Method for making a preconjugate

A method for making a preconjugate which includes immunogenic species of a polymorphic analyte. The method is carried out by reacting an activated binding moiety, and a polymorphic analyte at room temperature for between about 10 hours and about 60 hours. The attaching reaction results in an excess of the preconjugate which includes the immunogenic species of the polymorphic analyte. The preconjugate can be used to make an immunoreactive conjugate useful as a developer antigen in a competitive inhibition immunoassay for the polymorphic analyte.

BACKGROUND 
We have invented a new method for making and using a preconjugate. In 
particular, our invention is directed to a method for making a 
preconjugate from a polymorphic analyte. The preconjugate can be used to 
make an immunoreactive conjugate. 
There is a continuing and extensive need to detect and quantify various 
analytes in a test sample of a physiological fluid. An analyte can be a 
naturally occurring substance, such as an antibody, antigen or hormone or 
a metabolite or derivative thereof. An analyte can also be a man-made 
substance, such as a drug (including both therapeutic drugs and drugs of 
abuse) or a toxin or a metabolite or derivative thereof. The physiological 
fluid can be, for example, blood, serum, plasma, urine, amniotic, pleural 
or cerebrospinal fluid. 
Immunoassay methods have shown considerable utility for the detection and 
quantification of various analytes. An immunoassay involves an 
immunoprecipitation reaction. An immunoprecipitation reaction can occur 
when two reaction partners, each with a specific binding affinity for the 
other, are combined in a suitable liquid medium. The reaction partners can 
be an antigen and a specific binding partner for the antigen, such as an 
antibody. Generally, one of the reaction partners is present in an unknown 
amount in the test sample of the physiological fluid, and is the analyte 
to be detected and/or quantified. Typically, the liquid medium is a 
buffered aqueous solution. Once initiated, the immunoprecipitation 
reaction results in the formation of immunoprecipitates, or 
antibody-antigen complexes that are usually insoluble, but which can also 
be soluble, in the liquid medium. 
The presence of immunoprecipitates in the liquid medium can change optical 
properties, such as light scattering and light absorption properties of 
the liquid medium, by attenuation of incident light energy. These changes 
can be detected by an appropriate photometer in a photometric immunoassay. 
Photometric immunoassay techniques include both nephelometric and 
turbidimetric techniques. 
In nephelometric immunoassay, a photometer is used to measure the 
reflection or scatter of light by the immunoprecipitates towards a light 
detector. The immunoprecipitates can be aggregates of an analyte and a 
specific binding partner for the analyte, or aggregates of an 
analyte-conjugate and the specific binding partner. The amount of light 
scattered by the immunoprecipitates is directly proportional to the number 
of immunoprecipitates present, which typically increases as the 
immunoprecipitation reaction proceeds. This proportionality permits a 
quantitative determination of analyte concentration. 
In turbidimetric immunoassay, an attenuation or reduction of light energy 
passing through a liquid medium containing immunoprecipitates is measured 
by a light detector placed in the light path. The light energy reduction 
can be caused by reflection, scatter, and absorption of the incident light 
by the immunoprecipitates. The amount of light reduction caused by the 
immunoprecipitates is, again, directly proportional to the number of 
immunoprecipitates present, permitting a quantitative determination of 
analyte concentration. 
Many analytes, including numerous drugs, are haptens. A hapten is a low 
molecular weight (typically weighing less than about 7,000 Daltons) 
substance that is generally incapable of causing, by itself, a significant 
production of antibodies upon administration to an animal body, including 
a human body. This can occur because a hapten is too small to be 
recognized by the body's immune system. 
Traditional immunoassay methods for haptenic analytes are not practical 
because when a hapten-containing test sample is mixed with antibody, the 
hapten either does not combine with the antibody or does not form 
detectable immunoprecipitates. Fortunately, it is known that when a hapten 
is coupled to a larger, carrier molecule, the hapten can acquire antigenic 
properties. In other words, binding the hapten to the carrier molecule (to 
make an analyte-carrier molecule combination) permits the bound hapten to 
be recognized by an animal's immune system. Thus, an immunoprecipitation 
reaction can take place between the hapten (coupled to the carrier 
molecule), and an antibody to the hapten. 
The analyte-carrier molecule combination can be called a conjugate or 
analyte conjugate. The terms conjugate and analyte conjugate are used 
herein to mean the same thing, a haptenic analyte joined to a carrier 
molecule. A conjugate that is able to participate in a immunochemical 
reaction with a specific binding partner for the analyte (or for an 
analyte analog), can be called an immunoreactive conjugate. 
The ability of an immunoreactive conjugate to participate in an 
immunoprecipitation reaction has made possible the development of 
inhibition immunoassays (IIA) for diverse haptenic analytes in 
physiological fluids. As for immunoassays generally, an inhibition 
immunoassay is based on the principle that two specific binding partners, 
such as an antigen and its antibody, can engage in a specific affinity 
binding reaction to form a detectable aggregate. 
Generally, an inhibition immunoassay is carried out by combining: (1) an 
immunoreactive conjugate comprising a haptenic analyte and a carrier 
molecule; (2) a specific binding partner for the haptenic analyte, and; 
(3) an aliquot of a test sample of a physiological fluid. The specific 
binding partner, typically an analyte antibody, does not discriminate 
between the free analyte (if any) in the test sample and the analyte 
portion of the immunoreactive conjugate, allowing the immunoreactive 
conjugate and the specific binding partner to combine to form a detectable 
aggregate. 
The immunoreactive conjugate-specific binding partner aggregate can become 
detectable when it achieves a size large enough to affect optical 
properties of the liquid medium. Thus, large aggregates can attenuate 
transmission of incident light through the liquid medium. The amount of 
light attenuation by the liquid medium upon aggregate formation is 
inversely proportional to the amount of analyte present in the test 
sample. In this manner, an inhibition immunoassay can be used to detect 
and quantify various haptenic analytes in the test sample. 
The immunoreactive conjugate used in the inhibition immunoassay is usually 
not made by combining the haptenic analyte directly to the carrier 
molecule. Typically, a spatial separation between the carrier molecule and 
the analyte is required to prevent the larger carrier molecule from unduly 
hindering recognition of the analyte by analyte antibody. Hence, a 
derivative of the analyte is prepared. This analyte derivative can be 
called a preconjugate. The preconjugate can be coupled to the carrier 
molecule to make the conjugate. 
The preconjugate can comprise a binding moiety attached to an analyte of 
interest. The binding moiety in turn, can comprise a ligand and a spacer 
chain. Typically, the preconjugate is made by attaching the ligand and the 
analyte at opposite ends of the spacer chain. The spacer chain can reduce 
steric hindrance by the carrier molecule upon analyte antibody. 
Additionally, the spacer chain can enable the carrier molecule to undergo 
a specific affinity binding reaction with the ligand portion of the 
preconjugate, relatively unimpeded by the analyte portion of the 
preconjugate. Biotin:avidin are frequently used as ligand:carrier molecule 
binding partners. 
Immunoreactive conjugates have been prepared for a variety of analytes. A 
method for making an immunoreactive conjugate must be able to produce 
conjugate that has a consistent immunoreactivity from assay to assay. 
Additionally, the method must be able to produce a yield of the desired 
immunoreactive conjugate sufficient for numerous and repetitive 
immunoassay procedures, as can be required in a clinical or hospital 
environment. In particular, therapeutic drug monitoring programs can 
require large amount of a consistently immunoreactive conjugate. 
Unfortunately, a number of analytes have the characteristic of being 
polymorphic, that is of being comprised of both an immunogenic species and 
a nonimmunogenic species. Only the one or more immunogenic species of a 
polymorphic analyte can be used to make a useful immunoreactive conjugate. 
It can therefore be difficult to prepare a suitable immunoreactive 
conjugate from a polymorphic analyte. The immunogenic/nonimmunogenic 
species characteristic of a given polymorphic analyte may arise because 
such analytes have a plurality of isomeric forms and/or a plurality of 
reactive functional groups per analyte molecule. 
Thus, upon attempting to make a preconjugate by reacting a binding moiety 
with a polymorphic analyte, a plurality of preconjugates can result. When 
these preconjugates are combined with a carrier molecule, a plurality of 
conjugates can result. Some of these conjugates may be immunoreactive. 
Other conjugates resulting from the addition of carrier molecule to the 
same preconjugate mixture may have little or no immunoreactivity or may 
exhibit a variable immunoreactivity. 
