Radioisotopicassay using isotope transfer to chelator-target recognition molecule conjugate

A method of forming a therapeutic or diagnostic agent labeled with a radioactive metal ion, which comprises: contacting an unlabeled therapeutic or diagnostic agent, consisting of a substantially non-metal chelating portion and a chelating portion capable of chelating with the radioactive metal ion, with an ion transfer material having the radioactive metal ion bound thereto and having a binding affinity for the radioactive metal less than the binding affinity of the chelating portion for the radioactive metal ion, wherein prior to contacting the chelating portion is unchelated or is chelated with a second metal having a binding affinity with the chelating portion less than the binding affinity of the radioactive metal ion, whereby a radiolabeled therapeutic or diagnostic agent is formed by the contacting, and separating the radiolabeled therapeutic or diagnostic agent from the ion transfer material, is disclosed along with various components and kits useful in practicing this method and several variations thereof.

BACKGROUND OF THE INVENTION 
1. Field of the Invention: 
The present invention relates to methods of radioactively labeling 
diagnostic and therapeutic agents and is particularly related to systems 
in which a metal ion is bound to the labeled molecule through a chelating 
group. 
2. Description of the Prior Art: 
The use of radioactively labeled diagnostic and therapeutic agents has 
become routine practice in clinical and analytical laboratories throughout 
the world. Such radioactively labeled compounds are used both in vitro 
(for example, in radioimmunoassay systems) and in vivo (for example, both 
in diagnostic imaging techniques and in radiation therapy techniques). 
Initially, the number of radioisotopes that could be firmly attached to the 
typical organic molecules used as diagnostic and therapeutic agents was 
limited. The difficulty in forming stable carbon-metal bonds prevented the 
early utilization of many radioactive metals and typically limited 
radioisotopes used to label organic molecules to isotopes of phosphorus, 
carbon, hydrogen, and iodine. 
Recently, a new approach has enabled the labeling of such agents with metal 
ions. In this approach, a chelating moiety is covalently attached to the 
molecule of interest, and a radioactive ion is then chelated by the 
sequestering groups of the chelator. The chelating moieties which have 
generally been used for this purpose in the prior art have been analogues 
or derivatives of ethylenediaminetetraacetic acid (EDTA), although many 
variations have also occurred. For example, in 1968, W. F. Benisek and F. 
M. Richards suggested the covalent bonding of chelating groups based on 
methylpicotinimidate to the amino function of a protein molecule in order 
to facilitate crystallographic investigation of protein structure by 
binding a metal to the chelating site on the protein [J. Biol. Chem., 243 
4267-4271 (1968)]. Likewise, in U.S. Pat. No. 4,043,998, the compound 
1-(p-benzenediazonium)ethylenediaminetetraacetic acid, said to be a 
powerful chelating agent which can be bonded strongly to proteins through 
its diazonium group, was disclosed. In Science, 209, 295-297 (1980), B. A. 
Khaw et al. disclosed the use of a bifunctional chelating agent, 
diethylenetriaminepentaacetic acid (DTPA) to label an antibody with a 
radioactive isotope and the subsequent use of that labeled antibody to 
image experimental myocardial infarctions in dogs. The metal binding 
efficiencies of the resulting compounds were low, however, since 
attachment occurred through one of the carboxylate groups which would 
normally have participated in binding to the metal ion. Similarly, D. A. 
Scheinberg and O. A. Gansow taught in Science, 215 1511-1513 (1982), the 
use of DTPA and EDTA analogs covalently bonded to antibodies to image 
mouse erythroid tumors. 
Unfortunately, the radioactively labeled materials previously available 
suffered from several disadvantages. This was particularly true for 
imaging agents and other molecules labeled with an isotope of high 
specific activity. The short half-lives of the radioactive isotopes used 
and the radiation-induced degradation of the labeled molecules greatly 
reduced the shelf-lives of these materials and, when imaging agents are 
involved, greatly increased the amount of background radiation present. 
Furthermore, health hazards to the technicians handling these materials 
and hazards associated with disposing of the associated waste generated at 
various steps of synthesizing labeled compounds made the handling of 
radioactively labeled compounds difficult. 
Typically, as disclosed by Scheinberg and Strand in the article cited 
above, a bifunctional chelate was coupled to a target molecule, after 
which any metal ions present were removed by dialysis, typically against a 
solution containing low molecular weight chelating molecules such as EDTA. 
