Chloramphenicol derivatives

Chloramphenicol derivatives are provided for use in the preparing of antigen conjugates for the production of antibodies specifically for chloramphenicol. Specifically, the aryl amino group is derivatized to introduce a non-oxocarbonyl group which is used for amide formation with an antigen. The conjugate is then injected into a vertebrate for production of antisera which is isolated in conventional ways and find particular use in competitive protein binding assays.

BACKGROUND OF THE INVENTION 
In performing immunoassays, it is necessary to have a receptor which 
specifically recognizes the compound or compounds of interest while having 
weak or no binding to compounds of similar structure which may be 
encountered in the samples of interest. In order to obtain antisera, when 
haptens are involved, it is necessary that derivatives of the hapten be 
designed for conjugation to an antigen, where the resulting antisera will 
provide for the desired specificity. In many situations, the hapten of 
interest is highly functionalized, so that the synethetic procedure for 
the derivative must be designed to maintain the integrity of the 
structural features of the haptens. 
DESRIPTION OF THE PRIOR ART 
U.S. Pat. No. 3,817,837 describes an enzyme immunoassay. Hamburger and 
Douglass, Immunology 1969, 17(4), 599-602; Orgel and Hamburger, ibid, 
1971, 20(2), 233-9; Hamburger and Douglass, ibid, 1969, 17(4), 58791 and 
Hamburger, Science 152 (379), 203-5 (1966) describe various antibodies to 
chloramphenicol. 
SUMMARY OF THE INVENTION 
Chloramphenicol derivatives are prepared for conjugation to poly(amino 
acids) to prepare antigens for the production of antibodies and enzyme 
conjugates, where the enzyme conjugates and antibodies are used in 
combination for the determination of chloramphenicol. Particularly, the 
nitro group of chloramphenicol is reduced and the resulting aromatic amino 
group functionalized to provide a carbonyl functionality to react with the 
amino groups of the poly(amino acids) to provide a linking group. The 
conjugated antigens are employed in conventional ways for the production 
of antibodies specific for chloramphenicol. 
DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
The subject invention is concerned with the preparation of reagents for use 
in diagnostic immunoassays for chloramphenicol. Specifically, the nitro 
group of chloramphenicol is reduced to an amino group and the resulting 
amino group functionalized to provide for an oxo-carbonyl functionality 
for linking to available amino groups of the poly(amino acid). The 
carbonyl functionality will normally be separated from the ar-amino group 
by a chain of at least about 2 atoms and not more than about 8 atoms, 
perferably 3 to 5 atoms. The atoms may be carbon, nitrogen, chalcogen 
(oxygen and sulfur), usually carbon and oxygen, there normally being from 
0 to 1 heteroatom in the chain, where the heteroatoms are bonded solely to 
carbon atoms, with chalcogen normally bonded to saturated carbon. With 
oxo-carbonyl, a single bond will usually be formed by reductive amination 
with available amino groups of the poly(amino acid), while with carboxy 
groups, peptide bonds will normally be formed. The carboxy derivative can 
be activiated in a variety of ways to form peptide bonds. 
For the preparation of antibodies, the chloramphenicol derivative will be 
conjugated to an antigenic poly(amino acid), which may then be injected 
into vertebrates, particularly domestic animals, for production of 
antibodies. After a repeated number of injections based on a predetermined 
schedule, the antibodies may be harvested from the serum and may be used 
as obtained or further purified so as to concentrate the antibodies of 
interest. 
For the most part, the compositions of this invention will have the 
following formula: 
##STR1## 
wherein: 
R is an aliphatic linking group of from 2 to 12 atoms other than hydrogen, 
normally having from about 2 to 8 atoms in the chain, preferably 2 to 6, 
more preferably 3 to 5, wherein the atoms in the chain are carbon, 
nitrogen, and chalcogen of atomic number 8 to 16 (oxygen and sulfur), 
wherein the heteroatoms are bonded to other than hydrogen and chalcogen is 
bonded solely to saturated carbon; of particular interest are 
1-oxopolymethylenes with the oxo bonded to the nitrogen. 
