referred to as CC, and a hybrtd analyte 18 of the two other analytes, shown as havlng both a partlal dlamond shape and a partlal clrcular shape, here-Inafter referred to as AC. It wtll be assumed that antlbodles exlst whlch are speclflc to M , herelnafter referred to as anti-AA, and to CC, herelnafter referred to as antl-CC, but that the ant1-body to AC Is unstable and therefore non-exlstant for testlng pur-poses. However, both antt- M and antl-CC wlll cross-react wlth AC
due to the slmllarlty of molecular structure of the A and C sltes of the AC molecule to the AA and CC molecules.
y ;1~7;~ 8 In an inttlal procedure, whlch Ts performed prlor to or concurrent wlth the flrst reactlon step descrtbed hereln-below, antl- M 19 Is affIxed to a solld-phase 20 uslng a stan-dard Immunologtcal procedure therefor. Also, In a separate Immunologlcal proc0dure performed prl~r to the Instant test, antl-CC Is tagged for later detectlon. Procedures for taggtng antlbodles are well known and radloactlve, fluorescent, chem-lumlnescent, enzymatlc or other types of tags are approprlate for thls test procedure.
In the flrst reactlon step 22, the serum 12 Is combtned wlth the solld-phase antl-AA 20 and reacted 22 therewtth for an approprlate tlme and temperature. The tlme and temperature wlll vary wlth the partlcular antlbodles and analytes Involved In the test procedure, as weli as the concentratlons thereof. In the reactlon the solld phase antl- M 20 Immunochemlcally blnds 23 wlth the M 14 and removes all of the AA from the serum as It Is speclflc to that analyte. Addltlonally, because the antl-AA
19 wlll cross-react wlth the AC 18, all of the AC Is also removed from the serum.
In the next step of the test procedure, the reactants are separated 24, the solld-phase products 26 belng retalned and the llquld 28 belng dlscarded. Standard separatlon procedures, such as centrlfugatlon and asplratlon, are utlllzed. It Is to be noted that the dlscarded liquld 28 contalns all of the CC
analyte.
The retalned solld-phase 26 Is now reacted 32 wlth the tagged antl-CC 30. The reactlon 32 Is allowed to contlnue for an ~pproprlate tlme at an approprlate temperature. Agaln, the tlme and temperature wlll vary wlth the partlcular antlbodles and analytes Involved In the test procedure, as well as the con-centratlons thereof.
Thereafter, a standard separatlon step 34, Is per-formed whereln the solld-phase 36 Is retalned and the llquld 38 Is dlscarded. As Is schemattcally shown In FIG. 2, the tagged ~i antl-CC can only cross-react 39 wlth the AC whlch prevlously cross-reacted wlth the solld phase anti-AA 26; all of the CC havlng been dlscarded In the llquld portlon 28 of the flrst separatlon step ; 24.
It Is now seen that the remalnlng solId-phase 36 contalns 117'~35~3 a tag~ed sandwTch of molecules 40 In whlch the AC (the analyte of Interest) is sandwiched between two antibodles, nelther of whlch Is specific to that analyte but each of whlch wlll cross-react wlth It.
The solld-phase 36 may now be tested 42 for the tag, whlch test wlll glve an Indlcatlon of the presence of the analyts of Interest.
Callbratlon tests utlllzing known amounts of the an-alyte of Interest and the above-descrlbed test procedure are conducted stmultane~usly to create a calIbratton curve agalnst whlch to gauge the results of the test for the unknown sample.
Further tests utlllzlng known dlluttons of the unknown sample and the above-descrlbed test procedure may also be performed to obtaln test results whlch fall on sensltlve portlons of the call~ratlon curve.
A speclflc appllcatlon of the above-descrlbed test procedure for use In the detectlon of CK-MB antlgen In human serum can now be descrlbed.
As Is well known, human serum contalns three creatlne phospho-klnase Isoenzymes; creatlne phospho-klnase-MM (a skeletal tlssue extract, herelnafter referred to as CK-MM), creatlne phospho-klnase-BB (a braln tlssue extract, herelnafter referred to as CK-BB) and creatlne phospho-klnase-MB (a heart tlssue ex-tract, herelnafter referred to as CK-M8) whlch Is a hybrld form of CK-MM and CK-BB. A great deal of research has béen conducted to develop a test for CK-MB In that Its presence Is a speclflc Indlcatlon of myocardlal Infarctlon or slmllar heart dlsturbances.
Howaver, testlng for CK-MB by varlous methods has proved to be qulte dlfflcult as Its propsrtles are qulte slmllar to CK-MM and CK-BB, the presence of whlch can slgnlflcantly mask small amounts of CK-MB. Wlth partlcular regard to Immunometrlc assay tech-nlques for the detectlon of CK-MB, these have been made dlfflcult by the present Inablllty of researchers to prepare and develop a stable antlbody which Is speclflc to the CK-MB (herelnafter referred to as anti-CK-MB), and no such antl-CK-MB Ts commGrclally avallable at thls tlme. However, It is known that the antlbodles to both CK-MM and CK-BB therelnafter referred to as antl-CK-MM
and antl-CK-BB respectlvely) wtll cross-react wlth CK-MB, In addltton to thelr havlng speclflc reactlons wlth CK-MM and CK-BB