The chelate-conjugated molecules were then labeled with a radioactive 
metal solution, after which free metal was removed, for example, by 
ion-exchange chromatrography. The resulting labeled product was then 
stored and later used in the diagnostic or therapeutic process. Using such 
procedures, considerable handling of the radioactive material and 
generation of radioactive waste occurred, a disadvantage not overcome by 
any teachings of the prior art. 
SUMMARY OF THE INVENTION 
The present invention provides a universal method which can be used to 
radioactively label any diagnostic or therapeutic agent having a ligand 
portion thereof which is capable of binding with a radioactive metal ion. 
The labeling occurs immediately prior to the utilization of the agent and 
produces little or no radioactive waste. 
The invention provides a method of radioactively labeling a diagnostic or 
therapeutic molecule with a radioactive metal ion, which comprises: 
(A) contacting 
(1) an unlabeled therapeutic or diagnostic agent comprising 
(a) a substantially non-metal chelating portion attached to 
(b) a chelating portion capable of substantially chelating with said 
radioactive metal ion, with 
(2) an ion transfer material having said radioactive metal ion bound 
thereto and having a binding affinity of said chelating portion for said 
radioactive metal ion, 
wherein prior to said contacting said chelating portion is unchelated or is 
chelated with a second metal ion having a binding affinity with said 
chelating portion less than the binding affinity of said radioactive metal 
ion, whereby a radiolabeled therapeutic or diagnostic agent is produced by 
said contacting; and 
(B) separating said radiolabeled therapeutic or diagnostic agent from said 
ion transfer material. 
Additionally, the labeling method described above can be used as the first 
step of a diagnostic or therapeutic process, after which the normal steps 
of the process are carried out in their usual fashion. Typical of such 
processes are radioimmunoassay and in vivo diagnostic and therapeutic 
techniques. 
The invention provides, in addition to the aforementioned process, various 
elements and components to be used therein in the form of kits comprising 
these components and other components used in the various processes. 
In essence, the invention is based on the discovery that, if conditions are 
properly selected, hazards involving radioactive waste and radioactive 
products can be ameliorated by utilizing an ion transfer process as the 
last step prior to the ultimate use of a therapeutic or diagnostic 
molecule having a radiolabel. Thus, the necessity of handling radioactive 
material during the preparation of a diagnostic or therapeutic molecule is 
avoided and no waste radioactivity is generated in the clinical or 
analytical laboratory environment. Uses for the process, system, and 
components of the present invention are unlimited and include all of the 
uses to which prior art techniques involving radiolabeled diagnostic and 
therapeutic molecules have been put as well as other uses disclosed herein 
.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The terms "therapeutic or diagnostic agent" as used in the specification 
and claims of this application includes any substance or substances either 
alone or in mixtures which, when labeled with a radioactive metal ion, can 
be used in the treatment of a disorder of an animal or human body, in an 
in vivo diagnostic technique involving a human or animal body, or in an in 
vitro diagnostic technique for any analyte whose detection is desired. 
Typical of therapeutic agents are radioactive drugs containing 
beta-emitting radionuclides which are used for therapeutic purposes. These 
agents localize in pathological tissue and destroy it by ionizing 
radiation. In vivo diagnostic agents typically incorporate a 
gamma-emitting nuclide which, because of the physical or metabolic 
properties of the molecularly recognizable portion of the agent, localizes 
in a specific organ after administration. Diagnostic images reflecting 
organ structure and/or function can then be obtained by means of detection 
devices that detect the distribution of ionizing radiation emitted by the 
nuclide. In vitro diagnostic agents are exemplified by radioimmunoassay 
agents which are in wide-spread clinical use. These agents are employed in 
the measurement of minute quantities of various biological substances, 
such as hormones. 
Diagnostic and therapeutic agents of the invention have two functionally 
different portions of the molecule or molecular conjugate (although these 
may be at least in part the same structural portions in some molecules). 
These portions are (a) a substantially non-metal chelating portion 
attached to (b) a chelating portion capable of chelating with the 
radioactive metal ion being used. By "substantially non-metal chelating 
portion" is meant to include not only molecular portions which carry no 
metal-chelating groups, but also molecular portions which may carry 
certain groups capable of metal chelation but which do so with 
substantially less affinity than portion (b), the "chelating portion." 
Particularly preferred among the "substantially non-metal chelating 
portions" (a) are those which are molecularly recognizable portions. The 
phrase "molecularly recognizable portion" denotes any molecular portion of 
the total molecule which is capable of being recognized by a complementary 
system or molecule in the system in which the agent is being used. 