Polymorphic analytes include various vitamins, such as vitamin B.sub.12, 
steroids, antineoplastic and antibiotic compounds such as the 
aminoglycosides antibiotics, including gentamicin, tobramycin, and 
amikacin. Thus, gentamicin has A and C forms. Gentamicin A comprises a 
plurality of closely related or isomeric components, including at least 
gentamicin A.sub.1, gentamicin A.sub.2, gentamicin A.sub.3, and gentamicin 
A.sub.4. Gentamicin C comprises at least three closely related or isomeric 
components--gentamicin C.sub.1, gentamicin C.sub.2, and gentamicin 
C.sub.3. It is possible that not all of the gentamicin isomers are 
immunologically active. If so, only the one or more immunologically active 
isomers of gentamicin (the immunogenic species) can be used to prepare an 
immunologically active gentamicin conjugate useful in a competitive 
inhibition immunoassay for test sample gentamicin. 
Additionally, the aminoglycoside antibiotic tobramycin can have five or 
more reactive amine groups and four reactive hydroxyl groups per 
tobramycin molecule. Similarly, the aminoglycoside antibiotic amikacin can 
have four or more reactive amine groups and eight or reactive hydroxyl 
groups per amikacin molecule. As with gentamicin, the polymorphic 
characteristics of tobramicin and amikacin can result in a plurality of 
conjugates being formed, some of which will be immunoreactive while others 
may not be. 
Various methods have been attempted to synthesize a significant yield of a 
suitable immunoreactive conjugate from a polymorphic analyte for use in a 
competitive inhibition immunoassay for the polymorphic analyte. These 
methods are generally inefficient and laborious. 
Attempts have been made to separate out the useful immunoreactive species 
from the nonimmunoreactive species of a polymorphic analyte. The 
immunogenic species can then be used to make the immunoreactive conjugate. 
Such efforts have been largely unsuccessful or are difficult to achieve 
because of the structural and/or chemical similarity of the immunogenic 
and nonimmunogenic species of a particular polymorphic analyte. 
Attempts have also been made to selectively block one or more of the 
reactive functional groups of the polymorphic analyte, on the theory that 
the remaining unblocked reactive functional groups of the polymorphic 
analyte will permit a higher yield of the desired immunoreactive 
conjugate. Such selective blocking procedures have proved to be 
impractical, time consuming, expensive and can still result in a low yield 
of the desired immunogenic species preconjugate. 
A need therefore exists for a method of making a preconjugate from a 
polymorphic analyte that results in a high yield of preconjugate 
comprising an immunoreactive species of the polymorphic analyte. 
SUMMARY 
The present invention meets these needs. A method according to our 
invention results in a high yield of preconjugate comprising an 
immunogenic species of a polymorphic analyte. 
The preconjugate comprising the immunogenic species of the polymorphic 
analyte can be used to make an immunoreactive conjugate. The 
immunoreactive conjugate can be used as a developer antigen in a 
competitive inhibition immunoassay for test sample polymorphic analyte. 
DEFINITIONS 
The following definitions of various terms are provided to facilitate an 
understanding of the present invention. 
"Analyte" means the substance or group of substances to be detected and/or 
quantified in a physiological fluid. The term "analyte" encompasses 
analyte analog. 
"Analyte analog" means a substance that can specifically bind to a reaction 
partner for the analyte in much the same manner as the analyte itself. 
"Bidentate" or "bidentate conjugate" means a heterobifunctional conjugate 
with two chemical moieties, or bidentate members, attached by a spacer 
moiety, with each member being capable of specifically binding to a 
different macromolecule. Further definition and details regarding 
bidentate conjugates can be found in the U.S. Pat. No. 5,196,351, entitled 
"Bidentate Conjugate And Method Of Use Thereof", issued Mar. 23, 1993. 
"Binding moiety" means a ligand joined to a spacer compound. 
"Carrier molecule" means a compound that has a specific binding affinity 
for the ligand portion of the binding moiety. 
"Polymorphic analyte" means an analyte that has one or more isomers and/or 
more reactive functional groups per molecule of the analyte such that the 
polymorphic analyte comprises both an immunogenic species and a 
nonimmunogenic species. 
"Hapten" means a partial or incomplete antigen, typically a low molecular 
weight drug, that is generally incapable of causing by itself a 
significant production of antibodies. 
"Immunogenic species" means: (1) the isomer or isomers of a polymorphic 
analyte that can be used to make a preconjugate useful for making an 
immunoreactive conjugate, and/or; (2) the polymorphic analyte molecule or 
molecules with a plurality of functional groups that can be used to make 
an preconjugate useful for making an immunoreactive conjugate. 
"Ligand" means a molecule having a specific binding affinity for a carrier 
molecule. 
"Nonimmunogenic species" means: (1) the isomer or isomers of a polymorphic 
analyte that can be used to make a preconjugate, which preconjugate when 
used to make a conjugate, results in a conjugate that is less 
immunoreactive than the conjugate made from a preconjugate comprising an 
immunoreactive species of the polymorphic analyte, and/or; (2) the 
polymorphic analyte molecule or molecules with a plurality of functional 
groups that can be used to make an preconjugate which preconjugate when 
used to make a conjugate, results in a conjugate that is less 
immunoreactive than the conjugate made from a preconjugate comprising an 
immunoreactive species of the polymorphic analyte. 
"Polymorphic analyte" means an analyte that has one or more isomers and/or 
more reactive functional groups per molecule of the analyte such that the 
polymorphic analyte comprises both an immunogenic species and a 
nonimmunogenic species. 
"Spacer compound" means a substance attached to or capable of being 
attached simultaneously to both a ligand and a polymorphic analyte. 
A method according to the present invention for making a preconjugate from 
a polymorphic analyte can have two steps. The first step is to react in an 
attaching reaction a binding moiety and a polymorphic analyte. The 
polymorphic analyte comprises an immunogenic species and a nonimmunogenic 
species. The second step is to separate the preconjugate comprising the 
immunogenic species of the polymorphic analyte from the preconjugate 
comprising the nonimmunogenic species of the polymorphic analyte. 
The attaching reaction can result in a stoichiometric excess of the 
preconjugate comprising the immunogenic species of the polymorphic analyte 
relative to the amount of the preconjugate comprising the nonimmunogenic 
species of the polymorphic analyte. 
Preferably, the attaching reaction takes place in a reaction medium 
comprising a liquid capable of solubilizing the binding moiety and the 
polymorphic analyte. 
Also within the scope of the present invention is (1) a product by the 
process of the disclosed method and (2) an immunoreactive conjugate made 
from the preconjugate prepared by the attaching reaction. Such an 
immunoreactive conjugate can be made by contacting the preconjugate 
comprising the immunogenic species of the polymorphic analyte with a 
carrier molecule. 
The disclosed method can be used to make preconjugates useful for the 
preparation of immunoreactive conjugates from many different polymorphic 
analytes. 
DESCRIPTION 
We have discovered that under certain reaction conditions a binding moiety 
and a polymorphic analyte can be combined to consistently obtain a 
high-yield synthesis of a useful preconjugate. The preconjugate can be 
combined with a carrier molecule to make an immunoreactive conjugate. The 
immunoreactive conjugate can be used as a developer antigen in a 
competitive inhibition immunoassay for the polymorphic analyte. 
A method according to the present invention commences by reacting in an 
attaching reaction, a binding moiety and a polymorphic analyte. 
Preferably, the attaching reaction is carried out in a reaction medium 
that is capable of solubilizing both the binding moiety and the 
polymorphic analyte at all the concentrations of these reactants set forth 
herein. 
The second step of the method is to separate the preconjugate comprising 
the immunogenic species of the polymorphic analyte from the preconjugate 
comprising the nonimmunogenic species of the polymorphic analyte. The 
separation step removes substantially all of the preconjugate comprising 
the nonimmunogenic species of the polymorphic analyte from contact with 
the preconjugate comprising the immunogenic species of the polymorphic 
analyte. Thus, the separation step can yield essentially pure preconjugate 
comprising the immunogenic species and useful for making an immunoreactive 
conjugate. The preconjugate comprising the immunogenic species of the 
polymorphic analyte can be contacted with a carrier molecule to make the 
immunoreactive conjugate. 
The polymorphic analyte used to prepare the preconjugate, comprises at 
least one immunogenic species and at least one nonimmunogenic species. 
Thus, the polymorphic analyte has a plurality of isomeric forms and/or a 
plurality of reactive functional groups per analyte molecule. 
The polymorphic analyte can be selected from the group consisting of 
gentamicin, tobramycin, amikacin, vitamin B.sub.12, netilmicin, sisomycin, 
kanamycin, neomycin, vancomycin, erythromycin (including erythromycin A, 
B, C, E, F, N-demethylerythromycin A and the corresponding propionate 
esters), bleomycin, capreomycin, dactinomycin, lincomycin, oleandomycin, 
and derivatives, metabolites, and analogues thereof. 
The attaching reaction can result in a stoichiometric excess of 
preconjugate comprising the immunogenic species of the polymorphic analyte 
relative to the amount of preconjugate comprising the nonimmunogenic 
species of the polymorphic analyte. 
