CA2239969A1 - Reagents for measuring lead levels via the quantification of porphobilinogen - Google Patents

Abstract

Immunoassay methods and reagents for measuring the levels of lead in a test sample via the quantification of porphobilinogen use antibodies prepared with compounds of Formulas (II) and (III), wherein X and Y are linking groups consisting of 0 to 2 heteroatoms and 0 to 8 carbon atoms linked to an immunogenic carrier material P wherein P is preferably bovine serum albumin. Also described are the synthesis of fluorescein tracers of the structure of Formulas (V) and (VII), wherein A and C are linking groups consisting of 0 to 2 heteroatoms and 0 to 8 carbon atoms linked to a detectable moiety Q, and wherein Q is a detectable moiety, preferably fluorescein or a fluorescein derivative.

Description

CA 02239969 l998-06-08 W O 97/24621 PCT~US96/19544 REAGENTS FOR MEASURING LEAD LEVELS VIA THE QUANTIFICATION
OF PORPHOBILINOGEN

Field of the Invention The present invention relates to new and unique reagents for the direct qllAnt;fi~ tion of porphobilinogen formed from aminolevulinic acid in the presence of aminolevulinic acid dehydratase by immunoassay. The amount of porphobilinogen formed is inversely proportional to the levels of lead in a test15 sample. There is no indirect measurement of porphobilinogen complexes as is often used in colorirnetric determinations. The present lnvention also relates to immunogens, antibodies prepared from such immunogens, and labeled reagents for the quantification of porphobilinogen, preferably for use in a fluorescence polarization immunoassay.
Background of the Invention The rapid determination of trace metals in biological and environmental systems is incre~ingly important in identifying potential hazards and preserving the public health. The toxicity of certain metals such as2 5 lead is well-known. The absorption of even trace amounts of lead can cause severe damage to human organs. The numerous and widespread sources of lead in the environment, including the food supply, compounds the problems of screening affected groups. It is generally recognized that lead poisoning occurs in children at blood levels as low as 10-15 mg/dl as noted by S. Cummins 3 0 and L. Goldman in Pediatrics, 1992, 90, 995-996. Lead contamination of W O 97/24621 PCTrUS96/19544 environmental sources such as water, dust and soil require identification at even lower levels. In order to measure these amounts, the analytical techniques must be sensitive, contaminant-specific, and reliable.

S Previous te--hni-lues have relied upon atomic absorption spectroscopy as described in an article in ain. Chem., 1991, 37, 515-519 by Jacobson et al. or by measuring biological marlcers for exposure to lead as noted by A. Berlin et al. in Z. Klin. Chem. Klin. Biochem., 1974, S. 389-390. Atomic absorption spectroscopy (AAS) is limited in its general availability, high level of technical expertise 1 0 requirements for operation, throughput and expense of investing and maintaining the necessary instrumentation. The AAS method is generally carried out in a central laboratory with considerable amount of time between taking a test sample and obtaining the levels of lead in the test sample. The biological marker method is based upon an inhibition of the enzyme 1 5 aminolevulinic acid dehydratase (ALAD) where if the activity of ALAD from ablood sample had subnormal activity then that person may have been exposed to lead. The activity of ALAD after exposure to lead con~ining samples was further disclosed in US patent 5,354,652 to Silbergeld dated October 11, 1994.
This patent describes a colorimetric method for the quantification of lead. This2 0 method describes the chemical derivatization of porphobilinogen using a colorimetric agent (Erlich's reagent) where the amount of the resulting colored porphobilinogen derivative is quantified. The amount of porphobilinogen produced is inversely proportional to the col~e~E~onding lead levels. The principles of colorimetric determination of porphobilinogen produced from 2 5 ALAD was originally disclosed by K. D. Gibson et al. in Biochem., 1955, 61:618-629. Silbergeld also disclosed in this patent the use of an antibody to the ALAD-Lead complex, in this approach the ALAD-Lead specific antibody is used in an ELISA assay. A sample suspected of containing lead is mixed with ALAD, the amount of ALAD-Lead complex ~at is formed is detected with a W O 97/24621 PCTrUS96119544 horseradish peroxidase-anti-ALAD antibody. In a similar fashion, Jaffe has disclosed a method for measuring lead exposure by quantifying ALAD activity using an anti-ALAD antibody in patent application WO 95/04159. This method also measures the porphobilinogen produced from the ALAD enzyme 5 colorimetrically with Erlich's reagent. H~nkPn.~ et al. disclosed an indirect enzymatic method for the detection of lead using isocitrate dehydrogenase in US patent 5,368,707 dated November 29, 1994. This method measured the inhibition of the enzyme isocitrate dehydrogenase with subsequent electrochemical detection. A different approach for the detection of lead was I 0 disdosed by Jing in US patent 5,407,831, it is based on the selective chelation of lead and detection by atomic absorption spectroscopy.

The above described methods for measuring the levels of lead suffer from various disadvantages. The colorimetric method which employs Erlich's l 5 reagent has the disadvantage of the use of strong mineral acid and toxic heavy metals to give the desired color formation to quantify porphobilinogen. The method which employs the enzyme isocitrate dehydrogenase coupled with biosensor detection lacks the desired specificity (lines 28-31, page 3) which had to be overcome with laborious pretreatment (lines 25-47, page 5). Finally, the 2 0 method employing ALAD-Lead complex employs sandwich assay format where two antibodies of various specificity are employed in heterogeneous assay format. It is known that immunoassays based on heterogeneous format are complex in their manufacturability.

