GB2078953A - Two site cross-reaction immunometric sandwich assay method - Google Patents

Abstract

In a two site cross-reaction immunoassay sandwich testing method which has a particular application in the qualitative and quantitative determination of the level of the creatine phospho- kinase MB Isoenzyme (CK-MB) in serum, there are used 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 which is preferably bound to a solid phase is reacted with the unknown sample. Separation of the solid and liquid portions of the first reaction mixture is accomplished and the solid portion thereof is reacted with the second antibody which is tagged. The solid and liquid portions of the second reaction mixture are separated and the solid portion is tested for the tag as an indication of the presence of said analyte. In testing for creatine phospho-kinase-MB in human serum, there are used the cross-reacting antibodies to creatine phospho-kinase-BB and to creatine phospho-kinase-MM.

Description

SPECIFICATION Two site cross-reaction immunometric sandwich assay method Technical field 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.

Background ofprior art Conventional immunometric techniques depend upon the immunochemical reaction between a tagged antibody and the analyte to be assayed. Such antibodies are raised to specifically react with the particular analyte and may be tagged in a radioactive, fluorescent, chemluminacent, enzymatic or other manner.

However, many antibodies have cross-reactions with materials other than the specific analyte to be assayed whereupon the results of simple antibody-analyte tests become unreliable. To overcome this problem, sandwich type assay techniques have been developed which utilize two antibodies to sandwich the analyte therebetween.

Conventional sandwich techniques utilize three types of assays. In the first type, 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. Utilizing this sandwich technique, 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 utilizes antibodies that are specifically raised against the specific analyte to be measured. The unwanted cross-reactions of the specific anitbody 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-reactions from a fragment of the analyte.

The third sandwich technique uses the same specific antibody twice to detect the analyte of interest. In this technique, 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 except that the antibody is now tagged for assay purposes.

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 specific sandwich assay technique.

Breffsummarv 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 quantitative determination of the presence of the analyte of interest.

With particular regard to the detection of CK-MB 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 backround 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 immunological 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. This 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.

It is a further advantage of the present invention, as applied to the testing for CK-MB, that the test may be performed rapidly and with greater accuracy than test procedures presently available.

These and other objects and advantages of the present invention will no doubt become apparent to those of ordinary skill in the art after having read the following detailed description of the preferred embodiments which are illustrated in the several figures of the drawing.

Brief description of drawings Figure 1 is a flow chart depicting the two site cross-reaction immunometric sandwich assay method of the present invention; Figure2 is a schematic diagram of the assay method of the present invention; Figure 3 is a flow chart depicting the assay method of the present invention as applied to the detection of CK-MB; Figure 4 is a flow chart depicting an alternative embodiment of the assay method of the present invention.

Detailed description of the invention In standard sandwich assay techniques, two antibodies are utilized, each of which is raised against the specific analyte of interest. The utilization of the two specific antibodies gives a very high probability of detection of the analyte and a very low probability of error due to unwanted cross-reactants. However, such sandwich techniques cannot be utilized where an antibody that is specific to the analyte of interest has not been created or isolated. The present invention has application in just such a circumstance.

Where it has proven difficult to create and/or isolate a specific antibody for the detection of an analyte of interest, it may well be the case that known antibodies exist which, while specific to other analytes, will mutually cross-react only with the analyte of interest. Where two such antibodies exist, each being specific to a different analyte but each being capable of cross-reacting with the analyte of interest and there being no other analyte to which they both will cross-react, the 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).

As depicted in Figures 1 and 2, the general test procedure of the present invention is performed on a serum sample 12 containing three related analytes which are schematially depicted as a diamond shaped analyte 14, hereinafter referred to as AA, a circular shaped analyte 16, hereinadter 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. It will be assumed that antibodies exist which are specific to AA, hereinafter referred to as anti-AA, and to CC, hereinafter referred to as anti-CC, but that the antibody to AC is unstable and therefore non-existant for testing purposes.However, both anti-AA and anti-CC will cross-react with AC due to the similarity of molecular structure of the A and C sites of the AC molecule to the AA and CC molecules.

In an initial procedure, which is performed prior to or concurrent with the first reaction step described hereinbelow, anti-AA 19 is affixed to a solid-phase 20 using a standard immunological procedure therefor.

Also, in a separate immunological procedure performed prior to the intant test, anti-CC 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.