Preferably, the ratio of the stoichiometric excess of the preconjugate 
comprising the immunogenic species of the polymorphic analyte to the 
preconjugate comprising the nonimmunogenic species of the polymorphic 
analyte resulting from the attaching reaction is at least about 2:1. We 
have found that by our method it is possible to obtain such a ratio of 
about 3:1, 4:1 or 5:1. In a particularly preferred embodiment of the 
present invention, the ratio of the stoichiometric excess of the 
preconjugate comprising the immunogenic species of the polymorphic analyte 
to the preconjugate comprising the nonimmunogenic species of the 
polymorphic analyte resulting from the attaching reaction can be about 
9:1. These ratios were determined by, for example, visual examination of 
the relative sizes of the thin layer chromatography (TLC) spots of the 
attaching reaction products. 
The attaching reaction is preferably carried out at a temperature of at 
least about 10.degree. C. At a temperature below about 10.degree. C., the 
attaching reaction takes much longer to go to completion. More preferably, 
the attaching reaction is carried out at a temperature between about 
15.degree. C. and about 30.degree. C. Above about 30.degree. C. the 
reactants can begin to decompose. 
Additionally, the attaching reaction is preferably carried out for at least 
about 8 hours, and more preferably for between about 10 hours and about 60 
hours to ensure that the reaction has run essentially to completion. 
Preferably, the molar ratio of polymorphic analyte to binding moiety 
present at the beginning of the attaching reaction is at least about 
0.5:1. More preferably, this ratio is between about 0.5:1 and about 30:1, 
and most preferably between about 0.5:1 and about 5:1. In a particularly 
preferred embodiment, this ratio can be between about 1:1 and about 3:1. 
At a ratio of less than about 0.5:1 insufficient analyte is present to 
react efficiently with the binding moiety. When the ratio is above about 
30:1, the additional polymorphic analyte has an insignificant effect on 
the desired preconjugate yield. As these ratios approach equimolar ratios, 
the yield of the desired preconjugate can increase. Additionally, when the 
indicated molar ratios as used, the desired preconjugate can be obtained 
while conserving expensive reagents. Furthermore, the particular molar 
ratios specified has been found to result in immunoreactive conjugates 
with more reproducible and predictable immunoreactivity characteristics. 
The method can also include the step of activating the binding moiety in an 
activating reaction prior to the attaching reaction by mixing the binding 
moiety with a coupling reagent capable of attaching to and activating the 
binding moiety. When this step is carried out, the molar ratio of the 
binding moiety to the coupling reagent present at the beginning of the 
activating reaction is preferably at least about 1:0.9, and more 
preferably between about 1:1 and about 1:5. These molar ratios have been 
found to provide sufficient activated binding moiety for the attaching 
reaction step. When the ratio is greater than about 1:5, an excessive 
amount of coupling reagent which does not significantly contribute to 
binding moiety activation is in use. Most preferably, an excess of 
coupling agent over the binding moiety analyte of at least about 20% can 
be used to facilitate activating essentially all the binding moiety with 
an effective amount of the coupling reagent. 
A more detailed method for making a preconjugate comprising a binding 
moiety and an immunogenic species of a polymorphic analyte bound to the 
binding moiety, preferably has the steps of firstly activating a binding 
moiety by mixing the binding moiety with a coupling reagent capable of 
attaching to and activating the binding moiety. The next step is to react 
in an attaching reaction at a temperature between about 15.degree. C. and 
about 30.degree. C., for between about 10 hours and about 60 hours, the 
activated binding moiety and the polymorphic analyte comprising an 
immunogenic species and a nonimmunogenic species. The molar ratio of the 
polymorphic analyte to the activated binding moiety can be between about 
0.5:1 and about 30:1, and the molar ratio of the polymorphic analyte used 
in the attaching reaction to the coupling reagent used to activate the 
binding moiety can be between about 1:1 to about 1:5. The final step is 
separating preconjugate comprising the immunogenic species of the 
polymorphic analyte from preconjugate comprising the nonimmunogenic 
species. 
A method for making an aminoglycoside preconjugate, preferably has the 
steps of first activating a binding moiety in an activating reaction by 
mixing the binding moiety with a coupling reagent capable of attaching to 
and activating the binding moiety. The second step is to react in the 
attaching reaction at a temperature between about 15.degree. C. and about 
30.degree. C., for between about 10 hours and about 60 hours, the 
activated binding moiety and the polymorphic aminoglycoside. The 
polymorphic aminoglycoside comprises at least one immunogenic species and 
at least one nonimmunogenic species. The attaching reaction can result in 
a stoichiometric excess of the preconjugate comprising the immunogenic 
species of the polymorphic aminoglycoside. The molar ratio of the 
polymorphic aminoglycoside to the activated binding moiety can be between 
about 0.5:1 and about 30:1; the molar ratio of the binding moiety to the 
coupling reagent present at the beginning of the activating reaction can 
be between about 1:1 to about 1:5. The final step of the method is to 
separate the preconjugate comprising the immunogenic species of the 
polymorphic aminoglycoside from preconjugate comprising the nonimmunogenic 
species of the polymorphic aminoglycoside. The separation step removes 
substantially all of the preconjugate comprising the nonimmunogenic 
species of the polymorphic aminoglycoside from contact with the 
preconjugate comprising the immunogenic species of the polymorphic 
aminoglycoside. Thus, the separation step can yield essentially pure 
aminoglycoside preconjugate comprising the immunogenic species and useful 
for making an immunoreactive conjugate. The preconjugate comprising the 
immunogenic species of the polymorphic analyte can be contacted with a 
carrier molecule to make the immunoreactive conjugate. 
A suitable binding moiety can be made by joining a ligand to a spacer 
compound in a joining reaction. The ligand can be any small molecule 
(molecular weight less than about 7,000 Daltons) that is capable of 
undergoing a specific binding reaction with the carrier molecule. The 
ligand is a compound that is not identical to the analyte so that the 
analyte and the ligand have different specific binding partners. Thus, the 
ligand can be biotin, a hormone such as insulin, a steroid hormone, a 
thyroid hormone, a polypeptide, an oligonucleotide, a vitamin such as 
B.sub.12, or folic acid, a hapten such as 1-substituted-2, 
4-dinitrobenzene (also known as dinitrophenol, or DNP), digoxin or 
fluorescein. 
The carrier molecule is typically a large molecule (molecular weight 
greater than about 7,000 daltons) capable of undergoing a specific 
affinity binding reaction with the ligand. The carrier molecule can be a 
natural or synthetic macromolecule such as an antibody, avidin, an 
intrinsic factor, a lectin, or a complementary oligonucleotide. A 
preferred ligand-carrier molecule combination is biotin-avidin because of 
the ready available of these compounds and their suitability for use in 
the disclosed method. 
The spacer compound is interposed between an polymorphic analyte and the 
ligand, serving to spatially separate the analyte from the ligand. The 
spacer compound thereby functions to allow both the analyte and the ligand 
to simultaneously bind to their respective specific binding partners. 
Thus, the spacer compound connects the analyte to the ligand and regulates 
the ability of the analyte and ligand members of the preconjugate to 
simultaneously bind to their respective binding partners. Details 
regarding minimum, maximum, and preferred spacer compound lengths so as to 
enable simultaneous binding of two specific binding can be found in the 
copending U.S. patent application entitled "Novel Bidentate Conjugate and 
Method of Use Thereof", Ser. No. 07/536,058, filed Jun. 8, 1990, "(now 
U.S. Pat. No. 5,196,351, issued Mar. 23, 1993)", which application is 
incorporated herein in its entirety. 
Because the polymorphic analyte and the ligand portions of the preconjugate 
have different specific binding partners, the preconjugate can be referred 
to as a heterobifunctional preconjugate. 
Preferably, the binding moiety is activated by a coupling reagent prior to 
being reacted with the polymorphic analyte in the attaching reaction. An 
activated binding moiety can react more readily with the polymorphic 
analyte. The coupling reagent is preferably a dehydrating agent such as 
for example, carbonyldiimidazole (CDI), 1-ethyl-3-C3-dimethyl amino 
propyl(carbodiimide) (EDAC), dicyclohexylcarbodiimide (CPCC), or various 
suitable and known to the art phosphate compounds. 
The reaction medium can be a liquid capable of solubilizing the activated 
binding moiety, the polymorphic analyte, and the coupling reagent. In 
particular, an ability of the reaction medium to solubilize the binding 
moiety is an important characteristic of a suitable reaction medium. 
Suitable reaction media can include dimethylformamide, water, 
dimethysulfoxide, and various mixtures thereof. 
Where the polymorphic analyte selected is the aminoglycoside antibiotic 
amikacin, preferably the reaction medium contains a carbonate compound. 
Addition of a carbonate compound to the reaction medium was found to 
considerably facilitate work-up of the reaction products and isolation of 
the desired immunoreactive species amikacin preconjugate from other 
reaction products such as the nonimmunoreactive species amikacin 
preconjugate. More preferably the carbonate is a bicarbonate because a 
bicarbonate was found to be more effective than a carbonate. Most 
preferably, the carbonate is an alkali metal bicarbonate, such as sodium 
bicarbonate because such compounds are inexpensive, readily available, and 
have been found to assist isolation of the immunoreactive species amikacin 
preconjugate reaction product. 