2 5 Therefore, the challenge still exists to accurately and precisely measure levels of lead in a rapid and selective manner using automated instrumentation that is currently available in ~lini~l settings in a cost effective manner. This could be achieved by the direct measurement of porphobilinogen by immunoassay.

CA 02239969 l998-06-08 W O 97/24621 PCTrUS96/19544 Sltmmary of the Invention The present invention overcomes the disadvantages of previously described methods and therefore provides a new, direct method for the measurement of lead levels by uniquely and directly quantifying the amount of 5 porphobilinogen (PBG) produced from aminolevulinic acid (ALA) catalyzed by aminolevulinic acid dehydratase (ALAD), where the amount of porphobilinogen produced is inversely proportional to the amount of lead present in a test sample. This direct quantification of porphobilinogen is achieved in a hornogeneous immunoassay forma~ preferably employing 10 fluorescence polarization. This method provides the specificity, speed and convenience of homogeneous immunoassay to give precise, reliable and low cost quantification of porphobilinogen, especially well adapted to automated immunoassay analyzers. There is no indirect quantification of porphobilinogen via porphobilinogen complexes wllich are formed in processes for colorimetric I S determination.

The present invention also provides unique antibody reagents and labeled reagents for the quantification of porphobilinogen in a test sample.
These reagents do not contain toxic heavy metals such as mercury which are 2 0 utilized in the prior methods of the art in the chemical derivatization step.

The present invention further provides synthetic procedures for preparing unique immunogens which are employed for the production of such antibody reagents and for preparing such unique labeled reagents. According to 2 5 the present invention, the labeled reagents and the antibody reagents offer an advance in the art beyond previously known procedures. By the use of these reagents, the cumbersome and time-consuming method of colorimetric deterrnination of porphobilinogen is avoided, as is the chemical derivatization step.

W O 97/24621 PCT~US96/19544 Brief Description of the Drawings ~IGURE 1 illustrates the synthetic pathway for coupling a porphobilinogen (~BG) derivative to bovine serum albumin to produce immunogen (7) according to the method of the present invention.
FIGURE 2 illustrates the synthetic pathway for coupling a PBG derivative to bovine serum albumin to produce immunogen (14) according to the method of the present invention.
FIGURE 3 illustrates the synthetic pathway for coupling a PBG derivative to bovine serum albumin to produce immunogen (19) according to the method of the present invention.
FIGURE 4 illustrates the synthetic pathway for the preparation of a fluorescent tracer (21) according to the method of the present invention.
FIGURE 5 illustrates the synthetic pathway for the preparation of a fluorescent tracer (3(~) according to the method of the present invention.
E;IGURE 6 illustrates the synthetic pathway for the preparation of a fluorescent tracer (32) according to the method of the present invention.
FIGURE 7 illustrates the synthetic pathway for the preparation of a fluorescent tracer (34) according to the method of the present invention.
2 0 FIGU3~E 8 illustrates the synthetic pathway for the preparation of a fluorescent tracer (36) according to the method of the present invention.
~IGIJRE 9 is a graph which illustrates a calibration curve for the measuring levels of lead via the quantification of porphobilinogen on the Abbott IMx(g) analyzer.
2 5 FIGURE 10 is a graph which illustrates antibody dilution from animals inoculated with immunogens (7), (14) and(19).

W O 97/24621 PCT~US96/19544 Detailed Description of the Invention According to the present invention, lead levels are measured via the direct quantiQcation of porphobilinogen formed from aminolevulinic acid in 5 the presence of aminolevulinic acid dehydratase by immunoassay. A test sample is contacted with a labeled reagent or tracer and an antibody reagent, either simultaneously or sequentially in either order, and then measuring the amount of the labeled reagent which either has or has not participated in a binding reaction with the antibody reagent as a function of the amount of 10 porphobilinogen formed which is inversely proportional to the amount of lead in the test sample. In particular, the present invention relates to immunogens, antibodies prepared from such immunogens, and labeled reagents for use in the fluorescence polarization immunoassay for the quantification of porphobilinogen thereby measuring the levels of lead.

Lead is known to inhibit the formation of porphobilinogen (Formula 1) by ALAD and a measurement of porphobilinogen produced will indirectly measure the amount of lead present in a test sample. Pretreating a lead containing test sample frees the lead from within red blood cells and removes 2 0 interfering compounds such as proteins, endogenous ALAD, PBG, ALA and the like. Acids such as trichloroacetic acid (TCA), nitric acid, 5-sulfosalicylic acid or perchloric acid are commonly used for sample pretreatment. Such sample is centrifuged, pellet discarded and resulting supernatant, after neutralization, is incubated with ALAD to produce an amount of porphobilinogen (PBG) 2 5 inversely proportiona} to the lead concentration present in the test samp}e.This mixture is then assayed by fluorescence polarization immunoassay using the inventive reagents described herein to detect and quantify the amount of porphobilinogen present in the mixture and thereby measuring the amount of lead in the test sample.