~17;~58 respectlvely. The test procedure described hereinabove Is there-fore applicabte to the detectlon of CK-MB in accordance wlth the followlng descrlptlon.
Prtor to the actual assay of serum, antl-CK-BB Is afflxed to a solld phase as Is descrlbed hereinbelow. However, as Is also descrlbed herelnbelow an alternatlve embodlment of the above-descrlbed test procedure may be performed In whlch thls step ts not performed. Addltlonally, prlor to assay the antT-CK-MM
must be tagged, such as is described herelnbelow by the utlllza-tlon of radlo-actlve lodlne t 1).
Antl-CK-BB trabblt) for afflxatlon to a solld-phase Is commerlcally avallable from many sources. The solld-phase selected for llnkage wlth the antl-CK-B8 was Blo-Rad Immuno-Beads (Catalog No. 170-5602) although other solld phases could be util-Ized such as latex beads or cellulose beads or a membrane strlp.
Blo-Rad Immuno-Beads are polyacrylamlde beads havlng a dlameter of approxlmately 10 ~ tmlcrons) and whlch are deslgned for use as a solId phase In Immunoassay testlng. Followlng the procedure recommended by the manufacturer, the beads were flrst covalently llnked to goat antl-rabblt 199. 50mg of beads were then suspended In 25 to 50ml of PBS contalnlng 10~ calf serum, although other protelns could be used to mlnlmlze non-speclflc ~Indlng. There-after, the beads were washed wlth PBS buffer tpH approxlmately 7.4, 0.02M PO , 0.15M NaCI). The beads were then slurrled In 25 ml PBS contalnlng 1% BSA. Approxlmately 100 ~1 of antl-CK-BB was then reacted wlth the beads to llnk the antl-CK-BB to the beads.
The antlbody coated beads were then washed and separated from un-reacted antl-CK-BB wlth PBS buffer. The antl-CK-BB coated beads were then ready for utlllzatlon In the test procedure.
Prlor to testlng, the antl-CK-MM Is lodlnated wlth radlo-actlve lodlne tl251) to serve as a tag for the detectlon of CK-MB.
Such taggtng procedures are well known, see FRAKER, T. and SPECK, ~., BTochemlcal and Blo-physlcal Research Commlsslon, Volume BO, No. 4, Febr~ary, 1978. In the procedure, 100~9 of antl-CK-MM tgoat) was dlssolved In 100 ~1 of water. Stxteen ~1 of thls solutlon, to be used for tagging, was comblned wlth ZO ~ I tO.25M F~4) F'BS buffer tpH, 7.5) In a reactlon vlal. 4BO mlcrocurle of Nal was added to thls buffer. Thls was followed by iO~I t3.5mg/ml tn 0.05 phosphate buffer) of chloramJne-T solutton. Thls was mlxed for 60 seconds followed by lC~Il of sodium metabsisulfite solution (3.5mg/ml 0.05 phosphate buffer). The reaction mixture was fractionated over a Sephadex* G-50 column (washed and treated with goat serum), fractions numbered 18-23 were collected and pooled. The frac-tion pool was purified over a Dowex* lx8 column and collected.
This material was diluted to achieve an appropriate activity 1 evel .
Utilizing the previously prepared solid-phase anti-CK-BB and radiodinated anti-CK-MM, the test procedure for CK-MB is performed as depicted in FIG. 3.
Human serum 62 containing the isoenz~mes CK-MM64, CK- MB 66 and CK-BB is reacted 72 with the anti-CK-BB coated Immuno Beads 70. This reaction time and reaction temperature being parameters which may be varied depending upon the con-centration of the serum and the strength of the anti-CK-BB
on the Immuno-Beads. As is demonstrated in Exampl e 11 herein-below, these variables may be adjusted such that good test results are obtainable utilizing room temperature and reaction time of one hour.