Additionally, where the polymorphic analyte selected is the aminoglycoside 
antibiotic amikacin, we have found that a stoichiometric excess of 
preconjugate comprising the immunogenic species of the amikacin relative 
to the amount of preconjugate comprising the nonimmunogenic species of the 
amikacin is not generally obtained. 
The immunoreactive conjugate prepared from the preconjugate can be used as 
a developer antigen in a competitive inhibition immunoassay for a 
polymorphic analyte of interest. The immunoassay can be a photometric 
immunoassay such as, for example, a nephelometric or turbidimetric 
competitive inhibition immunoassay method. The consistent immunoreactivity 
of the conjugates prepared from the disclosed preconjugates were 
determined by standard competitive immunoassay procedures as set forth by 
the following examples.

EXAMPLES 
The following examples set forth illustrations of various features and 
embodiments of the present invention and are not intended to limit the 
scope of the claimed invention. In these Examples, all the preconjugates 
prepared were bidentate conjugates. 
Example 1 
(Preparation of benzyloxycarbonyl-6-aminohexanoic acid) 
Benzyloxycarbonyl-6-aminohexanoic acid used to make 
benzyloxycarbonyl-bis-6-amino hexanoic acid was prepared as follows. To a 
flask there was added a stir bar, 77.2 g (0.59M) of aminocaproic acid (98% 
pure, formula weight (FW) 131.18, melting point (MP) 210, freezing point 
(FP) 36.degree. C.) (Aldrich Chemical Co.) in 160 mL of water, and 90 mL 
of 6N sodium hydroxide. The solution was cooled in an ice bath to about 
5.degree. C. and maintained at that temperature while being stirred for 
the following steps. To the flask there was then added over 90 minutes, 84 
mL (100 g, 0.586 mM) of benzylchloroformate (95%, FW 170.60, FP 91.degree. 
C., density (d) 1.195, and n 1.5190) (Aldrich Chemical Co.) and 295 mL of 
2N sodium hydroxide. The benzylchloroformate in sodium hydroxide mixture 
was added to the flask in 10 equal portions of 8.5 mL of the 
benzylchloroformate followed by 29 mL of the sodium hydroxide. 
After addition of the tenth portion of 8.5 mL of the benzylchloroformate 
and 29 mL of the sodium hydroxide, the solution was stirred for one hour, 
then brought to room temperature and stirred for one more hour. A white 
solution with a pH of 9 was thereby formed. The solution was then poured 
into a 1 L beaker containing 200 g of ice. The pH of the solution was 
adjusted to pH 2 with 50 mL of concentrated hydrochloric acid (HCL). 
Sufficient water was then added so that the resulting solid white 
precipitate mass could be stirred, and the pH was brought back to pH 2 
with HCL. The white precipitate was filtered and washed with acidified 
water. The liquid filtrate was then acidified to precipitate additional 
white precipitate. 
The white precipitate was then resuspended in 1500 ml of water and 
triturated to break up lumps, followed by being stirred for 15 hours at 
room temperature. The white precipitate was then filtered, washed with 1.0 
L of water, and compressed on a Buchner funnel using the bottom of a 50 mL 
erlenmeyer flask. The white solid precipitate was then washed with 200 mL, 
100 mL, and 100 mL portions of hexane to remove water and 
benzylchloroformate, and then was dried. There was obtained 134.5 g 
(0.51M, 86% yield) of a white solid (mp 57.5.degree.-59.degree. C.), 
benzyloxycarbonyl-6-aminohexanoic acid. 
Example 2 
(Preparation of benzyloxycarbonyl-bis-6-aminohexanoic acid) 
Benzyloxycarbonyl-bis-6-aminohexanoic acid used to make 
benzyloxycarbonyl-tris-6-amino hexanoic acid was prepared as follows. 
Fifty-one point five grams (51.5 g) (0.196M) of 
benzyloxycarbonyl-6-aminohexanoic acid (FW 265.95) obtained following the 
procedure set forth in Example 1, was dissolved in 640 mL of toluene (ACS 
grade) and 20 mL (0.143M) of triethylamine (FW 101.19, BP 88.8.degree. C., 
FP 20.degree. C., d 0.726) (Aldrich Chemical CO.). The solution was then 
cooled to 0.degree. C. in an ice bath. While stirring the cold solution, 
21 mL (23.8 g, 0.219M) of ethyl chloroformate (FW 108.52, BP 93.degree. 
C., FP 36.degree. C., d 1.135) (Aldrich Chemical Co.) was added dropwise. 
The solution was then stirred for an additional 30 minutes, before being 
filtered to remove a white solid precipitate, triethylamine hydrochloride. 
To the filtered solution, cooled in the ice water bath, there was added 
over 45 minutes while stirring, 25.4 g (0.194M) of aminocaproic acid 
dissolved in 100 mL of cold 2N sodium hydroxide. The solution was then 
stirred for 1 hour at 0.degree. C., followed by being stirred for 2 hours 
at room temperature during which a white precipitate formed. The solution 
was then allowed to stand overnight at room temperature before decanting 
the toluene from the white solid precipitate. 
The white solid precipitate was then washed with 3 lots of 100 mL of ethyl 
ether. The white solid was then filtered and placed in acidic water pH 2, 
followed by being filtered, washed with hexane and dried to obtain 31 g 
(0.0818 mM, 41% yield, melting point 102.degree.-103.degree. C.) of a 
white solid, benzyloxycarbonyl-bis-6-aminohexanoic acid (FW 379.13). 
Silica gel thin layer chromatography of the white solid in 9/1 
chloroform/methanol gave a single spot, Rf 0.45. 
Example 3 
(Preparation of benzyloxycarbonyl-tris-6-aminohexanoic acid 
Benzyloxycarbonyl-tris-6-amino hexanoic acid used to make tris 
aminohexanoic acid was prepared as follows. Into a 500 mL round bottom 
flask there was placed 24 g (63.4 mM) of 
benzyloxycarbonyl-bis-6-aminohexanoic acid (FW 378.47) obtained by 
following the procedure of Example 2, a stir bar, 200 mL of dry 
tetrahydrofuran (THF) (FW 72.11, boiling point (BP) 67.degree. C., and 
density (d) 0.985) (Aldrich Chemical Co.), 60 mL of dry dimethylformamide 
(DMF), and 9 mL (6.534 g, 64.69 mM) of triethylamine (FW 101.19, BP 
89.degree. C., d 0.726) (Aldrich Chemical Co.) 
A drying tube was attached, the mixture was stirred to dissolve the solids, 
and then placed in a salt/ice bath to cool to -5.degree. C. Six mL (6.810 
g, 62.75 mM) of ethyl chloroformate (FW 108.52, BP 93.degree. C., d 1.135) 
(Aldrich Chemical Co.) was then added to the flask, stirred and the 
mixture was allowed to incubate for 15 minutes at -5.degree. C. The 
mixture was then filtered, and to the filtrate there was added over a 
period of 15 minutes, 16.6 g (126.54 mM) of aminocaproic acid (FW 131.18, 
MP 210.degree. C.) (Aldrich Chemical Co.), dissolved in 69 mL of 2N cold 
sodium hydroxide. The mixture was then stirred for 15 minutes at 
-5.degree. C., followed by being stirred for another 15 minutes at room 
temperature. The solvent was then removed completely by rotary 
evaporation. 
The remaining solid was transferred using water to a beaker and acidified 
to pH 2 using concentrated HCL to obtain a white solid. This solid was 
filtered and washed with water. While the solid was wet, it was 
recrystallized from methanol, and cooled overnight at 4.degree. C. The 
solid was then filtered and dried to obtain 17.54 g (58% yield) of a white 
solid, benzyloxycarbonyl-tris-6-aminohexanoic acid. Silica gel thin layer 
chromatography was used to confirm the presence of the single end product. 
Example 4 
(Preparation of Tris Aminohexanoic Acid) 
Tris aminohexanoic acid used to make biotin hexanoic acid was prepared as 
follows. Three grams (6.1 mM) of benzyloxycarbonyl-tris-6-amino hexanoic 
acid (FW 491.6), obtained by following the procedure of Example 3, was 
dissolved in 150 mL of methanol in a 500 mL round bottom flask with a stir 
bar. The flask was then flushed with nitrogen. Two spatulas full or about 
one half gram of 5% palladium catalyst on activated charcoal (Kodak or 
Aldrich Chemical Co.) was then added to the flask. The flask was then 
flushed with hydrogen gas at atmospheric pressure and room pressure. After 
being stirred for 3.5 hours, a silica gel thin layer chromatograph, in 8/2 
chloroform/methanol, was prepared and visualized with UV, iodine, and 
ninhydrin spray. The TLC showed disappearance of the starting material 
benzyloxycarbonyl-tris-6-amino hexanoic acid (Rf 0.6), and appearance of 
the amine product. 