HOOC rCOOH

H~ I ~NH2 Antibodies of the present invention are produced with immunogens 5 which are prepared with derivatives of the Formula II, III and IV:

HOOC~ COOH

I I H)l--N~CNH--X--P

HOOC\ COOH

I I I H ~ I ~ N H2 p HOOC~ COOH
I V ) ~C
p_z ~ NH2 wherein for II X is CO-(CH2)n-CO- with n from 0-6, for III Y is SO2-(CH2)n-CO-with n from Q-6, for IV Z is (CO)m-(CH2)n-CO- with m from 0 to 1 and n from 0-1 5 6; wherein for II, III and IV P is an immunogenic carrier material.

W O 97/24621 PCTrUS96/19S44 Labeled reagents of the presen~ invention are prepared with porphobilinogen compounds of the Formula V, VI, VII and VIII:

HOOC ~ rCOOH

H)~N~NH--A--Q
H
s HOOC~ rCOOH

V I H)~NI J~NH--B Q

HOOC
COOH
V I I 1~NJ~NH2 Q--C

~OOC
COOH
V I I I Q -D)~NJ~NH2 H

wherein for V A is ~CO-(CH2)n-CO- with n from 0-6, for VI B is -(CH2)n-CO- with n from 0-6, for VII C is SO2-(CH2)n-CO- with n from 0-6, for VIII Z is -(CO)m~
(CH2)n-CO- with m from 0 to 1 and n from 0-6; Q is a detectable moiety, 1 5 preferably a fluorescen~ moiety.

W O 97/24621 PCTrUS96/19544 The above immunogens were prepared as described below and as shown in Figures 1, 2 and 3. An imrnl~nogen of Formula II was prepared by N-acylation of porphobilinogen with the active ester of adipic acid mono ethyl ester followed by transformation of the free PBG acids to the corresponding tert-butyl5 esters. Hydrolysis of the ethyl ester to the free acid afforded the desired hapten.
Activation of the free acid and conjugation to bovine serum albumin followed by treatment with trifluoroacetic acid gave the desired immunogen as shown in Figure 1. An immunogen of Formula III was prepared from porphobilinogen by first protecting the side chain nitrogen as a carbamate followed by the I 0 conversion of the two free acids to tert-butyl esters. The resulting protected porphobilinogen was sulfonylated on the pyrrole nitrogen with benzyl.4-chlorosulfonylbutryate followed by hydrogenolysis of the benzyl ester which afforded the desired hapten. Activation of the free acid and conjugation to bovine serum albumin followed by treatment with trifluoroacetic acid afforded 15 the desired immunogen as shown in Figure 2. An immunogen of Formula IV
was prepared by the acylation of a PBG di-tert-butyl ester carbamate derivative,subsequent hydrolysis gave the desired hapten as the free acid. Activation of the free acid and conjugation to bovine serum albumin followed by treatment with trifluoroacetic acid gave the desired immunogen as shown in Figure 3.
Fluorescent labeled reagents for use in a fluorescence polarization immunoassay for the measuring lead levels via the quantification of porphobilinogen were synthesized as shown in Figures 4, 5, 6 and 7. A
porphobilinogen fluorescent derivative of Formula V was prepared by coupling 2 5 the active ester of 6-carboxyfluorescein with porphobilinogen to afford the desired tracer as shown in Figure 4. A fluorescent derivative of Formula VI was synfhesi7ed as shown in Figure 5. This derivative was prepared in the following manner: coupling of the nitroanion of tert-butyl 4-nitrobutryrate and an aldehyde with subsequent protection as an acetate followed by coupling with W O 97/24621 PCTrUS96/19544 an isocyanate formed the pyrrole nucleus. Deprotection of the primary alcohol followed by transformation to the corresponding tert-butyl ester and subsequent hydrogenolysis of the benzyl ester moiety afforded the desired hapten. The free acid was activated and coupled to 6-aminomethylfluorescein followed by 5 treatment with trifluoroacetic acid gave the desired porphobilinogen fluorescent tracer as shown in Figure 5. The preparation of a different fluorescent tracer of Formula VI was prepared using the same active ester used in Figure 5. This active ester was coupled with a fluorescent 6-aminocaproic acid derivative followed by treatment with trifluoroacetic acid afforded the 10 desired fluorescent porphobilinogen derivative shown in Figure 6. A
fluorescent tracer of Formula VII was prepared according to Figure 7 by couplingthe carboxy~lopyl sulfonamide derivative to 5-aminomethylfluorescein with subsequent treatment with trifluoroacetic acid gave the desired fluorescent tracer. The fluorescent tracer of Formula VIII was prepared according to Figure l S 8 coupling 5-aminomethylfluorescein with the 5-carboxypyrrole derivative followed by trea~nent with trifluoroacetic acid afforded the desired fluorescenttracer.

When following a fluorescence polarization immunoassay (FPIA) format 2 0 employing the reagents according to the present invention, the concentration, or level, of lead in a test sample can be accurately measured via the quantification of porphobilinogen. To perform a FPI~ for measuring the levels of lead, a calibration curve was generated for measuring the levels of lead (Figure 9).
According to the present invention, it has been found that superior fluorescence polarization immunoassay results for measuring the levels of lead via the quantification of porphobilinogen are obtained when employing (i) an antibody reagent comprising antibodies produced from a porphobilinogen W O97124621 11 PCTrUS96119544 (PBG) derived immunogen of Formula III where P is an immunogenic carrier as described above and (ii) a fluorescent labeled reagent of Formula VII where Qis a fluorescent moiety as described above. For the quantification of porphobilinogen, the antibody reagent comprises antibodies which are capable 5 of binding to or recognizing porphobilinogen wherein the antibodies are preferably produced with an immunogen prepared from the porphobilinogen derivative of Formula III where P is bovine serum albumin and Y is -SO2-(CH2)3-CO-, the labeled reagent is ~re~erably prepared from the derivative of Formula VII where Q is a fluorescent moiety and n=3.