Molecular recognition, as will be understood by those skilled in the art, 
includes the non-covalent binding in three dimensions between 
complementary portions of two molecules. A molecularly recognizable 
portion on an agent may be of low molecular weight (about less than MW 
2,000) or of high molecular weight. For example, it can be a 
polynucleotide sequence, such as RNA or DNA, to be recognized by its 
complementary sequence; an antigen portion (e.g., a drug, a pesticide, a 
metabolite, a physiologically occurring compound), to be recognized by its 
corresponding monoclonal or polyclonal antibody; an antibody portion, to 
be recognized by its corresponding antigen; a lectin portion, to be 
recognized by its sugar; a sugar portion, to be recognized by its lectin; 
a hormone portion, to be recognized by its receptor; a receptor portion, 
to be recognized by its hormone; an inhibitor portion, to be recognized by 
its enzyme; an enzyme portion, to be recognized by its inhibitor; a 
cofactor portion; to be recognized by a cofactor enzyme binding site; a 
cofactor enzyme binding site portion, to be recognized by its cofactor; a 
binding ligand, to be recognized by its substrate and vice versa (e.g., 
biotin-avidin); or any permutation or combination thereof. Among the most 
common molecularly recognizable portions are the three-dimensional protein 
arrangements in antigens and antibodies of various sorts, the cell wall 
structures present in various cells, and the nucleic acid sequences 
present in the DNA and RNA of organisms. It is preferred in many 
circumstances that the molecularly recognizable portion be either a 
natural constituent of a biological system or recognizable by a natural 
constituent of a biological system. Thus, in a competitive 
radioimmunoassay using a solid phase antibody which binds to a natural 
constituent present in the serum of a human or animal, the molecularly 
recognizable portion preferably would have the same structural features 
present in the natural component with which it was in competition for 
binding with the antibody. In a therapeutic agent designed to concentrate 
radioactivity in a specific tissue, the molecularly recognizable portion 
would be recognizable by a natural component of that tissue. However, it 
is the function of being molecularly recognizable that is important rather 
than the actual structure. For example, the molecularly recognizable 
portion could be an analog or an artifical component which binds more 
tightly than any natural component of a biological system and therefore is 
more selective for a particular tissue or other component of a biological 
system. Additionally, both the molecularly recognizable portion and the 
component which recognizes this portion may be entirely artificial, 
particularly in an in vitro diagnostic assay. As used in this application, 
the phrase "complementary substance" refers to the component which 
recognizes the molecularly recognizable portion of the agent, whether the 
complementary substance is of artificial or biological origin. 
Furthermore, the molecularly recognizable portion need be only a small part 
of the therapeutic or diagnostic agent and further need not correspond to 
an entire molecule present in any system. For example, when the 
molecularly recognizable portion is proteinaceous, it may be a relatively 
short sequence of amino acids found within a much larger sequence of amino 
acids as would be typical for a hapten or binding site which formed part 
of a large protein. 
The second essential portion of the agent is the "chelating portion." 
Chelates are coordination complexes that are formed between a metal ion 
and a ligand that contains at least two electron-donating groups arranged 
so that a ring structure is formed upon coordination. Especially stable 
are chelates containing 5- or 6-membered rings. Typical functional groups 
involved in chelation include acidic or anionic groups derived from 
carboxylic acids, oximes, hydroxyl compounds, phenols, sulfonic acids, and 
mercaptans. Uncharged functional groups capable of being involved in 
chelation include amines (primary, secondary and tertiary), carbonyl 
groups, thiocarbonyl groups, nitroso groups, and cyclic amines, such as 
those typically present in heterocyclic compounds. A ligand involved in 
complexation can be either charged or uncharged. 
The chelating portion of the agent typically will be formed by reacting a 
derivative of a known chelating agent with a molecule having a portion 
that forms the substantially non-metal chelating portion of the final 
therapeutic or diagnostic agent. Preferred are chelating portions which 
comprise a diamine wherein the two amine groups are substituted with two 
acetic acid moieties, with the two amino groups and/or the four acetic 
acid groups being capable of donating an electron pair to the same metal 
ion. Typically, the amino groups will be covalently attached to adjacent 
carbon atoms. Preferred are derivatives of ethylenediaminetetraacetic acid 
and other chelating groups having a binding constant for any radioactive 
metal ion at least as great as that of EDTA for the same metal ion. The 
ethylenediaminetetraacetic acid derivative 1,2-diaminocyclohexaneacetic 
acid and its derivatives and analogs are especially preferred. By 
derivatives and analogs is meant compounds having the basic skeletal 
structure and functional groups of these compounds but having additional 
functional groups which do not prevent the resulting compounds from 
functioning as chelating groups. Typical chelating molecules which can be 
modified to form the chelating portion of the agent are DCTA, EDTA, 
tartaric acid, alpha-benzoin oxime, 1,10-phenanthroline, and similar well 
known compounds. 