After being stirred for a total of 4 hours, the solution in the flask was 
flushed with nitrogen, heated to a temperature sufficient to dissolve the 
solids, and then filtered through a Whatman #1 filter (Whatman Co.) with 
diatomaceous earth to remove the palladium catalyst. The solution was then 
concentrated by rotary evaporation to 30 mL and combined with 30 mL of 
ethyl ether until the solution become cloudy and was placed in a cold room 
at 4.degree. C. overnight. Silica gel thin layer chromatography, in 1/1 
chloroform/methanol with 4% ammonium hydroxide, and visualized with iodine 
and ninhydrin spray. The TLC gave a single spot, Rf 0.3. The solution was 
then filtered to obtain 2.1 g (5.39 mM) (89% yield) of a white solid, tris 
aminohexanoic acid (FW 389.6). 
Example 5 
(Preparation of Biotin Tris-Amino-Hexanoic Acid) 
A biotin hexanoic acid derivative capable of being covalently linked to an 
aminoglycoside analyte, such as gentamicin, was synthesized and purified 
as follows. Into a round bottom flask there was placed 1252 mg (5.13 mM) 
of biotin (FW 244) and 60 mL of DMF with a magnetic stir bar. A drying 
tube was attached and the flask was heated in an oil bath for 15 minutes 
at a temperature between 70.degree. and 75.degree. C. To the flask there 
was then added 923 mg (5.70 mM) of 1,1'-carbonyldiimidazole (CDI) (FW 
162), followed by stirring and incubation for 30 minutes at a temperature 
between 70.degree. and 75.degree. C. 
The reaction mixture was then cooled to room temperature before adding 656 
mg (5.70 mM) of N-hydroxysuccinimide (NHS) (FW 115) to the flask. The 
mixture was then stirred for 18 hours at room temperature to obtain an 
activated biotin solution. Two grams (5.13 mM) of tris-aminohexanoic acid 
(FW 389.6), obtained by following the procedure of Example 4, was then 
dissolved in 60 ml of 0.2M sodium bicarbonate and added to the activated 
biotin solution. Besides tris-aminohexanoic acid, many longer or shorter 
chain organic acids can be prepared and used depending upon the desired 
steric distancing of an analyte from a carrier molecule in the final 
analyte-conjugate. 
The reaction was then allowed to proceed overnight, followed by adjustment 
of the reaction mixture to pH 2 by addition of 6M HCL and filtration of 
the solid reaction product to remove fluid. The solid obtained was 
triturated with 100 mL of 0.6N HCL, filtered, recrystallized from methanol 
and dried to obtain 2.8 g (89% yield) of a white solid, biotin 
tris-hexanoic acid (FW 615). Biotin tris-hexanoic acid is a binding moiety 
useful for making a preconjugate according to the method set forth in 
detail below. This particular binding moiety has an 18 carbon atom spacer 
chain. 
Silica gel thin layer chromatography, in 8/2 chloroform/methanol and 
visualized with iodine and ninhydrin spray, was used at various stages of 
the synthesis to show disappearance of the starting amine and appearance 
of the biotin acid derivative. 
Example 6 
(Preparation of a Biotinylated Gentamicin Preconjugate) 
A. A gentamicin-biotin preconjugate capable of undergoing a specific 
affinity binding reaction with avidin was prepared as follows. The 
abbreviation "mM" as used in this and in other Examples means millimole or 
one thousandth of a mole. Into a 25 mL round bottom flask there was placed 
100 mg (0.172 mM) of the biotin tris aminohexanoic acid binding moiety 
prepared by following the procedure of Example 5, dissolved in 15 mL of 
DMF (anhydrous 99%+, gold label, FW 73.10, BP 153.degree. C., d 0.945) 
(Aldrich) by application of heat. The reaction flask was then placed in an 
oil bath at 70.degree. to 75.degree. C. for 15 minutes. There was then 
added to the flask 30 mg (0.185 mM) of 1,1'carbonyldiimidazole (CDI) (MW 
162.2) (Sigma Chemical Co.), as the coupling reagent to activate the 
binding moiety. The temperature of the reaction solution was maintained at 
70.degree. to 75.degree. C. for 30 minutes, followed by being cooled to 
room temperature. 
To the reaction flask there was then added 20 mg (0.172 mM) of 
N-hydrosuccinimide (NHS) (97%, FW 115.09) (Aldrich), followed by stirring 
overnight at room temperature. The next step in the synthesis was dissolve 
71 mg (0.0855 mM) of gentamicin sulfate, potency: 591 .mu.g gentamicin per 
mg of gentamicin sulfate, 9.4% water) (Sigma) in 3 mL of water in a test 
tube. One hundred milligrams of sodium bicarbonate (FW 84.01) 
(Mallinckrodt) was then slowly added to the gentamicin sulfate/water 
solution in the tube. After the bubbling stopped, another 100 mg of sodium 
bicarbonate was added to the gentamicin solution in the tube. 
The gentamicin solution was then added to the activated biotin in the 
reaction flask to initiate an attaching reaction between the activated 
binding moiety and the gentamicin. Eight mL of water in 1 mL increments 
was then added to the reaction flask until the reaction solution became 
clear. The reaction solution was then stirred for 15 hours at room 
temperature. In another experiment the reaction solution was stirred at 
room temperature for 60 hours with equivalent results. The reaction 
solution was then evaporated to dryness and the remaining white residue 
was triturated with methanol and filtered. 
The filtrate was evaporated to dryness and the remaining solid residue was 
dissolved in 5 mL of methanol, followed by placement on a 1 cm by 30 cm 
column chromatography column of cellulose packed as a methanol slurry. The 
column was then eluted with 75 mL of methanol, followed by 100 mL of 
methanol/5% ammonium hydroxide, followed by 100 mL methanol/10% ammonium 
hydroxide. Progress of the column was followed by TLC of column elution 
fractions in methanol. Selected fractions were pooled, and evaporated to 
dryness to obtain 14.5 mg of a gentamicin-biotin preconjugate ready for 
conjugation to avidin. 
The molar ratio of gentamicin (polymorphic analyte) to biotin tris 
aminohexanoic acid (binding moiety) to CDI (coupling reagent) to NHS (the 
G:B:CDI:NHS ratio) at the beginning of the attaching reaction to form the 
preconjugate was 1:2:2.2:2. 
B. A second gentamicin-biotin preconjugate capable of undergoing a specific 
affinity binding reaction with avidin was prepared as follows. The 
relative molar ratios of gentamicin:biotin (or biotin tris aminohexanoic 
acid):CDI used were 29:1:1.2. Ten milligrams (0.017 mM) of biotin tris 
aminohexanoic acid was dissolved in 10 mL of DMF as the reaction medium, 
by being warmed in an oil bath at 70.degree. C. for 10 minutes. This was 
followed by addition of 3.5 mg (0.0216 mM) of CDI (MW 162) as the coupling 
reagent and, stirring in the oil bath for 30 minutes, and then by being 
stirred at room temperature for 1 hour. NHS (2.5 mg, 0.0217 mM, MW 115) 
was then added and the reaction mixture was stirred overnight. The next 
step was to slowly add 234 mg (0.50 mM) of gentamicin (MW 462) in 20 mL of 
dry DMF, to the reaction mixture followed by stirring overnight. The 
solvent was then evaporated. 
The remaining solid residue was dissolved in a minimum amount of methanol 
and loaded onto a silica gel column, 1 cm by 30 cm packed with 7 g of 
silica gel. Methanol followed by 10% ammonium hydroxide/methanol was used 
as the eluent, to obtain 0.134 g of a second preconjugate. 
C. A third gentamicin-biotin preconjugate capable of undergoing a specific 
affinity binding reaction with avidin was prepared as follows. The 
relative molar ratios of gentamicin:biotin (or biotin tris aminohexanoic 
acid):CDI used were 1.3:1:1.2. To a round bottom flask equipped with a 
drying tube there was added 2 g (3.44 mM) of biotin tris aminohexanoic 
acid dissolved in 150 ml of dry DMF. The flask was then placed in an oil 
bath at 70.degree. to 75.degree. C. for 15 minutes. As coupling reagent, 
667 mg (4.12 mM) of CDI was added, followed by stirring at 75.degree. C. 
for 30 minutes. 
After cooling to room temperature, 470 mg (4.10 mM) of NHS was added to the 
flask, and the reaction was allowed to proceed for 20 hours. Gentamicin in 
an amount of 2.134 g (4.16 mM) was then dissolved in 100 mL of dry DMF. 
The activated biotin was then added through a separating funnel to the 
gentamicin/DMF solution over a period of 30 minutes. This was followed by 
stirring for 24 hours at room temperature. 