The great advantage of this invention is that the porphobilinogen (PBG) produced from aminolevulinic acid (ALA) and the enzyme aminolevulinic acid dehydratase (ALAD) is quantified directly by immunoassay using unique, specific antibodies of this invention. In the previous art, the porphobilinogen 1 5 produced enzymatically was never measured directly by immunoassay, but indirectly measured by reacting porphobilinogen with ~rlich's reagent to produce a derivative of porphobilinogen which was subsequently measured by colorimetric methods. This latter approach has numerous disadvantages such as: use of concentrated acid, diminished precision and extended times needed 2 0 for analysis of each sample. The use of corrosive reagents limits the possibilities for automation and creates environmental problems of disposal.
Derivatization of porphobilinogen adds an extra step in the measuring process thereby decreasing the precision of an assay that measures porphobilinogen and makes reliable automation difficult. Extended reaction times necessary for 2 5 derivatization would decrease throughput of the number of assays. Therefore,our inventive reagents offer a vast leap over tl e previous art in (i) direct measurement of porphobilinogen by immunoassay (ii) use of highly specific antibodies to porphobilinogen which can operate in the presence of other porphyrin related compounds (iii) the method for the development of W O 97t24621 12 PCTrUS96/19544 porphobilinogen specific antibodies (iv) pairing of porphobilinogen specific antibodies and labeled reagents for use in the quantification of porphobilinogenand (v) use of inventive reagents in an automated system using existing clinicald~emistry instrumentation, such as Abbott IMx(g) or A~bott AxSYM(~) systems.
When perforrning a fluorescence polarization immunoassay for measuring the levels of lead via the quantification of porphobilinogen as described herein, the detectable moiety component of the tracer is a fluorescentmoiety such as fluoresceins, aminofluoresceins, carboxyfluoresceins, and the 1 0 like, preferably aminomethylfluorescein, aminofluorescein, 5-fluoresceinyl, 6-fluoresceinyl, 6-carboxyfluorescein, 5-carboxyfluorescein, thiourea-aminofluorescein, and methoxytriazinolyl-aminofluorescein, and the lilce fluorescent derivatives. The amount of tracer bound to the antibody varies inversely to the amount of porphobilinogen generated in the test sample.
15 Accordingly, the relative, and therefore characteristic, binding affinities of porphobilinogen and the tracer to the antibody binding site, are important parameters of the assay system. Generally, fluorescent polarization techniques are based on the principle that a fluorescent tracer, when excited by plane polarized light of a characteristic wavelength, will emit light at another 2 0 characteristic wavelength (i.e., fluorescence~ that retains a degree of the polarization relative to the incident stimulating light that is inversely related to the rate of rotation of the tracer in a given medium. As a consequence of this property, a tracer substance with constrained rotation, such as in a viscous solution phase or when bound to another solution component with a relatively 2 5 lower rate of rotation, will retain a relatively greater degree of polarization of emitted light than if in free solution. Therefore, within the time frame in which the ligand and tracer compete for binding to the antibody, the tracer and ligand binding rates should yield an appropriate proportion of free and bound W O 97/24621 13 PCT~US96/19544 tracer with the preservation of important performance parameters such as selectivity, sensitivity, and precision.

When performing a fluorescent polarization immunoassay for 5 measuring the levels of lead via the quantification of porphobilinogen according to the present invention, a test sample suspected of containing lead is incubated with ALAD then contacted with antiserum prepared with immlmogens according to the present invention in the presence of an appropriately selected fluorescein derivative thereof which is capable of 10 producing a detectable fluorescence polarization response to the presence of antiserum prepared with immunogens according to the present invention.
Plane polarized light is then passed through the solution to obtain a fluorescent polarization response and the response is detected as a measure of amount of porphobilinogen present in the test sample.

The porphobilinogen derivatives of the present invention are employed to prepare immunogens by coupling them to conventional carrier materials, and subsequently used to obtain antibodies. The porphobilinogen derivatives are also used to prepare labeled reagents which serve as the detection reagents 2 0 in immlmoassays for quantifying the amount of porphobilinogen and thereby measuring the levels of lead in a test sample.

The porphobilinogen derivatives of the present invention can be coupled to immunogenic carrier materials by various conventional techniques known 2 5 in the art where P is an immunogenic carrier material in Formula II or III As would be understood by one skilled in the art, the immunogenic carrier material can be selected from any of those conventionally known and, in most instances, will be a protein or polypeptide, although other materials such as carbohydrates, polysaccharides, lipopolysaccharides, poly(amino) acids, nucleic W O 97/24621 PCT~US96119544 acids, and the like, of sufficient size and immunogenicity can also be employed.Preferably, the immunogenic carrier material is a protein such as bovine serum albumin, lceyhole limpet hemocyanin, thyroglobulin, and the like. The immunogens according to the present invention are used to prepare antibodies, S both polyclonal and monodonal, according to methods known in the art for use in an immunoassay ~y~Lell- according to the present invention. Generally, a host ~nim~l, such as a rabbit, goat, mouse, guinea pig, or horse is inJected at one or more of a variety of sites with the immunogen, normally in mixture with an adjuvant. Further injections are made at the same site or different sites at 1 0 regular or irregular intervals thereafter with bleedings being taken to assess antibody titer until it is detPrmined that optimal titer has been reached. The antibodies are obtained by either bleeding the host ~nim~l to yield a volume of antiserum, or by somatic cell hybridization techniques or other techniques known in the art to obtain monoclonal antibodies, and can be stored, for 1 S example, at -20~C.