Z is hydrogen, hydroxyl, alkoxyl of from about 1 to 6 carbon atoms, more 
usually of 1 to 3 carbon atoms, an activating group capable of activating 
the non-oxo-carbonyl for forming peptide bonds in an aqueous medium with a 
poly(amino acid) e.g. p-nitrophenyl ester or N-oxy succinimide ester or Y, 
wherein Y is a poly(amino acid) residue, either a polypeptide or protein 
having 1 or more subunits, of at least about 5000, more usually at least 
about 10,000 molecular weight and may be 10,000,000 or more molecular 
weight, usually not more than 1,000,000, functioning as either an antigen 
or enzyme; 
m is 0 or 1, being 1 when Z is other than Y; and 
n is at least 1, being 1 when Z is other than Y and when Y being 1 to the 
molecular weight of Y divided by 2000, more usually divided by 3000, 
generally being from about 10 to 100 when Y acts as an antigen and is of 
molecular weight of from about 30,000 to 300,000 and of from about 2 to 
20, more usually 2 to 16, when Y functions as an enzyme. 
Preferred R groups include alkylene, alkenylene, alkyleneoxyalkylene 
(wherein heteroatoms are separated by at least 2 carbon atoms), N-lower 
alkyl (1-3 carbon atoms) alkyleneaminoalkylene (wherein the heteroatoms 
are separated by at least 2 carbon atoms). 
The compounds of primary interest are those where Z is Y and find use as 
antigens or enzymes, Y being a poly(amino acid), either antigenic or an 
enzyme. These compounds will for the most part have the following formula: 
##STR2## 
wherein R and m have been defined previously; 
Y.sup.1 is a poly(amino acid), functioning as an antigen or enzyme, of at 
least about 2000 molecular weight, more usually of at least about 10,000 
molecular weight and may be up to 10,000,000 molecular weight or greater, 
generally not exceeding about 600,000 molecular weight, more usually not 
exceeding about 300,000 molecular weight; 
n.sup.1 is at least 1, usually greater than 1, and generally not exceeding 
the molecular weight of Y.sup.1 divided by 1000, more usually divided by 
2000 and will usually be at least the molecular weight of Y.sup.1 divided 
by 100,000, more usually the molecular weight of Y.sup.1 divided 50,000, 
generally being from about 1 to 100, more usually from about 5 to 80, when 
Y.sup.1 is functioning as an antigen, and from about 1 to 30, more usually 
2 to 16, when Y.sup.1 is functioning as an enzyme. 
With intermediate molecular weight antigens, those having molecular weights 
in the range of about 20,000 to 600,000 the number of chloramphenicol 
groups which are bonded to the antigen will generally be from about 5 to 
100, more usually from about 20 to 90, while with low molecular weight 
antigens, those from about 2000 to 10,000 molecular weight, the number 
will generally be from about 1 to 20, more usually 2 to 10. 
As indicated previously, of particular interest are compounds where the 
oxo-carbonyl group (other than keto) and the non-oxo-carbonyl group are 
bonded to an amino group, which is part of a polypeptide or protein 
structure. One group of polypeptides and proteins is antigenic, so that by 
bonding the carbonyl derivative of chloramphenicol to the polypeptide or 
protein, antibodies can be formed to chloramphenicol. A narrower class of 
proteins, which also can be used as antigens, but will not normally be 
used as such, are enzymes which are employed as the detector in an 
immunoassay system. As antigens, inactive enzymes can be used. 
Polypeptides (referred to generally in the invention as poly(amino acid)) 
usually encompass from about 2 to 100 amino acid units (usually less than 
about 12,000 molecular weight). Larger polypeptides are arbitrarily called 
proteins. Proteins are usually composed of from 1 to 20 polypeptide chains 
called subunits, which are associated by covalent or noncovalent bonds. 
Subunits are normally of from about 100 to 300 or higher amino acid groups 
(or 10,000 to 35,000 or higher molecular weight). For the purposes of this 
invention, poly(amino acid) is intended to include individual polypeptide 
units and polypeptides which are subunits of proteins, whether composed 
solely of polypeptide units or polypeptide units in combination with other 
functional groups, such as porphyrins, as in haemoglobin or cytochrome 
oxidase. 
The number of chloramphenicol groups will vary depending upon whether the 
poly(amino acid) is an enzyme or antigen. The maximum number of groups 
will be limited by the effect of substitution on solubility, activity, and 
the like. For the formation of antibodies, a sufficient number of 
chloramphenicol groups should be present, so as to provide a satisfactory 
harvest of antibodies to chloramphenicol. Otherwise, the proportion of 
antibodies to chloramphenicol as compared to antibodies to other compounds 
may be undesirably low. With monoclonal antibodies a reasonable number of 
hybridomas should result which secrete useful antibodies. 