The substantially non-metal chelating portion of the molecule may be 
derived from any molecule of small or high molecular weight, any molecular 
complex, or any biological system (e.g., a virus, a cell or a group of 
cells). Among the common molecules which may be used as sources are amino 
acids, saccharides, nucleotides, proteins, polysaccharides, 
lipopolysaccharides, protein complexes, single- or double-stranded nucleic 
acids or segments thereof, whole viruses or viral compounds such as cores 
or capsids, bacteria, tissue cells, and the like. Among the most common 
proteins are the structural proteins, enzymes, immunoglobulins, and 
fragments thereof. Among the most common nucleic acids are DNA and RNA of 
various types, such as tRNA, mRNA, rRNA, and the like. Bacteria, either 
whole or fragments thereof, such as cell walls or other recognizable 
portions, include both gram positive and gram negative bacteria. Fungi, 
algae, viruses and other microorganisms (and fragments thereof) are also 
included as well as animal (e.g., mammalian) cells including red blood 
cells. 
Because the principal aspect of the present invention contemplates labeling 
a preformed therapeutic or diagnostic molecule consisting of a 
non-chelating portion and a chelating portion, the general techniques of 
producing such molecules are not considered part of the present invention, 
although certain types of chelating groups and methods of attaching them 
to the non-chelating portion of agents are discussed in later sections for 
purposes of illustration. 
As discussed in the section of this application entitled prior art, many 
therapeutic and diagnostic agents having chelating portions and 
non-chelating portions are already known. For example, Hnatowich et al., 
Science, 220, 613-615 (1983), which is herein incorporated by reference, 
disclose a method of covalently coupling the chelator 
diethylenetriaminepentacetic acid (DPTA) to proteins, such as 
immunoglobulins. Generally, a dianhydride of DTPA is reacted with a 
molecularly recognizable protein under straightforward conditions. This 
method may be used to attache a ligand to any molecule having an amino or 
hydroxyl group or a similar nucleophilic group. Since many molecules 
already contain one of these groups (and the remainder can generally be 
easily modified so that they do), this provides a general method of 
attaching a chelating group to any molecule of interest. Many similar 
methods, such as those disclosed in the references cited in the section of 
this application entitled "Description of the Prior Art," all of which are 
herein incorporated by reference, disclose further ligands and methods of 
modifying other molecules with them. 
In addition to those agents previously known to the prior art, many other 
diagnostic and therapeutic agents having a molecularly recognizable 
portion and a chelating portion can be synthesized by standard techniques 
of organic chemistry. For example, although the prior art has dealt with 
the attachment of chelating groups to proteins, it is also possible to 
attach chelating groups to non-proteinaceous molecules of interest, such 
as lipids, hormones, and sugars. Although chelating groups have not 
previously been attached to such molecules, many derivatives of these 
various classes of biological compounds are known which have covalent 
bonds formed through a carbon, oxygen, nitrogen or sulfur atom to an 
organic radical not normally part of the compound. Minor variations of the 
techniques used to snythesize these known compounds can be used to attach 
chelating groups to the recognizable molecules. 
Likewise, chelating molecules can be modified by standard chemical 
techniques to provide a functional group through which attachment to the 
recognizable molecule can take place. Several procedures are disclosed for 
the chelating groups that have been previously modified for attaching to 
proteins, as has been previously discussed. Furthermore, since many 
chelating molecules contain at least one radical derived from acetic acid, 
these molecules can easily be modified using standard techniques to create 
a functional group on the alpha carbon through which attachment can take 
place to recognizable molecule. The properly functionalized recognizable 
molecules and chelating groups can easily be attached one to the other by 
standard reactions of organic chemistry although, naturally, all the 
resulting compounds will not fall into the class of agents which exhibit 
the most preferred binding affinity. 
Chelating groups that are analogs of 1,2-diaminocyclohexaneacetic acid are 
particularly preferred for use in the practice of this invention. The 
chelating group is covalently bonded, generally though not necessarily 
through an appropriate bridging entity, to a diagnostic or therapeutic 
molecule of interest to create agents useful in the practice of the 
invention. The chelating portion provides a strong bonding site for metal 
ions and, by selecting the proper linking structure, can be coupled to a 
variety of sites on a wide range of molecules. 