Following the reaction phase 72, it is necessary to separate the liquid from the beads. To accomplish this, approx-imately 2ml of additional PBS buffer is added to the reactants as a wash 74. The reactants are then centrifuged 76 to achieve a button-like precipitate 78 . Centrifuging at room temperature for approximately ten minutes at a speed of approximately 2000 rpm has been found to yield good results.
Having achieved a button-like precipitate 7B at the bottom of the test vial the liquid 80 therein is aspirated and discarded, and the precipitate 78 is retained for the second v 30 phase of the test procedure.
It is noted that the anti-CK-BB coated beads will have reacted with, and therefore removed from solution, all of the CK-BB and CK-MB antigens. As the CK-MM antigen does not react wi th the anti-CK-BB, the CK-MM remains in solution and 35 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 radioiodinated anti-CK-MM 88 to the precipitate. The reactants are vibrated or vortexed to reconstitute 84 the precipitate into the reaction.
* trademark 1~7;~58 _9_ The reactlon tlme and reaction temperature are parameters whtch may be varied In accordance wlth the concentrations of the reactants. As Is demonstrated in Example 11 good test results may be obtained wtth the uttllzatlon of room temperature and a reaction ttme of approxlmately I hour.
After reactlon 86, a second separatTon Is per-formed In the same manner as the prlor separatlon. That ts approximately 2ml of PBS wash buffer 90 contalning 0.1~ Tween 20 Is added to the reactants and the reactants are centrtfuged 0 92 at room temperature for an approxlmate tlme of ten minutes at an approxlmate speed of 2000 rpm to yield a button~ e preclpltate 94 at the bottom of the test vlalA The llquld 96 - In the test vlal Is then aspirated 98 and dlscarded In an appro-prlate manner, in that It contalns excess radioactlve antl-CK-MiM. The button-llke precipltate 94 ls then tested 100 wlth a sclntlllation detector to lndtcate the presence of radlatlon as a posslble Indlcatlon In the test serum.
It Is Important to note that as all of the CK-MM was asplrated 82 and dlscarded In the llquld 80, there was no CK-MM
present In the second reactlon 86 wlth whlch the antl-CK-MM
could speclflcally react. The only reactlon possible for the antl-CK-MM was then to cross-react wlth the CK-MB that was bound to the beads by vlrtue of Its cross-reactlon wlth the antl-CK-BB.
As wlth all radlolmmunoassay technlques, It ts necessary to perform concurrent calIbratlon tests wlth known amounts of CK-M8 to form a callbratlon curve agalnst whlch to compare the results of the unknown test. It Is noted that there wlll always be some radlatlon from a zero level CK-MB
sample due to the attachment of the radlolodlnated antl-CK-M~i to the walls of the test vlal and remainlng in any molsture wlthln the preclpltate 94 after centrlfuglng 92. Thus, there wlll be radlatlon from a zero level CK-MB sample and It Is the increase In radlatlon over the zero level of radlatlon that provldes an Indlcatlon of the presence of CK-MB in the test sample.
Furthermore, lt Is well known in radlolmmunoassay testlng that the Increase In radloactlvlty does not vary llnearly with Increaslng amounts of analyte in the test serum. The 11 7;~35~