In ~ tiQn to fluorescence polarization immunoassays, various other immunoassay formats can be followed for the quantification of porphobilinogen according to the present invention. Such immunoassay 2 0 system formats include, but are not intended to be limited to, competitive, sandwich and immunometric techniques. Generally, such immunoassay :jyslellls depend upon the ability of an immunoglobulin, i.e., a whole antibody or fragment thereof, to bind to a specific analyte from a test sample wherein a labeled reagent comprising an antibody of the present invention, or fragment 2 S thereof, attached to a label or detectable moiety is employed to determine the extent of binding. Such detectable labels include, but are not intended to be limited to, enzymes, radiolabels, biotin, toxins, drugs, haptens, DNA, RNA, liposomes, chromophores, chemiluminescers, colored particles and colored microparticles, fluorescent compounds such as aminomethylfluorescein, 5-CA 02239969 l998-06-08 fluoresceinyl, 6-fluoresceinyl, 5-carboxyfluorescein, 6-carboxyfluorescein, aminofluorescein, thioureafluorescein, and methoxytriazinolyl-aminofluorescein, and the like fluorescent derivatives. As described herein, thetest sample can be a naturally occurring or artificially formed liquid, or an 5 extract thereof, and indudes, but is not intended to be limited to biological test samples such as whole blood, serum, plasma, urine, feces, saliva, cerebrospinal fluid, brain tissue, and the like. In addition, the test sample can be an ex~ract of a test sample, or any derivative thereof.

Typically, the extent of binding in such immunoassay system formats is determined by the amount of the detectable moiety present in the labeled reagent which either has or has not participated in a binding reaction with the analyte, wherein the amount of the detectable moiety detected and measured can be correlated to the amount of analyte present in the test sample. For I S example, in a competitive immunoassay system, a substance being measured, often referred to as a ligand, competes with a substance of close structural similarity coupled to a detectable moiety, often referred to as a tracer, for a lirnited number of binding sites on antibodies specific to the portion or portions of the ligand and tracer with structural similarity, shared with an immunogen 2 0 employed to produce such antibodies.

The present invention will now be illustrated, but is not intended to be limited by, the following examples. Bold-faced numerals contained in parenthesis refer to the structural formulae as used in the Figures:
~ 25 Abbreviations/Formulas: acetic acid = HOAc, acetonitrile = CH3CN, chloroform = CHC13, diethyl ether = Et20, dimethylform~mifle = DMF, ethyl acetate = EtOAc, l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide = EDAC, N-hydroxysuc~inimi~le = HOSu, methanol = MeOH, methylene chloride = CH2C12, tetrahydrofuran - THF.