One advantage of 1,2-diaminocyclohexaneacetic acid analogs is that they can 
be successfully used with polynucleotides and nucleic acids, unlike 
certain prior art aromatic chelating groups which cannot usually be used 
with polynucleotides because of intercalation. The cyclohexane-based 
dicyclohexanetetraacetic acid (DCTA) analogs generally do not interfere 
with any normal reactions of labeled polynucleotides or nucleic acids and 
can additionally be used with any of the other molecularly recognizable 
portions disclosed herein. The DCTA analogs also have binding affinities 
for metal ions several orders of magnitude higher than those of EDTA. 
Examples of therapeutic and diagnostic agents useful in this invention are 
also disclosed and discussed in U.S. Pat. No. 4,707,440 which is herein 
incorporated by reference in its entirety. 
The chemical structure of the preferred chelating groups in a diagnostic or 
therapeutic agent as described herein is exemplified by the following 
structural formula: 
##STR1## 
wherein R is the substantially non-metal chelating portion of the 
therapeutic or diagnostic agent, R.sup.1 is C.sub.1 -C.sub.4 alkyl or is 
--CH.sub.2 COOM, M is H or a catonic metal or a negative charge, and A is 
either a direct covalent bond or a bridging entity such as, e.g., of the 
type shown in the aforementioned co-pending application. Since spacing is 
the main consideration rather than the structure of the bridging entity, 
the chemical structure of the bridging entity is unimportant and is not 
limited as long as--among other things--molecular recognition is not 
unduly hindered. 
It is preferred to use a bridging entity to join the non-chelating molecule 
to the chelating molecule from which the chelating portion is derived. The 
selection of the bridging entity is, of course, varied depending on the 
type of molecules involved, the number and nature of the available bonding 
sites, the types of reactions which the labeled agent is to undergo, and 
other factors known to those skilled in the art. The linking group can be 
tailored to specific types of agents, for example, nucleotides, proteins, 
amino acids, enzymes, etc., to suit the needs of particular detection, 
imaging, or therapeutic techniques. 
Examples of generally useful linking groups include beta-thiopropionic acid 
hydrazide, betathioethylamine, and isothiocyanate. In particular, 
beta-thiopropionic acid hydrazide has been found to be highly suitable for 
the attachment of chelating groups to amine-containing molecules under 
mild conditions. The preferred bridging entity for a particular 
non-chelating molecule depends on the reactive functional groups present 
in that molecule. For example, molecules having a free amino group (such 
as proteins and peptides having one or more lysine residues) can be 
reacted with a carbonyl azide to form a peptide bond. Molecules having a 
free hydroxyl group (such as proteins having a tyrosine residue) can be 
reacted with an isothiocyanate or can be heated in the presence of the 
azide (which rearranges to form an isocyanate) to form a thiourethane or 
urethane. Molecules having a carbonyl group can form a Schiff base with an 
amino group of a modified chelating molecule which can then be reduced if 
desired to a secondary amine. Many variations of these bonding techniques 
exist and may be used as deemed appropriate. 
Examples of agents which can be used in the practice of the invention 
include those in which a molecularly recognizable portion is derived from 
a nucleotide or related compound. Methods of forming such compounds are 
described in detail and claimed in copending application Ser. No. 391,441, 
filed June 23, 1982, which is herein incorporated by reference. 
Accordingly, agents whose molecularly recognizable portion is derived from 
DNA, RNA, a nucleotide, a deoxynucleotide, nucleoside, or a 
deoxynucleoside can easily be prepared using the methods described therein 
for modifying the nucleotide or related molecule, together with the 
methods described herein for coupling to chelating groups. 
The ratio of the non-chelating portion of the agent to the chelating 
portion need not necessarily be 1:1. There may be many more chelating 
portions than non-chelating portions, or vice versa. In the case when the 
ratio of chelating portions to non-chelating portions is greater than 1, 
for example, 5-10 to 1 or even greater, the system amplifies the radiation 
provided by the primary recognition event by a factor equal to the ratio. 
It should again be noted that the aspect of the present invention relating 
to labeling an agent already containing a chelating portion in no way 
depends upon the structure of the molecules being manipulated but rather 
depends on their chelating ability and their ability to be recognized on a 
molecular scale in a biological or biochemical system. So long as 
chelation with radioactive metal ion is possible, molecular recognition 
can take place, and an ion transfer material is available which has a 
lower binding affinity for the radioactive metal than does the chelating 
portion of the agent, the invention can be practiced regardless of the 
structure of the molecule. 