varlatlon of radlatlon with concentratlon of analyte depends upon many varlables, such as quanttty of antlbody, dilutTon of antlbody, anttbody-analyte ratto and others. The stan-dard test procedure whlch Ts utillzed to overcome this test-Ing Impsdlment Is to perform a series of tests wlth varylng known dilutlons of the unknown test serum. A serles of results are then obtalned, one or more of whlch wlll lle on a senstttve portton of the callbratlon curve to glve an accurate Indlcatlon of the level of analyte In the sample. The Examples 1, 11 and 10 IV glven herelnbelow demonstrate the utlllzatlon of thls pro-cedure.
As would be obvlous to those skllled in the art, alternatlve solld-phase mater7als such as a membrane filter could be utlllzed Instead of the Immuno-Beads. In explorlng 5 thls alternatlve, a membrane 120 was created utlllzlng Whatman No. I fllter paper whlch was oxldlzed uslng the perlodate tech-nlque descrlbed by Ferrung, B., Maiollnl, R., and Masseyeff, R., Journal of Immunolo~lcal methods, Yolume 25, Page 49, 1979.
Strlps of the membrane were soaked In goat antl-rabblt I G so lutlon and utlllzed In much the same manner as the Immuno-beads are used In the test. The use of membrane as the solld-phase ellmlnates the need to centrlfuge, and the separatlon steps become the slmple washlng of the membrane to remove excess Im Y munoreagents. Results ustng a membrane solld-phase are presented 25 In Example Vl herelnbelow.
A varlatlon on the above descrlbed test procedure, see FIG. 4, may be conducted In the followlng manner. Rather than afflxlng the flrst antlbody 130 to a solld-phase 132, the flrst antlbody 130 Is added 136 dlrectly to the unknown serum 134.
Thereafter, the solId-phase 132, whlch has not been coated wlth the flrst antlbody 130, but whlch Is prepared for coatlng as descrlbed herelnabove, 15 added 138 to the reactants. The reactlon 138 Is then permltted to proceed In the manner des-crlbed herelnabove and the remalnlng steps of the test procedure are conducted as prevlously descrlbed followlng reactlon 72 of FIG. 3. It Is found that sufflclent amounts of the solld-phase antlbody-antlgen complex attach to the beads 132 to glve ad-equate test results. Examples IV and V presented herelnbelow are speclfic examples of the results achleved utlllzlng thls ~ 17;~58 "
variatlon of the test procedure.
The followlng examples demonstrate the tnstant test-tng method uslng several combTnatlons of test condttions.
EXAMPLE I
Thls experiment was carried out wlth antl-CK-BB coated immuno-beads. 10Otll of sample was mlxed wlth 200~1 of beads.
After overnlght Incubatlon at room temperature, 0.5 ml PBS
buffer wlth 10% calf serum was added to the test tube. The beads were separated by centrtfugatlon. They were then reacted 1~ for 5 hours at room temperature wlth radlolodlnated antt-CK-MM.
After Incubatlon, beads were washed wlth I ml PBS buffer wlth 10% calf serum. The beads were then centrlfuged and counted.
Results Normal human serum count (NHS) -- 1090 counts per mln.
5 Callbrators tlOO ~I per test) Ratlo Sample Counts NHS COUNTS
E. I ~g/ml of CK-MB powder In serum1.17 D. 10 ~g/ml of CK-MB powder In serum 3.20 C. 50 ~g/ml of CK-MB powder In serum 5.71 B. 100 yg~ml of ~K-M3 p wd~ In s~rum 6.65 The callbrator samples were made by dlssolvlng a powder contatnlng 6.8% CK-MB In normal human serum at varlous dllutlons.
Patlent Samples (known to have hlgh level of CK-MB) Hlgh level 10 ~1 3.66 ~1 7.44 ~1 1~.1 100 ~1 14~9 As Is seen, a good response curve was obtalned wlth CK-MB
calIbrators, and patlent sample, whlch was identlfled as havtng hlgh CK-MB content by other technlques, could be measured utll-lzlng four dllutlons to assure a readlng level that was correl-atable on the callbratlon curve.