In the first reaction step 22, 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. In the reaction 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.

In the next step of the test procedure, the reactants are separated 24, the solid-phase products 26 being retained and the liquid 28 being discarded. Standard separation procedures, such as cenrifugation 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.

Thereafter, a standard separation step 34, is performed wherein the solid-phase 36 is retained and the liquid 38 is discarded. As is schematially shown in Figure 2, the tagged anti-CC can only cross-react 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.

It is now seen that 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 gauge the results of the test for the unknown sample. Further tests utilizing known dilutions of the unknown sample and the abovedescribed test procedure may also be performed to obtain test results which fall on sensitive portions of the calibration curve.

A specific application of the above-described test procedure for use in the detection of CK-MB antigen in human serum can now be described.

As is well known, human serum contains three creatine phospho-kinase isoenzymes. creatine phosphokinase-MM (a skeletal tissue extract, herreinafter referred to as CK-MM), creatine phospho-kinase-BB (a brain tissue extract, hereinafter referred to as CK-BB) and creatine phospho-kinase-MB ( heart tissue extract, hereinafter referred to as CK-MB) which is a hybrid form of CK-MM and CK-BB. A great deal of research has been conducted to develop a test for CK-MB in that its presence is a specific indication of myocardial infarction or similar heart disturbances. However, testing for CK-MB by various methods has proved to be quite dificult as its properties are quite similsarto CK-MM and CK-BB, the presence of which can significantly mask small amounts of CK-MB.With particular regard to immunometric assay techniques for the detection of CK-MB, these have been made difficult by the present inability of 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. However, it is known that the antibodies to both CK-MM and CK-BB (hereinafter rederred to as anti-CK-MM and anti-CK-BB respectively) will cross-react with CK-MB, in addition to their having specific reactions with CK-MM and CK-BB respectively. The test procedure described hereinabove is therefore applicable to the detection of CK-MB in accordance with the following description.

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 anti-CK-MM must be tagged, such as is described hereinbelow by the utilization of radio-active iodine (1251).

Anti-CK-BB (rabbit) for affixation to a solid-phase is commerically available from many sources. The solid-phase selected for linkage with the anti-CK-BB 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 u (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. 50mg of 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 P04, 0.15M NaCI). The beads were then slurried in 25 ml PBS containing 1% BSA. Approximately 100 Fl 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.The anti-CK-BB coated beads were then ready for utilization in the test procedure.

Prior to testing, the anti-CK-MM is iodinated with radioactive iodine (1251) to serve as a tag for the detection of CK-MB. Such tagging procedures are well known, see FRAKER, T. and SPECK, J., Biochemical and Bio-physical Research Commission, Volume 80, No. 4, February, 1978. In the procedure, 100ug of anti-CK-MM (goat) was dissolved in 100 coil of water. Sixteen yl of this solution, to be used for tagging, was combined with 20 F I (0.25M P04) PBS buffer (pH, 7.5) in a reaction vial. 480 microcurie of Nai 125 was added to this buffer. This was followed by 10,us (3.5mg/ml in 0.05 phosphate buffer) of chloramine-T solution.This was mixed for 60 seconds followed by 1 0u1 of sodium metabisulfite 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 fraction pool was purified over a Dowex 1 x8 column and collected. This material was diluted to achieve an appropriate activity level.

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 Figure 3.

Human serum 62 containing the isoenzymes CK-MM 64, 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 concentration of the serum and the strength of the anti-CK-BB on the Immuno-Beads. As is demonstrated in Example 11 hereinbelow, these variabies 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, approximately 2 ml 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 78 at the bottom of the test vial the liquid 80 therein is aspirated and discarded, and the precipitate 78 is retained for the second 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 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 radioiodinated 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 11 good test results may be obtained with the utilization of room temperature and a reaction time of approximately 1 hour.

After reaction 86, 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 often minutes at an approximate speed of 2000 rpm to yield a button-like 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 button-like precipitate 94 is then tested 100 with a scintillation detector to indicate the presence of radiation as a possible indication in the test serum.

It is important to note that as all of the CK-MM was aspirated 82 and discarded in the liquid 80, there was no CK-MM present in the second reaction 86 with which the anti-CK-MM could specifically react. The only reaction possible for the anti-CK-MM was then to cross-react with the CK-MB that was bound to the beads by virtue of its cross-reaction with the anti-CK-BB.