W O 97/24621 PCT~US96119544 Analytical HPLCs were run using a Waters 8 x 100 mm mBC18R P
cartridge with a flow rate of 2 mL/min and 1 = 225 nm and preparative HPLCs were run using a Waters 40 x 100 mm mBC~8RP cartridge with a flow rate of 45 mL/min and 1 = 225 nm unless otherwise noted.
1P~ F l SYNIHESIS OF PORPHOBILINOGEN IMMUNOGEN (7) To a solution of adipic acid mono ethyl ester (22.8 g, 131 mmol3 in 1 0 dimethylform~mi~le (DMF) (100 mL) was added dicyclohexylcarbodiimide (DCC) (28.3 g, 137 mmol) and N-hydroxysuc~ inimi~1e (HOSu) (15.7 g, 137 mmol). The reaction was stirred for 20 hours under N2, filtered and solvents removed in vacuo. The oil was taken up in EtOAc (100 mL), filtered and dried in vacuo.
The crude material was purified by flash chromatography (50% EtOAc/50%
l 5 Hexane) to afford 30.6 g (85%) of Adipic active ester. lH NMR (d6 DMSO): d 4.05 (q, J=7.14 Hz, 2H), 2.80 ~s, 4H), 2.75-2.65 ~m, 2H), 2.40-2.27 (m, 2H), 1.70-1.55 (m, 4H), 1.16 (t, J=7.14 Hz,3H); mass spec IM+H]+ 272.
Adipic active ester (2.67 g, 9.83 mmol) in DMF (30 mL) was added to a solution of PBG (1) (800 mg, 3.28 mmol) in 0.1û M NaH2PO4 buffer (30 mL)J1M
2 0 Na2CO3 ~2.4 mL). The reaction was covered with foil, stirred for 6 hours under N2 and then solvents removed in vacuo. Preparative HPLC purification (25%
CH3CN/75% H2O + 0.1% formic acid) gave 840 mg (67%) of PBG-Adipic ethyl ester (2). lH NMR (d6 DMSO): d 10.24 (s, lH), 7.93 (t, J=6.41 Hz, lH), 6.39 (s, lH), 4.12 (d, J=4.48 Hz, 2H), 4.05 (q, J----7.12 Hz, 2H), 3.27 (s, 2H), 2.60-2.00 (m, 8H), 1.58-2 5 1.38 (m, 4H), 1.17 (t, J=7.12 Hz, 3H); mass spec [M+H~+ 383; HPLC (25%
CH3CN/75% H2O + 0.1% formic acid): 5.66 min, >97%.
tert-Butyl isourea (4.7 g, 23.5 mmol) was added to a 5~C solution of PBG-Adipic ethyl ester (2) (640 mg, 1.67 mmol) in DMF (6 mL) under argon, the reaction was stirred for 44 hours at room temperature. After adding wo 97/24621 17 rcT/uss6/lss44 tetrahydrofuran (THF) (15 mL), the reaction was filtered and solvents were removed in vacuo. Purification by preparative HPLC (60% CH3CN/40% ~2~ +
0.1% formic acid) afforded 300 mg (36%) of PBG-di-tcrt-butyl-Adipic etllyl ester(3). IH NMR (CDC13): d 8.67 (s, lH), 6.62 (t, J=5.38 Hz, lH), 6.41 (s, lH), 4.29 (d, ~=5.56 Hz, 2H), 4.11 (q, J=7.12 Hz, 2H), 3.32 (s, 2H), 2.72-2.10 (m, 8H), 1.70-1.55 (m, 4H), 1.44 (s, 9H), 1.43 (s, 9H), 1.24 (t, J=7.13 Hz, 3H); mass spec [M+H]+ 495; HPLC
(60% CH3CN/40% H2O + 0.1% formic acid): 8.09 min, >93%.
To a 5~C solution of PBG-di-tert-butyl-Adipic ethyl ester (3) (300 mg, 0.607 mmol) in EtOH (7 mL) was added a solution of H2O (7 mL)/KOH (102 mg, 1.82 1 0 mmol); the reaction was warmed to room temperature, covered with foil, stirred for 3 hours under argon and solvents removed in vacuo. The residue was dissolved in H2O (150 mL), acidified to pH = 2.8 with 1.5 M H3PO4 and extracted with EtOAc (4 x 100 mE). The combined extracts were washed with brine, dried over Na2SO4, filtered and dried in vacuo to give 220 mg (78%) of 1 5 PBG-di-tert-butyl-Adipic hapten (4). IH NMR (CDCl3): d 8.90 (s, lH), 6.89 (t, ~=5.61 Hz, lH), 6.44 (s, lH), 4.30 (d, J=5.73 Hz, 2H), 3.33 (s, 2H), 2.68 (t, J=7.93 Hz, 2H), 2.45 (t, J=7.93 Hz, 2H), 2.33 (t, J= 6.63 Hz, 2H), 2.20 (t, J=6.87 Hz, 2H), 1.70-1.55 (m, 4H), 1.44 (s, 9H), 1.43 (s, 9H); mass spec [M+H]+ 467; HPLC (60% CH3CN/40%
H2O + 0.1% formic acid): 4.07 min, >72%.
2 0 A solution of EDAC (183 mg, 0.955 mmol) in DMF (6 mL) was added to a solution of PBG-di-tert-butyl-Adipic hapten (4) (110 mg, 0.236 mmol), HOSu (110 mg, 0.956 mmol) in DMF (2 mL); the reaction was covered with foil and stirred for 20 hours under argon. After removing solvents in vacuo, the residue was dissolved in a Et2O/0.05 M NaH2PO4 buffer (pH = 5.5) solution and extracted 2 5 with Et2O (4 x 50 mL). The combined extracts were washed with brine, dried over Na2SO4, filtered and dried in vacuo. Yield was 105 mg (80%) of PBG-di-tert-butyl-Adipic active ester (5). HPLC (60% CH3CN/40% H2O + 0.1% formic acid): 5.76 min, >77%.