After evaporation of the solvent under reduced pressure, the residue was 
dissolved in a small quantity of methanol, and loaded onto the top of a 
2.5 cm by 60 cm column containing 70 g of silica gel packed as a methanol 
slurry. The excess, unreacted biotin was eluted using 1400 mL of methanol. 
The column was then eluted with 5% ammonium hydroxide in methanol. Column 
fractions were monitored using TLC (5% NH.sub.4 OH/CH.sub.3 OH). Fractions 
exhibiting a positive reaction to cinnamaldehyde spray were pooled to give 
1.5 g of a third gentamicin-biotin preconjugate. 
For all the gentamicin-biotin preconjugates prepared as set forth above, it 
was clear that the reaction between the activated biotin ester and the 
gentamicin resulted in an excess of the amount of the gentamicin 
preconjugate that could be used to make an immunoreactive gentamicin 
conjugate (i.e. biotin binding moiety joined to an immunoreactive species 
of the gentamicin), as compared to the amount of gentamicin preconjugate 
that could not be used to make an immunoreactive gentamicin conjugate 
(i.e. biotin binding moiety joined to a nonimmunoreactive species of the 
gentamicin). 
Thus, for example, a visual inspection of the relative size of the TLC 
reaction product spots, showed that the spot of immunoreactive gentamicin 
species preconjugate was larger than the TLC spot of the nonimmunoreactive 
gentamicin species preconjugate. Examination of the relative size of the 
TLC spots (followed by conjugation with avidin of at least the major 
product (large TLC spot) and subsequent immunoreactivity study) showed 
that the yield of the desired preconjugate was in excess as compared to 
the yield of the undesired preconjugate. Specifically, for the reaction 
parameters specified the relative area of the immunoreactive gentamicin 
species preconjugate TLC spot:nonimmunoreactive gentamicin species 
preconjugate TLC spot varied from about 2:1 to about 5:1. 
Gentamicin:biotin ratios of 5:1, 10:1, 15:1. 20:1, 25:1, and various 
intermediate concentration ratios can be used in the method set forth 
above. It can be reasonably expected that the reaction products obtained 
by such alternate analyte:binding moiety ratios would yield preconjugates 
that could be used to make immunoreactive conjugates. 
Additionally, the gentamicin (polymorphic analyte):CDI (coupling reagent) 
ratios can, it is reasonably postulated, be varied to any ratio between 
about 0.5:1 and about 30:1 with results comparable to those set forth 
above. 
Example 7 
(Preparation of Gentamicin-Biotin-Avidin Conjugates) 
I. A gentamicin-biotin-avidin conjugate useful as an inhibitor in 
competitive inhibition immunoassays for gentamicin was prepared as 
follows. Into a 50 mL tube there was placed 203.1 mg of avidin dissolved 
in 20 mL of 0.1M phosphate buffer, pH 7.4, with 100 .mu.L of HABA. Next, 
21 mg of the preconjugate made by following the procedure of Example 6A. 
above, in methanol, was added to the avidin solution in 200 .mu.L aliquots 
(the color changed from pink-orange to light yellow), and allowed to stand 
for 1 hour. The mixture was then transferred to a dialysis bag (6.4 mm, 
12,000-14,0000 MW cutoff) using 1 mL of citrate buffered saline (CBS), pH 
6.0. Dialysis was carried out at 4.degree. C. in CBS, pH 6.0 at a volume 
of 2000 mL, with 5 changes over 3 days to recover 29 mL of an 
immunoreactive gentamicin-(C.sub.18,N.sub.3)-biotin-avidin conjugate. 
II. A second gentamicin-biotin-avidin conjugate was prepared by dissolving 
0.134 of the preconjugate made by following the procedure of Example 6B. 
above in 5 ml of methanol. Twenty milligrams of avidin (Boehringer Manhein 
GmbH) was then dissolved in 1 mL of phosphate buffer, 0.1M, pH 7.4, with 
20 .mu.L of 2-(4 hydroxyphenylazo) benzoic acid indicator. The 
preconjugate solution was then added to the avidin solution in 50 .mu.L 
portions, until 450 .mu.L had been added. The color changed from 
pink-orange to light yellow. The mixture was then left at room temperature 
for 1 hour, followed by dialysis against 500 mL of 0.05M phosphate buffer, 
pH 7.4. Four changes of the buffer were made over a two day period, as the 
dialysis continued. 
III. A third gentamicin-biotin-avidin conjugate was prepared by dissolving 
1.4 g of the preconjugate made by following the procedure of Example 6C. 
above in 10 ml of methanol. One hundred milligrams of avidin (Boehringer) 
was then dissolved in 5 mL of phosphate buffer, 0.1M, pH 7.4, followed by 
addition of 100 .mu.L of 2-(4 hydroxyphenylazo) benzoic acid indicator. A 
100 .mu.L portion, followed by a 50 .mu.L portion of the preconjugate 
solution was then added to the avidin solution. Five mL of the phosphate 
buffer was then added, followed by being left at room temperature for 1 
hour. The mixture was then left at room temperature for 1 hour, followed 
by dialysis against 500 mL of 0.05M phosphate buffer, pH 7.4. Four changes 
of the buffer were made over a two day period, as the dialysis continued. 
Example 8 
(Preparation of Monoclonal Antibodies to Gentamicin) 
Hybridomas capable of making monoclonal antibody to two forms or species of 
gentamicin were prepared. The materials used were as follows. The myeloma 
cells used were derived from the P3X63-Ag8.653 myeloma line, a 
non-secreting mouse myeloma line developed by Kearney et al., J. Immunol., 
123:1548 (1979). The spleen cells used were taken from Balb/c mice 
immunized by the procedure below. The growth media was DME low glucose 
(Irvine Scientific), supplemented with 10% fetal calf serum (Hyclone), and 
2 mM 1-glutamine (Irvine Scientific). The used media was growth media from 
a three day culture of 653.1 cells, centrifuged and filtered to remove 
cells. The CHAT Media was 50% growth media and 50% conditioned media with 
100 units/ml of penicillin-streptomycin solution (irvine Scientific), 
4.times.10.sup.-7 M aminopterin (Sigma), 1.times.10.sup.-4 M hypoxanthine 
(MA Bioproducts), 1.6.times.10.sup.-5 M thymidine (MA Bioproducts), and 10 
units/ml insulin (Eli Lily). The conditioned media was 50% growth 
media-50% used media and 2.5.times.10.sup.-5 M b-mercaptoethanol (Sigma). 
Polyethylene glycol (PEG) with a molecular weight between about 1300 and 
1600 (Sigma) was used. The injection media was DME low glucose with 100 
units/ml penicillin-streptomycin solution. One-half milliliter of Pristane 
(2,6,10,14-tetramethylpentadecane, available from Aldrich) was injected 
intraperitoneally into each Balb/c mouse two weeks prior to hybridoma 
injection. 
The hybridomas were made using the method developed by Kohler and Milstein, 
Nature 256:495 (1975). The spleen from the immunized mouse was aseptically 
removed after cervical dislocation and was ground in a tissue sieve until 
a single-cell suspension was obtained. After washing, the cells were mixed 
with the washed 653.1 myeloma cells in a 2:1 ratio of spleen to myeloma 
cells and then pelleted. The supernatant was removed and the PEG added 
slowly over one minute. PBS was added to bring the total volume to 22 ml 
and the cells were then pelleted for 8 minutes after the start of PEG 
addition. The pellet was resuspended in 200 ml of CHAT media and 0.2 ml of 
the suspension was added to each well of ten 96-well microtiter plates. 
The wells were supplied with fresh CHAT on days 6 and 7 post fusion. 
Testing of the wells for growth using radioimmunoassay (RIA) began on day 
10 and continued over the next 3-4 days. Wells with a count greater than 
the negative control were retested on the following day. If the reading 
remained greater than the negative control on the second day of testing, 
the colony was considered positive and was cloned. Cloning was carried out 
by limiting dilution in conditioned media into two 96-well plates, one 
with 5 cell/well and one plate with 1 cell/well. One week after cloning, 
single colony wells were tested by RIA. If all wells tested positive, the 
line was considered pure and was recloned a second time for stability. If 
all the wells did not test 100% positive, a positive well was used for the 
second cloning. The plates were tested again 7 days after the cloning. 
This procedure was repeated until all the clones tested 100% positive. The 
cells were then expanded in growth media and injected in injection media 
into the peritoneal cavity of Pristane-primed Balb/c mice at a 
concentration of about 3.times.10.sup.6 hybridoma cells per mouse. 
Prior to injection, supernatant from the cultured cells was used for 
isotyping by the Ouchterlony gel diffusion method, Acta Path Microbiol 
Scand 26:507 (1949). Ascites fluid was harvested from the mice about 10 
days after the mice had been injected with the hybridoma cells. The 
ascites fluid was then titered by RIA and the IgG isotype content was 
measured using a Beckman ICS rate nephelometer. 