Likewise, the structure of the ion-transfer material is unimportant so long 
as the binding affinity (i.e., the binding function) is within the 
limitations disclosed. Although, generally speaking, it is sufficient for 
the practice of this invention to use an ion transfer material whose 
binding affinity for the radioactive metal ion is merely less than the 
binding affinity of the therapeutic or diagnostic agent for the same ion, 
it is preferred that the ratio of binding affinities be less than 0.1, 
more preferably less than 0.01, and most preferably less than 0.001, in 
order to ensure effective transfer of the radioactive metal ion from the 
ion transfer material to the agent. 
Suitable ion transfer materials include both inorganic and synthetic 
organic products. Inorganic ion transfer materials include both the 
naturally occurring materials (e.g., mineral zeolites such as sodalite and 
clinoptilolite, the green sands, and clays such as the montmorillonite 
group), and synthetic products such as the gel zeolites, dehydroxides of 
polyvalent metals such as hydrated zirconium oxide, and the insoluble 
salts of polybasic acids with polyvalent metals such as zirconium 
phosphate. Preferred ion transfer materials are the synthetic organic 
cation exchange resins. These include weak-acid, cation-exchange resins 
and strong-acid resins. The weak-acid resins are generally based on 
acrylic or methacrylic acid that has been crosslinked with a difunctional 
vinyl monomer, such as divinyl benzene. Other weak-acid groups, such as 
phenolic or phosphonic functional groups, may also be used. The weak-acid 
resins are generally used at a pH above 4. The strong-acid resins are 
generally based on sulfonated copolymers of styrene and divinyl benzene. 
These materials are particularly preferred because of their ability to 
exchange cations across the entire pH range. The most preferred ion 
exchange materials are sufficiently porous to provide a large surface area 
on which exchange can take place. Pore sizes are preferably sufficient to 
allow easy passage of the agent through the pores and most preferably are 
several times the largest diameter of the molecule in question. However, 
if the diagnostic or therapeutic agent is particularly large, transfer may 
occur on exterior surfaces only. 
Many commercially available ion transfer materials are known and may be 
used in the practice of this invention if the guidelines set forth herein 
are followed. For example, Dowex 50 and materials having similar 
properties are particularly suitable. 
In general, the labeling process of the present invention is accomplished 
by contacting the therapeutic or diagnostic agent as defined herein with 
an ion transfer material having the radioactive element bound thereto. The 
contacting may consist either of passing a solution containing the agent 
over a column of the ion transfer material or by suspending the ion 
transfer material in a solution of the agent. Although these methods of 
contacting are preferred, any other method of intimately contacting a 
solution containing the agent with the ion transfer material is suitable. 
The amount of radioactivity bound to the ion transfer material, the 
duration of the contact time, and the ratio of the amount of the 
diagnostic agent to the amount of the ion transfer material, as well as 
other conditions, vary depending on the amount of radioactivity needed for 
the particular situation in which the agent is to be used, as is well 
understood to those skilled in the art. If the conditions and contacting 
times are not known, they can easily be determined by simple 
experimentation. After a sufficient contacting time, the radioactivity 
labeled agent is separated from the ion transfer material by any suitable 
technique. Typically, the ion transfer material will be present in the 
form of a column and the agent can be separated by elution. Elution can 
occur using the solvent in which contacting took place, or a second eluent 
may be used if such treatment more easily dislodges the agent from the ion 
transfer material. If not already known, suitable eluents may be 
determined by simple experimentation since elution of radioactivity is 
easily followed. It is particularly preferred that an eluent not 
permanently change a molecularly recognizable portion of the agent so that 
the recognition event can no longer take place. However, a temporary 
change, for example in conformation, causes no harm if the recognizable 
structure can later be regained. Thus elutions with solvents or solutions, 
or under conditions which result in a reversible conformational change in 
the structure of a peptide, for example, are acceptable. Nevertheless, 
elutions of agents of biological origin at or near physiological 
conditions (e.g., pH, ionic strength, temperature, etc.) is preferred, 
particularly if the eluent is to be directly in one of the diagnostic or 
therapeutic procedures which are later discussed. 