The first group of protein materials or polypeptides which will be 
considered are the antigenic polypeptides. These may be joined to the 
carbonyl group of the chloramphenicol analog through an amino group. The 
product can be used for the formation of antibodies to chloramphenicol. 
The protein materials which may be used will vary widely, and will nomally 
be from 1000 to 10 million molecular weight, more usually 20,000 to 
600,000 molecular weight. 
Enzymes will normally be of molecular weights in the range of about 10,000 
to 600,000, usually in the range of about 12,000 to 150,000, and more 
usually in the range of 12,000 to 80,000. Some enzymes will have a 
plurality of enzyme subunits. It is intended when speaking of enzyme 
molecular weights to refer to the entire enzyme. There will be on the 
average at least about 1 chloramphenicol per enzyme, when the labeling is 
not limited to a specific amino group, and rarely more than 30 
chloramphenicols per enzyme, usually not more than 20 chloramphenicols per 
enzyme. For example, with lysozyme the average number of chloramphenicol 
groups would be in the range of about 2 to 5. For glucose-6-phophate 
dehydrogenase the average number will be in the range of 2 to 20. 
While the chloramphenicol analog may be bonded through the non-oxo-carbonyl 
group to hydroxyl or mercapto groups, which are present in the poly(amino 
acids), for the most part the bonding will be to amino. Therefore, the 
compounds are described as amides, although esters and thioesters may also 
be present. The aldehyde derivative will be bonded solely to amino to form 
alkylamine groups through reductive amination. 
Amino acids present in proteins which have free amino groups for bonding to 
the carbonyl-modified-chloramphenicol include lysine, N-terminal amino 
acids, etc. The hydroxyl and mercaptan containing amino acids include 
serine, cysteine, tyrosine and threonine. 
Various protein and polypeptide types may be employed as the antigenic 
material. These types include albumins, serum proteins, e.g. globulins, 
ocular lens proteins, lipoproteins, etc. Illustrative proteins include 
bovine serum albumin, keyhole limpet hemocyanin, egg albumin, bovine 
gamma-globulin, etc. Small neutral polypeptides which are immunogenic such 
as gramicidins may also be employed. Various synthetic polypeptides may be 
employed, such as polymers of lysine, glutamic acid, phenylalanine, 
tyrosine, etc., either by themselves or in combination. Of particular 
interest is polylysine or a combination of lysine and glutamic acid. Any 
synthetic polypeptide must contain a sufficient number of free amino 
groups as, for example, provided by lysine. 
The second group of protein molecules are the detectors. These are the 
enzymes to which the carbonyl modified chloramphenicol may be conjugated. 
As indicated, the chloramphenicol conjugated enzyme is useful for 
immunoassays. A description of the immunoassay technique will follow. 
Various enzymes may be used such as peptidases, esterases, amidases, 
phosphorylases, carbohydrases, oxidases, e.g. dehydrogenase, reductases, 
and the like. Of particular interest are such enzymes as lysozyme, 
perosidase, .alpha.-amylase, .beta.-galactosidase, dehydrogenases, 
particularly malate dehydrogenase and glucose-6phosphate dehydrogenase, 
alkaline phosphatase, .beta.-glucuronidase, cellulase and phospholipase. 
In accordance with the I.U.B. Classification, the enzymes of interest are: 
1. Oxidoreductases, particularly Groups 1.1, and more particularly 1.1.1, 
and 1.11, more particularly, 1.11.1; and 3. Hydrolases, particularly 3.2, 
and more particularly 3.2.1. 
The substituted enzymes will for the most part have the following formula: 
##STR3## 
wherein: 
m and R have been defined previously; 
Y.sup.2 is an enzyme substituted at other than the active site, and having 
at least 30, preferably at least 50 percent of its original activity prior 
to conjugation; and 
n.sup.2 will usually be of from 1 to 30, more usually from 2 to 20, 
preferably 2 to 14, more preferably 2 to 12, but generally on the average 
not more than about 60 percent of the total lysine groups available in the 
enzyme, although small enzymes such as lysozyme may have all available 
lysine groups conjugated. 
In forming the various amide products which find use in the subject 
invention, the carboxylic acid will normally be activated. This can be 
achieved in a number of ways. Two ways of particular interest are the 
reaction with a carbodiimide, usually a water soluble dialiphatic or 
dicycloaliphatic carbodiimide in an inert polar solvent, e.g. 
dimethylformamide, acetonitrile or hexamethylphosphamide. The reaction is 
carried out by bringing the various reagents together under mild 
conditions and allowing sufficient time for the reaction to occur. 