The immunization protocol for generation of a Gent 3B1 monoclonal antibody 
was as follows. A female Balb/c mouse was injected intraperitoneally with 
20 .mu.g of the gentamicin BSA antigen in Freund's complete. One month 
later, 20 .mu.g of gentamicin BSA was injected intravenously. Two weeks 
after that, 20 .mu.g of gentamicin BSA was given in a combination of 
intravenous and intraperitoneal injection. Three days thereafter, the 
immunized mouse's spleen was removed and fusion was performed. The 
hybridomas so prepared were capable of producing monoclonal antibody with 
a specific affinity for gentamicin. 
The immunization protocol for generation of GV AS5 monoclonal antibody was 
as follows. A female Balb/c mouse was injected intravenously with 1 .mu.g 
of the gentamicin BSA antigen in Freund's complete. On day three, the 
mouse was injected with 139 .mu.g of gentamicin BSA intravenously. On day 
four, the mouse was injected with 130 .mu.g of gentamicin BSA 
intravenously. On day five, 139 .mu.g of the gentamicin was injected 
intravenously. On day six, 139 .mu.g of the gentamicin was again injected 
intravenously. On day seven, the immunized mouse's spleen was removed and 
fusion was carried out. The hybridomas so prepared were capable of 
producing monoclonal antibody with a specific affinity for gentamicin. 
Two different monoclonal antibodies against gentamicin were prepared 
because gentamicin exists in several similar but not identical chemical 
species or isomers. Thus, an assay against gentamicin that uses monoclonal 
antibodies against two species of gentamicin permits a more accurate 
quantification of the amount of total gentamicin present. 
Example 9 
(Immunoreactivity of the Gentamicin-Biotin-Avidin Conjugate with a 
Gentamicin Antiserum) 
The immunoreactivity of the gentamicin-biotin-avidin conjugates, obtained 
by following the procedure of Example 7, with a gentamicin antiserum was 
measured as follows. The gentamicin-biotin-avidin conjugate solutions were 
diluted in ICS.TM. diluent (Beckman) with 0.1% BSA to obtain five 
dilutions with 0.5, 0.4, 0.3, 0.2, and 0.1 mg/mL of avidin respectively. 
The ascites fluid containing anti-gentamicin monoclonal antibody obtained 
by following the procedure of Example 8 was filtered and diluted. Next, 
the gentamicin antiserum was diluted in the ICS diluent to obtain five 
antiserum dilutions of 1/5, 1/10, 1/15, 1/17.5, and 1/20 respectively. 
The immunoreactivity assay was carried out on a ICS.TM. manual nephelometer 
(Beckman). The results obtained are shown in Table 1 below, indicating the 
clear and significant immunoreactivity of the gentamicin-biotin-avidin 
conjugates prepared with the gentamicin monoclonal antibody in the 
antiserum. OR indicates an over or out of instrument range reading. 
TABLE 1 
______________________________________ 
Absorption Rate Units Upon Cross-Titering a 
Prepared Gentamicin-Biotin-Avidin Conjugate with a 
Gentamicin Monoclonal Antibody Containing Antiserum 
Conj. Conc. Antiserum Conc. 
(mg/mL) 1/10 1/15 1/17.5 
1/20 
______________________________________ 
0.5 OR 3230 2530 2070 
0.4 OR 3330 2530 2040 
0.3 OR 3190 2645 2090 
0.2 3140 2940 2790 2350 
0.1 1230 1070 925 1010 
______________________________________ 
Example 10 
(Use of the Gentamicin-Biotin-Avidin Conjugate in a Competitive Inhibition 
Immunoassay with Known Amounts of Gentamicin) 
Photometric immunoassays for gentamicin were carried out using the 
conjugates prepared by following the procedure of Example 7, using known 
amounts of gentamicin. Six calibrators with known amounts of gentamicin 
(0, 1, 2, 4, 8, and 12 .mu.g of gentamicin per mL) were prepared. A 
Synchron CX.RTM. 5 clinical analyzer (Beckman) was used to measure the 
change in liquid medium turbidity as the competitive inhibition 
immunoassay reaction took place. 
The rate of change of the cuvette liquid medium turbidity over time upon 
addition of the prepared immunoreactive conjugate was measured for each 
calibrator. The rate signals were plotted on a vertical axis against the 
gentamicin concentrations of the calibrators on the horizontal axis. 
Automatic comparison by the Synchron CX.RTM. 5 clinical analyzer of such 
calibration values obtained, with the initial rate of change of cuvette 
liquid medium turbidity caused by an unknown amount of gentamicin in a 
sample, permitted detection and quantification of the amount of gentamicin 
present per unit volume of the test sample. 
The photometric rate signals detected were plotted on a vertical axis 
against the gentamicin concentrations of the six calibrators on the 
horizontal axis to establish a plot of light attenuation versus gentamicin 
concentration. The results obtained showed that the 
gentamicin-biotin-avidin conjugate prepared is useful for the detection 
and quantification of gentamicin in a competitive inhibition immunoassay. 
Example 11 
(Use of the Gentamicin-Biotin-Avidin Conjugate in a Competitive Inhibition 
Immunoassay with Unknown Amounts of Gentamicin) 
The preconjugates prepared by the procedure set forth in Example 7 were 
used in separate competitive inhibition immunoassays carried out on a 
Synchron CX.RTM. 4 clinical analyzer (Beckman) to determine the amount of 
unknown gentamicin in serum samples from 51 different patients. In the 
immunoassay, 0.30 mg/ml of the conjugate was used. 
The assay was repeated on the same 51 patient samples and compared with the 
results obtained on the same patient samples using: (1) a Synchron.RTM. 
turbidimetric clinical analyzer (Beckman) with a gentamicin immunoassay 
kit having a different gentamicin conjugate present; (2) an Array.TM. 
automated nephelometric analyzer, and; (3) an Abbott TDX.TM. florescent 
polarimization immunoassay (FPIA) instrument. The monoclonal antibody used 
for the immunoassays was the GVAS5 monoclonal antibody, diluted 1:4. 
Example 12 
(Preparation of Immunoreactive Tobramycin-Biotin-Avidin Conjugates) 
A. Immunoreactive tobramycin-biotin-avidin conjugate were prepared as 
follows. Biotin tris aminohexanoic acid (10 mg, 0.017 mM, MW 582) was 
dissolved in a flask with 2 ml of dry DMF by being warmed in an oil bath 
at 75.degree. C., following by being kept at this temperature for a 
further 10 minutes. CDI (3.5 mg, 0.0216 mM, MW 162) was then added to the 
flask, and the 75.degree. C. temperature was maintained for a further 30 
minutes, followed by stirring at room temperature for 2 hours. NHS (2.5 
mg, 0.0217 mM, MW 115) was then added to the flask, and the room 
temperature stirring continued overnight. 
Tobramycin (24 mg, 0.051 mM, MW 467.5) was then dissolved in 5 mL of dry 
DMF, and the activated biotin was added dropwise to the dissolved 
tobramicin with stirring at room temperature. The room temperature 
stirring was continued overnight. The solvent was then evaporated to 
dryness. The remaining solid residue was dissolved in a minimum amount of 
methanol, placed on a 1 cm by 30 cm chromatography column packed with a 
silica gel methanol slurry, and eluted with methanol and methanol/10% 
ammonium hydroxide. The appropriate fractions were collected, as 
determined, by TLC, to obtain 10 mg of a tobramycin-biotin preconjugate. 
In this experiment the molar ratios of tobramycin to biotin to CDI/NHS used 
were 3:1:1.3. 
The conjugate was then made by dissolving 50 mg of avidin in 2.5 mL of pH 
7.4 phosphate buffer, 0.1M. HABA was used as previously set forth, to 
determine formation of the tobramicin-biotin-avidin conjugate. The 
conjugate was dialyzed against CBS with six changes of the buffer. 
B. A second tobramycin-biotin-avidin conjugate was prepared as set forth 
above in this Example, but using molar ratios of tobramicin to biotin to 
CDI/NHS of 30:1:1.3. 
An anti-tobramycin goat polyclonal antibody was used to determine the 
immunoreactivity and usefulness of the two conjugates prepared in 
competitive inhibition immunoassays for unknown amounts of tobramycin in 
test samples. Standard and calibration curves were established. It was 
determined that the two prepared tobramycin conjugates were both 
immunoreactive and suitable for use in immunoassays for tobramycin. 
C. A third tobramycin-biotin-avidin conjugate was prepared as follows. The 
biotin tris aminohexanoic acid binding moiety (415 mg, 0.71 mM) was 
dissolved in a round bottom flask with a drying tube and 45 ml of dry DMF 
by being warmed in an oil bath at 70.degree. to 75.degree. C., following 
by being kept at this temperature for a further 15 minutes. CDI (140 mg, 
0.86mM) was then added to the flask, and the 75.degree. C. temperature was 
maintained for a further 30 minutes with stirring. After being cooled to 
room temperature, NHS (97 mg, 0.84 mM) was then added to the flask, an d 
the room temperature stirring of the solution continued for 20 hours. 