Any radioactive metal ion capable of producing a therapeutic or diagnostic 
result in a human or animal body or in an in vitro diagnostic assay may be 
used in the practice of the present invention. Suitable ions including the 
following: 
______________________________________ 
Antimony-124 Iodine-125 Scandium-44 
Antimony-125 Iodine-131 Scandium-46 
Arsenic-74 Iridium-192 Selenium-75 
Iron-55 Silver-110m 
Barium-103 Iron-59 Silver-111 
Barium-140 Sodium-22 
Beryllium-7 Krypton-85 Strontium-85 
Bismuth-206 Strontium-89 
Bismuth-207 Lead-210 Strontium-90 
Lutecium-177 Sulphur-35 
Cadmium-109 
Cadmium-115m Manganese-54 Tantalum-182 
Calcium-45 Mercury-197 Technetium-99 
Mercury-203 Tellurium-125m 
Cerium-139 Molybdenum-99 Tellurium-132 
Cerium-141 Terbium-160 
Cerium-144 Neodynium-147 Thallium-204 
Cesium-137 Neptunium-237 Thorium-228 
Chlorine-36 Nickel-63 Thorium-232 
Chromium-51 Niobium-95 Thulium-170 
Cobalt-56 Tin-113 
Cobalt-57 Osmium-185 + 191 
Titanium-44 
Cobalt-58 
Cobalt-60 Palladium-103 Tungsten-185 
Erbium Platinum-195m Vanadlum-48 
Europium-152 Praseodymium-143 
Vanadium-49 
Promethium-147 
Gadolinium-153 
Protactinium-233 
Ytterbium-169 
Gold-195 Yttrium-88 
Gold-199 Radium-226 Yttrium-90 
Rhenium-186 Yttrium-91 
Hafnium-175 Rubidium-86 
Hafnium-175 + 181 
Ruthenium-103 Zinc-65 
Hafnium-181 Ruthenium-106 Zirconium-95 
______________________________________ 
The following non-limiting example illustrates the preparation of a 
diagnostic or therapeutic agent using a Dowex 50 column. The column is 
first equilibrated with a dilute solution of a buffer, for example, 0.05M 
ammonium acetate and then loaded with a radioactive ion, for example, 
nickel-63, by passing a solution of the ion through the column. After the 
column is prepared (when presented in kit form, as later described, the 
column would be prepared by one other than the ultimate user during 
preparation of the kit), the agent having a chelating portion is passed 
through the column and eluted as the radiolabeled metal chelate. 
The labeling procedure described above is particularly useful in 
combination witb established therapeutic and diagnostic techniques which 
use an agent having the properties described in this application. For 
example, a diagnostic agent useful in radioimmunoassay (RIA) can be 
labeled immediately prior to its use, thus greatly reducing non-specific 
binding caused by radiation damage which would occur with an agent which 
had been labeled and stored for a long period of time. RIA is a well-known 
technique and will not be described in detail here. For particulars, 
reference is made to Chard, "An Introduction to Radioimmunoassay and 
Related Techniques," North-Holland Publishing Company, 1978, which is 
herein incorporated by reference. Any of the many variations of RIA can be 
used, such as homogeneous phase RIA, heterogeneous or solid phase RIA, 
single antibody or double antibody methods, and direct (forward) or 
reverse sandwich assays. Particularly preferred are solid phase systems 
wherein the antibody (IgG or IgM) is covalently coupled to an insoluble 
support so that both the antibody and the bound complex after incubation 
can be readily separated from the soluble free fraction. A wide variety of 
solid phase supports have been described, which include particles of 
dextran or cellulose, continuous surfaces such as polystyrene or 
polypropylene discs, walls of plastic tubes, glass discs, glass particles, 
and the like. Particulate solid phases are widely used for a variety of 
different assays and can be used in the practice of the present invention. 
Antibodies are attached to the particles by any of a number of techniques 
designed to yield a non-reversible covalent or non-covalent link between 
protein and particle, for example, directly or by cyanogen bromide 
activation. Other alternatives are the use of antibodies entrapped in te 
interstices of a polyacrylamide gel or bound to magnetic particles. An 
assay tube is set up containing either sample or standard, along with the 
tracer and an appropriate amount of solid phase bound antibody, plus a 
detergent to prevent aggregation of the particles and non-specific 
absorption of the tracer. After an incubation period during which the 
tubes are continuously mixed, the solid phase is sedimented by 
centrifugation; the supernatant is removed and the solid phase subject to 
two or more washes with buffer in order to remove free tracer trapped 
within and between the particles. The counts on the solid phase (bound 
fraction) are then measured. Imunoradiometric assays, as described in 
Chard at page 423, can also be used. When a second antibody is used, the 
second antibody can be either IgM or IgG. The present invention is not 
limited to any of these techniques in particular. 