W O 97/24621 18 PCTrUS96/19544 PBG-di-tert-butyl-Adipic active ester (5) (37 mg, 0.066 mmol) in DMF (16 mL) was added to a 5~C solution of BSA (210 mg) in 0.1 M NaH2PO4 buffer (pH =
7.7, 20 mL); the reaction was warmed to room temperature, covered with foil and stirred for 40 hours under N2. Purification by gel filtration [60 g G-25 S Sephadex, 20% MeOH/80% H2O (100 mM ammonium acetate)], fractions collected and lyophilized. Yield was 280 mg of PBGdi-tert-l:~utyl-Adipic BSA
Immunogen (6).
Trifluoroacetic acid (T~A) (10 mL) was added to a suspension of PBG-di-tert-butyl-Adipic BSA Tmmllnogen (6) (280 mg) in CH2Cl2 (10 mL), the reaction was l 0 stirred for 20 minutes under N2 and then solvents removed in vacuo. The residue was dissolved in H20 (80 mL), neutralized with lM NH40H and mixture lyophilized. Dissolved solid in H2O (lOO mL), neutralized with lM
NH40H and then lyophili7e~1 The resulting solid was dialyzed in H20 (4 L) for 6 hours and lyophili~etl to give 240 mg of PBG-Adipic-BSA Immunogen (7).
l S
EXA~PT~F ?
SYNTHESIS OF PORPHOBILINOGEN IMMUNOGEN (14) A solution of di-tert butyl dicarbonate (BOCzO, 2.15 g, 9.4 mmol) in 15 mL
2 0 tetrahydrofuran (THF) was added to a solution of porphobilinogen hydrate (1)(2.3 g, 9.4 mmol) in 30 mL 0.5 M aqueous sodium carbonate (15 mmol) and 15 mL THF. The reaction mixture was stirred under nitrogen for 4 hours, another 10 mL 0.5 M sodiurn carbonate and a 10 mL solution of BOC2O (2.1 g, 9.4 mmol) in THF were added to the reaction mixture and stirred for another 4 hours.
HPLC indicated <2% starting material and >95~O desired product. The crude mixture was poured into 200 mL water/5 mL 1 M NaOH after removing solvents in vacuo and extracted with 50 mL Et2O. The aqueous layer was acidified with 1 M phosphoric acid, extracted 3 x 10~) mL EtOAc, dried combined extracts over sodium sulfate and removed solvents in vacuo to afford 2.86 g W O 97/24621 PCTrUS96/19544 (94%) N-BOC PBG diacid (8). IH NMR (DMSO-d6) d 12.02 (br s, lH), 11.98 (br s, lH), 10.14 (br s, lH), 6.81 (s, lH), 6.36 (s, lH), 4.01 (d, J=5.56 Hz, 2H), 3.45 (s, 2 H), 2.77 (t, J =7.09 Hz, 2~I), 2.56 (t, J=7.09 Hz, 2H), 1.43 (s, 9H); mass spec (M)+ 324.
ter~-Butyl isourea (19.6 g, 98 mmol) was added to a 10~C solution of N-BC:)C
PBG diacid (8) (2.8 g, 8.6 mmol) in 15 mL dimethylformamide (DMF) under nitrogen and stirred 16 hours at room temperature. Reaction mixture was poured into 45 mL distilled w~ter and 50 mL phosphate buffer (500 mM, pH=6.0), extracted 3 x 125 mL Et2O, washed combined extracts with 70 mL brine and removed solvents in vacuo. Purification by preparative HPI C afforded 1.7 1 0 g (45%) di tert-butyl ester N-BOC PBG (9). lH NMR (CDC13) d 8.58 (br s, 1~), 6.45 (s, lH), 5.26 (br s, lH), 4.15 (d, J=6.07 Hz, 2H), 3.30 (s, 2 H), 2.69 (t, J=8.47 Hz, 2H), 2.45 (t, J=8.47 Hz, 2H), 1.43 (sl br s, 27 H); mass spec (M)~ 438.
Potassium hexamethyldisilylazide (0.50 M, 1.2 mL, 0.60 rnmol) was added to a 0~C solution of di tert-butyl ester N-~OC PBG (9, 219 mg, 0.50 mmol) in 5 mL
1 5 THF under nitrogen and stirred for 30 minutes at 0~C. A solution of benzyl 4-chlorosulfonylbutyrate (166 mg, 0.6 mmol) in 1 mL THF was added to the reaction mixture and stirred overnight. Workup consisted of pouring the reaction into a mixture of 50 mL water, 5 mL saturated sodium bicarbonate and extracting with 3 x 75 mL EtOAc, removing solvents in vacuo and purifying the 2 0 residue by preparative HPLC to afford 128 mg (37%) of the desired benzyl N1-sulfonyl compound (10). IH NMR (CDCl3) d 7.39 (br s, 5H), 6.84 (s, lH3, 5.33 (br s, lH), 5.11 (s, 2H), 4.36 (d, J=6.2 Hz, 2H), 3.52 (s, 2H), 3.31 (t, ~= 7.5 Hz, 2H), 2.63 (t, J=7.9 Hz, 2H), 2.51-2.39 (m, 4H), 2.03 (p, J=7.3 Hz, 2H), 1.43 (sl br s, 27 H); mass spec (M+H)+ 679.
A mixture of Nl-sulfonyl compound (10) (110 mg, 0.16 mmol), 25 mL
absolute ethanol and 10% palladiurn on carbon (Pd/C, 60 mg) was stirred under 1 atm hydrogen for 16 hours. Piltered off catalyst and removed solvents in vacuo to afford 92 mg (84%) desired Nl-sulfonyl compound (11). IH NMR
(CDC13) d 7.90 ~v br s, lH), 6.87 (s, lH), 5.35 (br s, lH), 4.37 (d, J=5.8 Hz, 2H), 3.56 W O 97/24621 PCT~US96/19S44 (br s, 2H3, 3.36 (t, J-7.3 Hz, 2H), 2.63 (t, J=7.6 Hz, 2H), 2.46 (t, J=7.6 Hz, 2H), 2.39 (t, J=7.2 Hz, 2H), 2.02 (p, J=7.2 Hz, 2H), 1.44 (sl br s, 27 H); mass spec (M)+ 589. EDAC (115 mg, 0.60 mmol) was added to a solution of Nl-sulfonyl compound (11) (70 mg, 0.12 mmol), HOSu (136 mg, 1.2 mmol) and 3 mL DMF
5 then stirred under nitrogen for 14 hours. The reaction mixture was poured into70 mL Et2O, washed 1 x 30 mL phosphate buffer (500 mM, pH=6.0) and removed solvents in vacuo to afford 67 mg (82%) of desired active ester (12). lH NMR
(CDCl3) d 6.88 (s, lH), 5.32 (br s, lH), 4.38 (d, J=5.9 Hz, 2H), 3.53 (s, 2 H), 3.40 (t, J=7.2 Hz, 2~I), 2.83 (s, 4H), 2.74 (t, J=7.2 Hz, 2H), 2.63 (t, J=7.2 Hz, 2H), 2.20-2.08 (m, l 0 4H), 1.43 (sl br s, 27 H); mass spec (M+H)+ 686.
A solution of active ester (12) (44 mg, 0.064 mmol)/2 mL DMF was added to a solution of bovine serum albumin (BSA, 220 mg), 22 mL phosphate buffer (50 mM, pH=7.8) and 6 mL DMF, then stirred for 15 hours at room temperature.
Purification of the protected immunogen by G-25 column chromatography and 1 5 lyophilization of fractions containing purified immunogen afforded 278 mg of desired protected immunogen (13).
Trifluoroacetic acid ~12 mL) was added to a suspension of the protected immunogen ~13) (277 mg) in methylene chloride (12 mL) and stirred for 30 minutes at room temperature. The reaction mixture solvent were removed solvents in VRCUO and stirred with 40 mL aqueous ammonium acetate (100 mM) for 15 hours to give a homogeneous solution which was lyophilized. The material was dissolved in 50 mL water, lyophilized and repeated two times to give 210 mg desired porphobilinogen immunogen (14).