Thls example shows effect of reduclng Incubatton tlme and varylny the amount of beads to 200~1 and 500 ~1. The wash buffer used was 2 ml wlth 5g calf serum and was added before each centrtfugatlon. The Incubatlon tlme was one hour for each of the reactlons at room temperature. Other test condltlons were Iden-13 7~58 -~2-tical to those of Example 1.
Results 200 ~I Beads 500 ~i Beads Counts (cpm) Ratio Sample Counts NHS COUNTS
Hlgh 50 ~1 2.35 2.85 100 ~1 3.31 3.77 Callbrater 50 ~g/ml 2.24 3.25 100 ~g/ml 2.8 4.50 The rattos obtalned, although slgnlfIcantly lower than the overnlte Incubatlon, were stlll qulte acceptable. The effect of reductlon in Incubatton tlme thus can be compensated by In-creasing the amount of beads.

The method descrlbed In Example 11 was used wTth 200 ~1 of beads and an Incubatlon tlme of 1.5 hours for each reactlon.
The tracer used In thls experlmen+ had hlgher non-speclfic blndlng.
Several serlal samples of a pattent just admltted to a hospltal for a myocardial 7nfarct were tested.
Sample Count NHS 3835 counts per mlnute Ratlo Sample Counts NHS COUNTS
Callbrators 50 ~g/ml 2.2 100 ~g/ml 2.7 PATIENT A:
11 a.m. 2.24 12 noon 2.75 I p.m. 3.00 4 p.m. 3.15 8 p.m. 3.54 NORMAL PATIENTS:
B .94 C 1.06 D .99 The rlsing leveis of CK-MB shown in thls Example demonstrate the well known increase In CK-M8 levels In the patlent followtng a x~

myocardial infarct or similar incident.
EXAMPLE IV
- In this example, polyacrylamide beads were used as a precipitating antibody. In the first step, 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 ~ 1 of these beads being comparable in a number of beads to 250~1 of beads used in previous examples. These beads were not precoated with anti-CK-BB. After a one hour incubation period, the 0 reactants were centrifuged and aspirated. A second incubation of one hour was carried out in the normal manner. Thereafter

2 ml of PBS buffer with 1% BSA and 1% Tween* was added and react-ants were centrifuged, aspirated and the solid-phase counted.
The results were as follows:
Sample Count RatioSample Counts NHS COUNTS
Calibraters 50 ~g/ml 3.1 100 ~g/ml 4.2 The above sample contained roughly 5 mlU/ml and 10 mlU/ml CK-MB which is roughly the limit of normal range.
EXAMPLE V
Negative samples were run using the method discussed in Example IV.
RESULTS
SAMPLE COUNTSStandard Deviation 13 negative samples 2260 305 NHS, 16 times 2017 199 12 negative samples 2172 300 with high total CK
Calibrater 50 ~g/ml 4281 100 ~g/ml 5936 Thus negative samples, even those with high total CK
levels, would all be well under the 50 ~g/ml calibrator sample and are seen to be quote close to the NHS samples.
* trademark 117~58 EXAMPLE VI
The use of a membrane as a soild-phase is demonstrated by using antl-CK-~a ;mmobiltzed on Whatman No. I filter paper as described hereinabove.
The antibody coated membrane (3/8~ x 1 ) was placed In a mixture of sample and 2ml PES 5% BSA bufferO After a flrst incubatton of I hour, the llquids was asplrated and the membrane was thoroughly washed. Radlolodlnated antl-CK-MM was added. After a second lncubatlon of one hour the llquld was asplrated. The membrane was washed thoroughly and transferred to another tube.
The results were as follows:
RESULTS
Sample NHS 967 counts per mlnute Ratlo Sample Counts NHS COUNTS
Callbrater 400 lJg/ml 3.3 100 ~g/ml 1.9 Thus, a membrane solid-phase Is seen to produce oognlzable results although they are not as pronounced as were the results utllizing the beads as tie solld-phase.
It can therefore be seen that the present Inventlon darives Its unlqueness from the utlllzatlon of the cross-reactlve capabllltles of the antlbodles that are selected for use. Thls is contradlstlnctlon to standard Immunologlcal assay technlques In whlch the cross-reactlve capabllltles of the antlbodles are mlnlmlzed or avolded through the use of multiple antlbodles whlch are each speclflc to the analyte of Interest. Utlllzatlon of the Instant test technlque for the detectlon of CK-MB In human serum provldes a rapld and effectlve means for the detection and Iden-tlflcatlon of myocardlal Infarcts and slmilar heart dlsturbances In patlents Immedlately followlng thelr occurrence.
Whereas the preferred embodlment of the present Inven-tlon has been described above, It Is contemplated that other alter-atlons and modlficatlons may become apparent to those skllled tnthe art after havlng read the above dtsclosure. It Is therefore Intended that the appended clalms be interpreted as coverlng all such alteratlons and modiflcatlons as fall withln the true splrlt and scope of the inventlon.