As with all radioimmunoassay techniques, it is necessary to perform concurrent calibration tests with known amounts of CK-MB to form a calibration curve against which to compare the results of the unknown test. It is noted that there will always be some radiation from a zero level CK-MB sample due to the attachment of the radioiodinated anti-CK-MM to the walls of the test vial and remaining in any moisture within the precipitate 94 after cenrifuging 92. Thus, there will be radiation from a zero level CK-MB sample and it is the increase in radiation over the zero level of radiation that provides an indication of the presence of CK-MB in the test sample.

Furthermore, it is well known in 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.

As would be obvious to those skilled in the art, alternative solid-phase materials such as a membrane filter could be utilized instead of the Immuno-Beads. In exploring this alternative, a membrane 120 was created utilizing Whatman No. 1 filter paper which was oxidized using the periodate technique described by Ferrung, B., Maiollini, R., and Masseyeff, R.,Journaloflmmunologicalmethods, Volume 25, Page 49, 1979. Strips of the membrane were soaked in goat anti-rabbit 19 G solution and utilized in much the same manner as the immuno-beads are used in the test. The use of membrane as the solid-phase eliminates the need to centrifuge, and the separation steps become the simple washing of the membrane to remove excess immunoreagents. Results using a membrane solid-phase are presented in Example VI hereinbelow.

Avariation on the above described test procedure, see Figure 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 Figure 3. It is found that suficient amounts of the solid-phase 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.

The following examples demonstrate the instant testing method using several combinations of test conditions.

Example I This experiment was carried out with anti-CK-BB coated immuno-beads. 100u1 of sample was mixed with 200p1 of beads. After overnight incubation at room temperature, 0.5 ml PBS buffer with 10% calf serum was added to the test tube. The beads were separated by cenrifugation. They were then reacted for 5 hours at room temperature with radioiodinated anti-CK-MM. After incubation, beads were washed with 1 ml PBS buffer with 10% calf serum. The beads were then centrifuged and counted.

Results Normal human serum count (NHS) --1090 counts per min.

Calibrators (100 l per test) Ratio Sample Counts NHS COUNTS E. 1 llg/ml of CK-MB powder in serum 1.17 D. 10 Fg/ml of CK-MB powder in serum 3.20 C. 50 Fg/ml of CK-MB powder in serum 5.71 B. 100 g/ml of CK-MB powder in serum 6.65 The calibrator samples were made by dissolving a powder containing 6.8% CK-MB in normal human serum at various dilutions.

Patient Samples (known to have high level of CK-MB) High level 10 l 3.66 25u1 7.44 50 l 12.1 100yl 14.9 As is seen, a good response curve was obtained with CK-MB calibrators, and patient sample, which was identified as having high CK-MB content by other techniques, could be measured utilizing four dilutions to assure a reading level that was correlatable on the calibration curve.

Example II This example shows effect of reducing incubation time and varying the amount of beads to 200u1 and 500 1. The wash buffer used was 2 ml with 5% calf serum and was added before each centrifugation. 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 200 u1 Beads 500 Ill Beads Counts (cpm) NHS 913 1475 Ratio Sample Counts NHS COUNTS High 50 lli 2.35 2.85 100 irk 3.31 3.77 Calibrater 50 g/ml 2.24 3.25 1001lg/ml 2.8 4.50 The ratios obtained, although significantly lowerthan the overnite incubation, were still quite acceptable.

The effect of reduction in incubation time thus can be compensated by increasing the amount of beads.

Example 111 The method described in Example II was used with 200 u1 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 NHS 3835 counts per minute Ratio Sample Counts NHS COUNTS Calibrators 50 Fg/ml 2.2 100ug/ml 2.7 PatientA: 11 a.m. 2.24 12noon 2.75 1 p.m. 3.00 4 p.m. 3.15 8 p.m. 3.54 Normal Patients: B .94 C 1.06 D .99 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.

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 R I of these beads being comparable in number of beads to 250u1 of beads used in previous examples. These beads were not precoated with anti-CK-BB. After a one hour incubation period, the reactants were centrifuged and aspirated. A second incubation of one hour was carried out in the normal manner. Thereafter 2 mi 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 Ratio Sample Counts NHS COUNTS Calibraters 501lg/ml 3.1 100uglml 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 Counts Standard Deviation RUN 1 NHS 2015 13 negative samples 2260 305 RUN 2 NHS,16times 2017 199 12 negative samples 2172 300 with high totai CK Calibrater 50 Wg/ml 4281 100ug/ml 5936 Thus negative samples, even those with high total CK levels, would all be well under the 50 Fg/ml calibrator sample and are seen to be quote close to the NHS samples.