Another way is to use esters of the carboxy modified chloramphenicol which 
are operative in water for acylating amine functions. Illustrative of 
groups bonded to carboxy to provide activated esters which can be used in 
water are p-nitrophenyl and N-succinimidyl. For the aldehyde conjugation, 
a reductive amination is carried out in a polar, usually aqueous medium, 
employing sodium cyanoborohydride as the reducing agent. 
The antibodies which are prepared in response to the conjugated antigens of 
this invention have strong specific binding to the parent drug, the 
conjugated antigen, the compound or derivative thereof used to conjugate 
to the antigen, and the chloramphenicol labeled compounds, e.g. enzyme 
conjugates. 
As previously indicated, the subject enzyme conjugates and antibodies find 
use in immunoassays. The enzyme conjugates of the subject invention are 
particularly useful in the method described in U.S. Pat. No. 3,817,837. In 
performing an effective immunoassay, there are many considerations. Since 
the aforementioned assay is spectrophotometric, one desires that there be 
a substantial change in signal with changing concentration of the analyte 
in the range of interest of the analyte. Thus, the antigenic conjugate 
must provide antibodies which when employed in combination with the enzyme 
conjugate, results in a sensitive response to variations in the 
chloramphenicol concentration. 
In addition, there are a number of considerations about the antigen. 
Normally, one immunizes a number of animals with the antigen. Initial 
bleeds tend to have low titer of low binding affinity, but within a 
relatively short time a plateau of titer and affinity is reached. A good 
antigen provides a high titer and a high average affinity with most or all 
the animals immunized. One of the significant advantages of a high 
affinity high titer is that one can use smaller amounts of the antisera in 
that the antibody of interest is a larger proportion of the total amount 
of gamma-globulin. 
There is the further consideration of cross-reactivity. When determining a 
drug, one does not wish other drugs or naturally occurring compounds to 
affect the observed signal. Where other compounds are able to bind to 
various degrees to the antisera, the other compounds can have a 
substantial affect on the signal. This can be particularly true with 
metabolites, which, are not in themselves active in the same manner as the 
drug precursor. Thus, in many situations, the antigen precursor must be 
designed to provide antibodies which will not significantly bind to 
metabolites of the analyte of interest. 
EXPERIMENTAL 
The following examples are offered by way of illustration and not by way of 
limitation. 
All temperatures not otherwise indicated are centigrade. Percents and parts 
not otherwise indicated are by weight, except for mixtures of liquids 
which are by volume. Abbreviations which are employed are as follows: 
THF--tetrahydrofuran; tlc--thin layer chromatography; h--hour; 
DCC--dicyclohexyl carbodiimide; NHS--N-hydroxy succinimide; HOAc--acetic 
acid; BSA--bovine serum albumin; EDAC--ethyl dimethylaminopropyl 
carbodiimide.

EXAMPLE I 
Preparation of the 1-p-adipamidophenyl-2-dichloroacetamido-1,3-propanediol 
conjugate of bovine serum albumin. 
A. A solution of oxalyl chloride (7.94 g, 62.5 mmol) and adipic acid 
monomethylester (4.0 g, 25 mmol) in benzene (20 ml) was heated to reflux 
under argon for 30 min, then allowed to stir over night under argon for 16 
h. The reaction mixture was distilled at slightly reduced pressure to 
remove benzene and oxalyl chloride, then at 73.degree. and 0.2 mm to 
afford 4.46 g (100%) of a colorless oil. 
B. To a solution of N-1,3-dihydroxy-1-(p-anilino)propyl dichloroacetamide 
(703 mg, 2.39 mmol) in THF (17 ml) and Et.sub.3 N (0.5 ml) at 0.degree. 
under argon was added adipic acid chloride monomethylester (853 mg, 4.78 
mmol) in THF (3 ml) dropwise over 30 min and the reaction was allowed to 
stir warming to room temperature over 90 min. Tlc indicated that two new 
products, bisacylated and monoacylated had been formed in nearly equal 
amounts. The reaction mixture was poured into ice/HCl (pH=1), extracted 
with ethyl acetate (3.times.100 ml), washed (saturated brine), dried 
(Na.sub.2 SO.sub.4) and concentrated. Nmr confirmed that the two spots on 
tlc were due to mono and bisacylated starting material. This reaction 
mixture was hydrolyzed without further purification. 