Claims (10)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. In a two site immunometric sandwich immuno-assay method for the detection and measurement of an analyte of interest in serum where the analyte of interest has at least two immunochemically differing binding sites, and is present along with other molecules which have one immunochemically identical binding site to the analyte of interest, the improvement in combination therewith com-prising the utilization in said immunoassay method of two antibodies, neither of which antibodies is specifically produced against said analyte of interest and each of which said antibodies will cross-react with only one of said differing sites of said analyte of interest, wherein said analyte of interest becomes immunochemically sandwiched between said two antibodies, and wherein one of said two antibodies is tagged for detection as an indication of the presence of said analyte of interest.

2. In a two site immunometric sandwich immuno-assay method for the detection and measurement of creative phospho-kinase-MB (CK-MB) in human serum, the improvement in combination therewith comprising the utilization of antibody to CK-BB as a first antibody and antibody to CK-MM
as a second antibody to measure CK-MB specifically when present in serum along with other CK isoenzymes including but not restricted to CK-MM, CK-BB, CK-BB.lgG complex, wherein said antibody to CK-MM is tagged for detection and measurement of the presence of CK-MB.

3. The two site immunometric sandwich assay method as recited in claim 2 wherein said tag is radio-active iodine 125I.

4. A two site immunometric sandwich assay method for the detection and measurement of an analyte in serum, said analyte having at least two chemically different binding sites, said method comprising:

(a) selecting a first antibody not specific to the analyte of interest but which will cross-react with only one of the differing binding sites of the analyte of interest, and affixing said first antibody to a solid-phase to form a bound antibody;
(b) selecting a second antibody not specific to the analyte of interest which differs from said first antibody and which also cross-reacts with only one of the differing binding sites of said analyte, and tagging said second antibody to form a tagged antibody, said first and second antibodies being further selected such that only the analyte of interest will cross-react with both of them;
(c) reacting said bound antibody with a serum suspected of containing said analyte to form a first reaction solution;
(d) separating the solid-phase portion of said first reaction solution from the liquid portion thereof;
(e) reacting said solid-phase portion of said first reaction solution with said tagged antibody to form a second reaction solution;
(f) separating the solid-phase portion of said second reaction solution from the liquid portion thereof;
and (g) testing said solid-phase portion of said second reaction solution to detect the gag of said second antibody as an indication of the presence of said analyte of interest.

5. A two site immunometric sandwich assay method as recited in Claim 4 wherein said solid-phase is a mate-fiat selected from the group consisting of polyacrylamide beads, latex beads, cellulose beads and a membrane strip.

6. A two site immunometric sandwhich assay method as recited in Claim 4, wherein said tag is radioactive iodine 125I.

7. A two site immunometric sandwich assay method as recited in Claim 4, wherein said separating steps are accomplished by centrifuging the solutions to precipitate said solid-phase and by aspirating to remove said liquid portion therefrom.

8. A two site immunometric sandwich assay method as recited in Claim 4 and further comprising repeating steps (a) - (g) several times substituting for said serum in each repetition of steps (a) - (g) a sample containing a different known amount of said analyte; creating a calibra-tion curve from the test results of the repetitions;
and comparing the test results for said serum to said calibration curve.

9. A two site immunometric sandwich assay method as recited in Claim 8 wherein steps (a) - (g) are repeated with several known dilutions of said serum; and comparing the results of these tests with with said calibration curve to obtain a quantitative determination of the amounts of said analyte in said serum.

10. The two site immunometric sandwich assay of Claim 4, wherein said analyte is creatine phospho-kinase MB (CK-MB) in human serum which also contains creatine phospho-kinase -MM (CK-MM) and creative phospho-kinase-BB
(CK-BB), said first antibody is specific to CK-BB and said second antibody is specific to CK-MM.