Example VI 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 placed in a mixture of sample and 2ml PBS 5% BSA buffer.

After a first incubation of 1 hour, the liquids 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: Results Sample NHS 967 counts per minute Ratio Sample Counts NHS COUNTS Calibrater 400 Rg/ml 3.3 100,ug/ml 1.9 Thus, a membrane solid-phase is seen to produce cognizable results although they are not as pronounced as were the results utilizing the beads as the solid-phase.

It can therefore be seen that the present invention derives its uniqueness from the utilization of the cross-reactive capabilities of the antibodies that are selected for use. This is 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 occurrence.

Whereas the preferred embodiment of the present invention has been described above, it is contemplated that other alterations and modifications may become apparent to those skilled in the art after having read the above diclosure. It is therefore intended that the appended claims be interpreted as covering all such alterations and modifications as fall within the true spirit and scope of the invention.

Claims (26)

1. A two site cross-reaction immunometric sandwich assay method forthe detection and measurement of an analyte in serum which comprises: (a) selecting a first antibody which will cross-react with an analyte of interest and affixing said first antibody to a solid-phase to form a bound antibody; (b) selecting a second antibody which differs from said first antibody and which also cross-reacts with 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 tag of said second antibody as an indication of the presence of said analyte of interest.

2. A two site cross-reaction immunometric sandwich assay method as recited in Claim 1 wherein said solid-phase is a material selected from the group consisting of polyacrylamide beads, latex beads, cellulose beads and a membrane strip.

3. Atwo site cross-reaction immunometric sandwich assay method as recited in Claim 1,wherein said tag is radioactive iodine '251.

4. Atwo site cross-reaction immunometric sandwich assay method as recited in Claim 1, wherein said separating steps are accomplished by centrifuging the solutions to precipitate said solid-phase and by aspirating to remove said liquid portion therefrom.

5. A two site cross-reaction immunometric sandwich assay method as recited in Claim 1 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 whereby a calibration curve is created; and comparing the test results for said serum to said calibration curve.

6. Atwo site cross-reaction immunometric sandwich assay method as recited in Claim 5 wherein steps (a) - (g) are repeated with several known dilutions of said serum; and comparing the results of these tests with said calibration curve to obtain a quantitative determination of the amounts of said analyte in said serum.

7. A two site cross-reaction immunometric sandwich assay method for the detection and measurement of an analyte in serum which comprises: (a) selecting a first antibody which will cross-react with an analyte interest; (b) selecting a second antibody which differs from said first antibody and which also cross-reacts with said analyte and tagging said second 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 first antibody with a serum suspected of conaining said analyte to form a first reaction solution; (d) adding a solid-phase which is specific to said first antibody to said first reaction solution and further reacting said first reaction solution therewith; (e) separating the solid-phase portion of said first reaction solution from the liquid portion thereof;; (f) reacting said solid-phase portion of said first reaction solution with said tagged antibody to form a second reaction solution; (g) separating the solid-phase portion of said second reaction solution from the liquid portion thereof; (h) testing said solid-phase portion of said second reaction solution to detect the tag of said second antibody as an indication of the presence of said analyte of interest.

8. A two site cross-reaction immunometric sandwich assay method as recited in Claim 7 wherein said solid-phase is a material selected from the group consisting of polyacrylamide beads, latex beads, cellulose beads and a membrane strip.

9. The two site cross-reaction immunometric sandwich assay method as recited in Claim 7 wherein said tag is radioactive iodine 1251.

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

11. The two site cross-reaction immunometric sandwich assay method as recited in Claim 7 and further comprising repeating steps (a) - (h) several times substituting for said serum in each repetition of steps (a) (h) a sample containing a different known amount of said analyte whereby a calibration curve is created; and comparing the test results for said serum to said calibration curve.

12. The two site cross-reaction immunometric sandwich assay method as recited in Claim 11 wherein steps (a) - (h) are repeated with several known dilutions of said serum; and comparing the results of these tests with said calibration curve to obtain a quantitative determination of the amounts of said analyte in said serum.