C. To a solution of the above mixture (1.29 g, 2.23 mmol) in methanol (15 
ml) at 0.degree.-4.degree. was added an aqueous 5% sodium carbonate 
solution (18 ml) dropwise over 1 h. Stirring at 4.degree. was continued 
for 27 h. Tlc indicated incomplete hydrolysis, therefore 5% sodium 
carbonate (4.5 ml) was added dropwise and the reaction was allowed to stir 
at 4.degree. for an additional 15.5 h. At this time an additional 4.5 ml 
of 5% sodium carbonate was added and stirring was continued for 4 h. The 
reaction mixture was poured into ice/HCl (20 ml), was made acidic (pH=1), 
extracted with ethyl acetate (4.times.100 ml), washed (brine), dried 
(Na.sub.2 SO.sub.4) and concentrated to afford 1.05 grams of crude 
material. Preparative thin layer chromatography on silica gel plates 
eluting with methylene chloride (83%) and methanol (17%) afforded 160 mg 
(16%) of the mono-N-adipoyl product, and 309 mg of partially hydrolyzed 
material. The latter was redissolved in methanol (5 ml) at 4.degree. and 
to it was added 5% sodium carbonate solution (5.5 ml). This reaction 
mixture was allowed to stir for 4 days, was quenched with ice/HCl (pH=1), 
extracted with ethyl acetate, dried (Na.sub.2 SO.sub.4) and concentrated. 
Preparative tlc on silica eluting with methylene 
chloride/methanol/dichloroacetic acid (84/16/0.1, v/v/v) afforded 160 mg 
(16%) of the desired product. 
D. To a solution of the above product (C) (160 mg, 0.38 mmol) in dry THF 
(20 ml) under argon at 0.degree. was added DCC (165 mg, 0.8 mmol) and NHS 
(46 mg, 0.4 mmol) and the reaction mixture was allowed to stir for two 
days. Tlc (CH.sub.2 Cl.sub.2 /MeOH/HOAc, 85/15/0.1) indicated incomplete 
reaction and therefore DCC (83 mg, 0.4 mmol) and NHS (23 mg, 0.2 mmol) 
were added. Stirring at 0.degree.-4.degree. was continued for 24 h. at 
which time the reaction mixture was filtered, evaporated, washed with 
hexane, evaporated, dissolved in THF (10 ml) and added dropwise to a 
solution of BSA in phosphate buffer (20 ml) (pH=8.5) at 0.degree. and the 
reaction was allowed to stir for 30 h. Centrifugation at 10K and 4.degree. 
for 30 min. followed by dialysis against water (1.times.4L), followed by 
Sephadex G-25 (100 g, 500 ml) chromatography, then followed by dialysis 
against water afforded 449 mg (88%) of protein conjugate. The hapten 
number, determined by UV extinction coefficients was found to be between 
21 and 25 (.lambda..sub.max =246). 
EXAMPLE II 
Preparation of the 1-p-adipamidophenyl-2-dichloroacetamido-1,3-propanediol 
conjugate of glucose-6-phosphate dehydrogenase. 
1-p-(Adipamidophenyl)-2-dichloroacetamido-1,3-propanediol (0.0053 g), 
0.0018 g of NHS and 0.0029 g of EDAC were weighed into a dry 2-necked 
flask. After further drying the flask and its content overnight, 250 .mu.l 
of dimethylformamide was added into the flask and the solution formed was 
stirred for four hours at 25.degree. C. Sixty-five .mu.l of this solution 
was slowly added over a period of 5.25 hours to a 4.degree. C. solution 
containing 0.6 mg of glucose-6-phosphate dehydrogenase (Beckman Co., 
Fullerton, Ca), 30 mg glucose-6-phosphate disodium salt (Sigma Co.) and 20 
mg of nicotinamide adenine dinucleotide reduced (Sigma Co.) in 0.5 ml of 
0.055 M Tris buffer, pH 8, and 0.2 ml of carbitol. The pH of the reaction 
mixture throughout the 5.25 hour period was maintained in the range of 8.5 
to 9.5 using 0.1 N sodium hydroxide. At the end of the reaction period, 
the reaction mixture was chromatographed on a 19.times.1.9 cm column of 
Sephadex G-50 (Pharmacia, Piscataway, N.J.) and eluted with 0.055 M Tris 
buffer, pH 8, containing 0.05% sodium azide and 0.005% thimerosol. 1.4 ml 
fractions were collected and those fractions containing high enzyme 
activity (usually fractions 7 to 10) were pooled and were used as the 
adipamido-chloramphenicol glucose-6-phosphate dehydrogenase conjugate. 