Similarly, the method can be applied in vivo diagnostic and therapeutic 
techniques by labeling the agent immediately prior to its use. This aspect 
of the invention is especially important because of the high levels of 
radioactivity associated with such agents, especially therapeutic agents, 
which result in rapid degradation of any molecularly recognizable portion 
of the molecules and loss of specificity. By using the technique of this 
invention in combination with establishing in vivo techniques for using 
radioactive agents, destruction of any molecularly recognizable portion of 
the agent, which reacts with a complementary substance in a human or 
animal body to cause selective localization in a target region, is greatly 
reduced. Accordingly, it is possible in many cases to use a lower total 
mount of the radioactive isotope in a diagnostic technique because of 
increased specificity. This technique is particularly suited to use with 
monoclonal antibodies to which a chelating group is attached. 
The present invention lends itself readily to the preparation of kits 
comprising one or more of the elements necessary to perform the labeling 
process. Thus, a kit may comprise a carrier being compartmentalized to 
receive in close confinement therein one or more container means or series 
of container means such as test tubes, vials, flasks, bottles, syringes, 
or the like. A first of said container means or series of container means 
may contain the therapeutic or diagnostic agent as described herein. A 
second container means or series of container means may contain an ion 
transfer material capable of binding the radioactive metal ion of interest 
for the particular application of interest. Two embodiments for the second 
container means are possible with regard to the radioactive metal itself. 
In one embodiment, the ion is bound to the ion transfer material during 
the process of manufacturing the kit. The user of such a kit is therefore 
not required to handle radioactive material in fluid form at any point 
prior to obtaining the diagnostic or therapeutic agent in the eluting 
fluid, which can be chosen so that it is immediately useable. 
Alternatively, the kit may provide an ion transfer material not having any 
radioactive metal ion bound thereto. This greatly simplifies preparation, 
storage, and handling of the kit itself. The radioactive metal ion is then 
bound to the ion transfer material by the user of the kit. The ion 
transfer material may then be utilized to label several doses or aliquots 
of the therapeutic or diagnostic agent. Such a kit and procedure is 
particularly suited for isotopes of very short lifetimes, such as are 
often used in in vivo procedures. Medical technicians who would normally 
use solution chemistry to label a therapeutic agent comprising a chelating 
portion and an antibody, for example, can accomplish the same result using 
the techniques of this invention and a kit adapted to that use with less 
waste radioactivity and contaminated glassware. 
It is preferred that the second container means be fitted with fluid inlet 
and outlet means whereby the agent (unlabeled with radioactivity), when 
inserted into the inlet means, intimately contacts the ion transfer 
material while passing through or being contained within said container 
means prior to exiting through the outlet means. It is particularly 
preferred that the inlet and outlet means be fitted with confining means 
such as a screen, which prevent the exit of the ion transfer material fron 
the container. In a particularly preferred embodiment of the present 
invention, the second container means containing the ion transfer material 
having the radioactive ion bound thereto is columnar or tubular in form, 
with the inlet and outlet means being at opposite ends of the tube. Thus, 
a user can easily label any dianostic or therpeutic agent having a 
chelating portion thereon by adding the agent through the inlet means and 
removing the agent as it exits the outlet means. Typically, passage of the 
agent through the container means would occur in solution, whereby the 
agent would intimately contact ion transfer material therein. The 
radiolabeled agent can be recovered either by force of pressure or suction 
or by allowing it to drain from the lower exit means or by passing an 
eluting fluid through the column, as is well understood by those skilled 
in the art. One suitable technique would be to use a disposable syringe or 
other administering means suitable for use in the diagnostic or 
therapeutic procedure for which a radioactive agent is desired which is 
fitted with connecting means by which it can be attached to the exit means 
of the ion transfer material container. The agent can then be withdrawn 
into the syringe with minimum danger of loss or contamination. Typically, 
the kit would also contain a third container means having therein an 
eluant suitable for eluting the agent from the column. If the kit is 
intended for a particular in vitro diagnostic technique, for example, a 
competitive radioimmunoassay procedure, a fourth container means can 
contain a complementary substance capable of binding with any molecularly 
recognizable portion of the agent, for example, a solid phase antibody 
capable of binding both with the anlyte and the diagnostic agent. If the 
unlabeled agent is present in a dry form (e.g., lyophilized), a fifth 
containing means containing a solvent may be supplied. A typical complete 
kit of the invention will contain at least the first two container means 
and associated substances and may optionally contain any other related 
materials useful for the procedure under consideration.