13. A two site cross-reaction immunometric sandwich assay method for the assay of creatine phospho-kinase-MB (CK-MB) in human serum which also conains creatine phospho-kinase-MM (CK-MM) and creatine phospho-kinase-BB (CK-BB), comprising: (a) affixing an antibody to CK-BB to a solid-phase to form a bound antibody; (b) tagging an antibody to CK-MM; (c) reacting said bound antibody with a serum suspected of containing CK-MB 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 the second reaction solution from the liquid portion thereof;; (g) testing said solid-phase portion of said second reaction solution to detect the tag of said antibody to CK-MM as an indication of the presence of CK-MB.

14. A two site cross-reaction immunometric sandwich assay method as recited in Claim 13 wherein said solid-phase is a material selected from the group consisting of polyacrylamide beads, latex beads, cellulose beads and a membrane strip.

15. A two site cross-reaction immunometric sandwich assay method as recited in Claim 13 wherein said tag is radioactive iodine 1251.

16. A two site cross-reaction immunometric sandwich assay method as recited in Claim 13 wherein said separating steps are accomplished by centrifuging the solutions to precipitate said solid-phase and by aspirating to remove said liquid portion therefrom.

17. Atwo site cross-reaction immunometric sanwich assay method as recited in Claim 13 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 whereby a calibration curve is created; and comparing the test results for said serum to said calibration surve.

18. A two site cross-reaction immunometric sandwich assay method as recited Claim 17 wherein steps (a) - (g) are repeated with several known dilutions of said serum; and comparing the results of these tests with said calibration curve to obtain a quantitative determination of the amounts of said analyte in said serum.

19. A two site cross-reaction immunometric sandwich assay method for the assay of creatine phospho-kinase-MB (CK-MB) in human serum which also contains creatine phospho-kinase-MM (CK-MM) and creatine phospho-kinase-BB (CK-BB) comprising: (a) tagging an antibody to CK-MM; (b) reacting an antibody to CK-BB with a serum suspected of containing CK-MB to form a first reaction solution; (c) adding a solid-phase which is specific to said antibody to CK-BB to said first reaction solution and further reacting said first reaction solution therewith; (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 CK-MM 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 said tag of said antibody to CK-MM as an indication of the presence of CK-MB.

20. A two site cross-reaction immunometric sandwich assay method as recited in Claim 19 wherein said solid-phase is a material selected from the group consisting of polyacrylamide beads, latex beads, cellulose beads and a membrane strip.

21. A two site cross-reaction immunometric sandwich assay method as recited in Claim 19 wherein said tag is radioactive iodine 125j,

22. A two site cross-reaction immunometric sandwich assay method as recited in Claim 19 wherein said separating steps are accomplished by centrifuging the solutions to precipitate said solid-phase and by aspirating to remove said liquid portion therefrom.

23. Atwo site cross-reaction immunometric sandwich assay method as recited in Claim 19 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 whereby a calibration curve; and comparing the test results for said serum to said calibration curve.

24. A two site cross-reaction immunometric sandwich assay method as recited in Claim 23 wherein steps (a) - (g) are repeated with several known dilutions of said serum; and comparing the results of these tests with said calibration curve to obtain a quantitative determination of the amounts of said analyte in said serum.

25. A two site cross-reaction immunometric sandwich assay method for the detection and measurement of an analyte in serum which comprises: (a) selecting a first antibody which will cross-react with an analyte of interest; (b) selecting a second antibody which differs from said first antibody and which also cross-reacts with said analyte, 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 first antibody with a serum suspected of containing said analyte to form a first reaction solution; (d) separating the first antibody and other reactants immunochemically attached thereto of said first reaction solution from the remainder thereof;; (e) reacting said first antibody and other reactants immunochemically attached thereto of said first reaction solution with said second antibody to form a second reaction solution; (f) separating the second antibody and other reactants immunochemically attached thereto of said second reaction solution from the remaindertherreof; and (g) testing said second antibody and other reactants immunochemically attached thereto of said second reaction solution to detect the quantity of said second antibody attached thereto as an indication of the presence of said analyte of interest.

26. A two site cross-reaction immunometric sandwich assay method as recited in Claim 25 wherein a solid-phase is utilized to immunochemically bind with said first antibody as an aid in separating said first antibody and other reactants immunochemically attached thereto from the remainder of the reactants of said first reaction solution, and said second antibody is tagged for detection.