The compositions prepared above were used in an assay for chloramphenicol. 
The assay employed the following reagents: 
TABLE I 
______________________________________ 
Buffer: 0.055 M Tris-HCl, pH8.1 (RT), 0.05% NaN.sub.3, 
0.005% Thimerosal 
Assay Buffer: 
Buffer, 0.5% NaCl, 0.01% (v/v) Triton 
X-100, pH8.1 (RT) 
Reagent A: 
Buffer, 1.0% RSA, G6P(Na.sub.2), NAD, pH5 (RT) 
antichloramphenicol optimized for response 
Reagent B: 
Buffer, 0.9% NaCl, 1.0% RSA, 0.032 M G6P(Na), 
pH6.2, sufficient enzyme to give a maximum 
rate of 700 .DELTA.OD. 
______________________________________ 
Protocol 50 .mu.l of the sample is drawn up into a diluter and dispensed 
with 250 .mu.l of the assay buffer into a 1 ml Croan cup. A 50 .mu.l 
aliquot of the diluted sample is drawn up and dispensed with a 250 .mu.l 
portion of assay buffer into a second Croan cup. Into the second Croan cup 
is introduced 50 .mu.l of the antibody reagent with 250 .mu.l of the assay 
buffer, followed by the addition of 50 .mu.l of the enzyme reagent and 250 
.mu.l of the assay buffer. Immediately after the enzyme addition, the 
entire sample is aspirated into the flow cell. After 15 sec. a first 
reading is taken, followed by a second reading after a 30 sec. interval. 
The results are reported as the difference in absorbance .times.2.667. 
Four sheep were immunized with the antigen according to Example I. The 
antisera produced at the C bleed were tested for optimal assay response 
(O.D.) and for effective titer. The optimal assay response is the optimal 
separation in O.D. units between 2.5 .mu.g/ml and 40 .mu.g/ml 
chloramphenicol calibrators. This response shows the best range of the 
standard curve obtainable in accordance with the above described protocol. 
The larger the optimal assay response, the better the precision and 
accuracy. Effective titer is the amount of antiserum required per assay to 
give the optimal assay response. The higher the titer, the lower the 
required quantity of antiserum, the less expensive the assay production 
costs and the less extraneous material introduced into the assay medium. 
The following table reports the results of the four antisera. 
TABLE II 
______________________________________ 
Optional Assay 
Antiserum Response O.D. 
Effective Titer (.mu.1) 
______________________________________ 
3430C 135 0.8 
3431C 133 0.8 
3432C 148 1.2 
3433C 134 2.0 
______________________________________ 
Cross reactivity was determined against metabolites of chloramphenicol as 
well as other drugs. Of the three known metabolites of chloramphenicol, 
1-p-nitrophenyl-2-amino-1,3-propanediol; 
1-p-aminophenyl-2-dichloroacetamido-1,3-propanediol; and 
1-p-aminophenyl-2-amino-1,3-propanediol, only the second metabolite showed 
significant activity, which is defined as the concentration of a compound 
which, when spiked into a 15 .mu.g/ml control, will give a response in the 
assay equal to the response of 130% (i.e. 19.5 .mu.g/ml) of the 
concentration of the 15 .mu.g/ml control, the cross reactivity 
concentration for the second compound was 2 .mu.g/ml. Besides this 
metabolite, the chloramphenicol/succinate salt and thiamphenicol showed 
similar cross reactivity. Based on an independent study of a comparison 
between the above assay and HPLC with patient samples, which showed good 
correlation statistics between the two techniques, the effect of the cross 
reactivity on the subject assay to correctly quantitate chloramphenicol is 
believed to be minimal. 
The compositions of the subject invention provide for reagents which 
provide a sensitive accurate assay for chloramphenicol, distinguishing 
chloramphenicol from closely related metabolites. The antigenic conjugate 
provides for the efficient production of antibodies having high affinity 
and high titer for chloramphenicol. The combination of the antibodies and 
enzyme conjugates result in an accurate rapid assay for chloramphenicol in 
serum. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity of understanding, it 
will be obvious that certain changes and modifications may be practiced 
within the scope of the appended claims.