Tobramycin (1000 mg, 2.14 mM) was then dissolved in 50 mL of 0.5M sodium 
bicarbonate, and the activated biotin was added through an addition funnel 
to the dissolved tobramicin over a period of 30 minutes with stirring at 
room temperature. The room temperature stirring was then continued for 24 
hours. The solvent was then evaporated to dryness under reduced pressure. 
The remaining white solid residue was (3.45 g) was extracted twice with 
100 mL of hot methanol (100 ml for each extraction), filtered, and 
evaporated to obtain 3.19 g of a white solid. 
The 3.19 g of white solid was dissolved in 50 ml of methanol and absorbed 
into 5 g of silica gel. After removal of the solvent under reduced 
pressure, the silica gel was transferred to the top of a silica gel column 
(2.5 cm by 60 cm) containing 70 g of silica gel packed as a methanol 
slurry. The column was then eluted with 700 ml of m ethanol to remove 
excess biotin tris aminohexanoic acid, followed by elution with 600 ml of 
methanol with 10% ammonium hydroxide. Column fractions were monitored by 
TLC using methanol with 10% ammonium hydroxide, and fractions judged 
appropriate by positive reaction to cinnamaldehyde spray, were pooled to 
yield 370 mg of a third preconjugate. Conjugation to avidin was carried 
out as previously set forth, followed by a determination by the methods 
already given, that the third conjugate was also immunoreactive and 
suitable for use in a competitive inhibition immunoassay for tobramycin. 
The molar ratios of tobramycin to biotin to CDI/NHS used to prepare the 
third preconjugate were 3:1:1.2. 
For all the tobramycin-biotin preconjugates prepared as set forth above, it 
was clear that the reaction between the activated biotin ester and the 
tobramicin resulted in an excess of the amount of the tobramicin 
preconjugate that could be used to make an immunoreactive tobramicin 
conjugate (i.e. biotin binding moiety joined to an immunoreactive species 
of the tobramicin), as compared to the amount of tobramicin preconjugate 
that could not be used to make an immunoreactive tobramycin conjugate 
(i.e. biotin binding moiety joined to a nonimmunoreactive species of the 
tobramycin) 
Thus, for example, a visual inspection of the relative size of the TLC 
spots of reaction product, showed that the spot of immunoreactive 
tobramycin species preconjugate was larger than the TLC spot of the 
nonimmunoreactive tobramycin species preconjugate. Examination of the 
relative size of the TLC spots (followed by conjugation with avidin of at 
least the major product (large TLC spot) and subsequent immunoreactivity 
study on at least the major product) showed that the yield of the desired 
preconjugate was in excess as compared to the yield of the undesired 
preconjugate. Specifically, for the reaction parameters specified the 
relative area of the immunoreactive tobramycin species preconjugate TLC 
spot:nonimmunoreactive tobramycin species preconjugate TLC spot varied 
from about 2:1 to about 5:1. 
Tobramycin:biotin ratios of 5:1, 10:1, 15:1. 20:1, 25:1, and various 
intermediate concentration ratios can be used in the method set forth 
above. It can be reasonably expected that the reaction products obtained 
by such alternate analyte:binding moiety ratios would yield preconjugates 
that could be used to make immunoreactive conjugates. 
Additionally, the tobramycin (polymorphic analyte):CDI (coupling reagent) 
ratios can, it is reasonably postulated, be varied to any ratio between 
about 0.5:1 and about 30:1 with results comparable to those set forth 
above. 
Example 13 
(Preparation of a Biotinylated Amikacin Preconjugate) 
A biotinylated amikacin preconjugate was prepared as follows. Biotin tris 
aminohexanoic acid activated with CDI/NHS in DMF, was reacted overnight 
with amikacin dissolved in 0.5M sodium bicarbonate, followed by elution of 
the preconjugate by column chromatography. 
Preconjugates so prepared were conjugated to avidin and were found to 
exhibit similar immunoreactivities towards anti-amikacin antibodies and 
were determined to be useful as inhibitors in competitive inhibition 
immunoassays for amikacin in serum test samples. 
Several amikacin-biotin preconjugates suitable for preparing immunoreactive 
conjugates were made by activating a biotin binding moiety with CDI/NHS in 
DMF followed by addition of amikacin in 0.5M NaHCO.sub.3. The coupling of 
amikacin and biotin was carried out overnight. The molar ratios of the 
reactants used was amikacin:biotin:CDI/NHS 5:1:1.2. Column chromatography 
as carried out using a 10% NH.sub.4 OH/MeOH elution. 
Amikacin was reacted with a biotin tris aminohexanoic acid binding moiety 
to prepare an amikacin preconjugate as follows. 
Biotin tris aminohexanoic acid binding moiety (MW 582, 1 g, 1.72 mM) was 
dissolved in 100 ml dry DMF by warming at 75.degree. C. CDI (334 mg, 2.08 
mM) was then added and stirred at this temperature for 30 minutes, 
followed by stirring at room temperature for 2 hours. Then NHS (235 mg, 
2.04 mM) was then added and stirring was continued overnight at room 
temperature. 
Amikacin (MW 585, 5 g, 8.55 mM) was dissolved in 80 mL of 0.5M NaHCO.sub.3 
and the activated biotin ester was then added slowly through a funnel. 
After 15 minutes 40 mL of 0.5M NaHCO.sub.3 was added along with 10 mL of 
water. Stirring was then continued overnight at room temperature. 
The solvent was then evaporated completely to obtain 12.15 g of a solid. 
This solid was extracted with 200 ml MeOH (100 ml each) heated and 
filtered. The filtrate was evaporated until dried to obtain 6.29 g of a 
solid. The solid was dissolved in methanol and adsorbed onto 8.5 g of 
silica gel and was loaded on a silica gel column packed in MeOH. Eluted 
fractions were followed by TLC. Appropriate fractions were pooled and 
evaporated to obtain 570 mg of the desired amikacin-biotin preconjugate. 
The ratio of amikacin:biotin:CDI:NHS used as 5:1:1.2:1.2 
The experiment set forth immediately above was repeated using an 
amikacin:biotin:CDI:NHS ratio of 0:1:1:3:1.3, and replacing the sodium 
bicarbonate by DMF. The column eluent used as 10% ammonium hydroxide 
(NH.sub.4 OH) in methanol. It was found that not using a carbonate such as 
sodium bicarbonate made the amikacin preconjugate reaction product more 
difficult to isolate. 
Amikacin:biotin ratios of 5:1, 10:1, 15:1. 20:1, 25:1, and various 
intermediate concentration ratios can be used in the method set forth 
above. It can be reasonably expected that the reaction products obtained 
by such alternate analyte:binding moiety ratios would yield preconjugates 
that could be used to make immunoreactive conjugates. 
Additionally, the amikacin (analyte):CDI (coupling reagent) ratios can, it 
is reasonably postulated, be varied to any ratio between about 0.5:1 and 
about 30:1 with results comparable to those set forth above. 
Example 14 
(Conjugation of an Amikacin-Biotin Preconjugate to Avidin) 
100 mg avidin (Boehringer Manheim GmbH) was dissolved in 5 mL of phosphate 
buffer, 0.1M, pH 7.4. HABA was used as a color indicator. 580 mg of the 
amikacin preconjugate dissolved in 25 ml of 1:1 MeOH/H.sub.2 O, was added 
in 100 .mu.L aliquots, for a total of 600 .mu.L. The color of the solution 
changed from pink-orange to light yellow. 5 ml of phosphate buffer was 
then added and the solution was dialyzed against CBS with three changes. 
The disclosed method for making immunoreactive conjugates has many 
advantages, including the following: 
1. A consistently high yield of polymorphic analyte preconjugates useful 
for preparing immunoreactive conjugates can be obtained. 
2. A stoichiometric excess of preconjugate comprising the immunogenic 
species of the polymorphic analyte relative to the amount of preconjugate 
comprising the nonimmunogenic species of the polymorphic analyte, can be 
obtained by the present method, except for the aminoglycoside antibiotic 
amikacin. 
3. The aminoglycoside antibiotic amikacin can be easily isolated from other 
reaction products by using a carbonate in the reaction medium. 
4. The disclosed methods can be carried out under mild conditions. 
Although the present invention has been described in detail with regard to 
certain preferred embodiments, other embodiments, version, and 
modifications are within the scope of the disclosed invention. For 
example, the polymorphic analytes are not restricted to only certain 
aminoglycoside antibiotics. Furthermore, the preconjugate can be made 
using a variety of coupling reagents and binding moieties. 
Accordingly, the spirit and scope of the following claims should not be 
limited to the descriptions of the specific embodiments of the present 
invention set forth above.