2 5 ~A~PI.F 3 SYNTHESIS OF PORPHOBILINOGEN IMMUNOGEN (19) Trichloroacetyl chloride (207 mL, 1.85 mmol) was added to a mixture of di tert-butyl ester N-BOC PBG ~9, 675 mg, 0.50 mmol), anhydrous potassium W O 97/24621 PCTrUS96119544 carbonate (2.24 g, 16 mmol) and 15 mL anhydrous Et2O, the reaction mixture was stirred for 30 minutes under nitrogen at 0~C. Poured mixture into 200 mL
Lt2O/100 mL water, 100 mL saturated sodium bicarbonate, separated, extracted aqueous layer 1 x 50 mL Et2O and dried combined organic extracts over sodium 5 sulfate. Removal of solvents in vacuo afforded 805 mg of crude C-5 substituted trichloromethyl ketone (15). Mass spec (M+ NH4)~ 602.
Aqueous sodium hydroxide (1.0 M, 3.5 mL, 3.5 mmol) was added to a solution of crude C-5 substituted trichloromethyl ketone (15) (805 mg, 1.5 mmol), 30 mL acetone and 6 mL water, the reaction mixture was stirred for 25 1 (1 minutes then removed acetone in vacuo. The crude mixture was poured into 100 mL water, extracted with 50 mL Et2O, adjusted pH of aqueous layer to 2.5 with 1.0 M phosphoric acid and extracted 3 x 100 mL EtOAc. The combined EtOAc extracts were washed 1 x 30 mL brine and removed solvents tn vacuo to give a dark oil which was purified by preparative HPLC to afford 122 mg (51%
1 5 from 9) desired C-5 substituted carboxylic acid (16). lH NMR ~CDC13) d 9.88 (br s, 2H), 5.5Z (br s, lH), 4.28 (d, J=5.5 Hz, 2H), 3.40 (s, 2 H),3.02 (t, J=7.8 Hz, 2H), 2.49 (t, J=7.8 Hz, 2H), 1.44 (sl br s, 27 H); mass spec (M+H~+ 483.
EDAC (289 mg, 1.25 mmol) was added to a solution of C-5 substituted carboxylic acid (16) (122 mg, 0.25 mmol), HOSu (288 mg, 2.5 mmol) and 3.3 mL
2 0 DM~ then stirred under nitrogen for 14 hours. The reaction mixture was poured into 100 mL Et2O, washed 1 x 50 mL phosphate buffer (500 mM, pH=6.0), washed 1 x 50 mL water and removed solvents in vacuo to afford 143 mg (98%) of desired active ester (17). IH NMR (CDCl3) d 10.02 (br s, lH), 5.50 ~br s, lH), 4.24 (d, J=5.5 Hz, 2H), 3.42 (s, 2 H), 3.05 (t, J=7.6 Hz, 2H), 2.82 (s, 4H), 2.51 (t, J=7.6 Hz, 2 5 2H), 1.45 (sl br s, 27 H); mass spec (M+H)+ 597.
A solution of active ester (17) (65.5 mg, 0.113 mmol)/2 mL DMF was added to a solution of bovine serum albumin (BSA, 150 mg), 50 mL phosphate buffer (50 mM, pH=8.8) and 10 mL DMF, then stirred for 3 days at room temperature.
Purification of the protected immunogen by G-25 column chromatography and W O 97/24621 22 PCT~US96/19544 lyophilization of fractions containing purified immunogen afforded 207 mg of desired protected immunogen (18).
Trifluoroacetic acid (10 mL) was added to a suspension of the protected immunogen (18) (206 mg3 in methylene chloride (10 mL) and stirred for 30 5 minutes at room temperature. The reaction solvents were removed in vacuo and resulting residue wa~ stirred with 40 mL aqueous ammonium acetate (100 mM) for 15 hours to give a homogeneous solution which was lyophilized. The material was dissolved in 50 mL water, lyophilized and repeated two times to give 153 mg desired porpho~;linogen immunogen (19).

1PT .F 4 SYNTHESIS OF PORPHOBILINOGEN TRACER (21) To a solution of 6-carboxyfluorescein (34 mg, 0.090 mmol) in 1 5 dimethylform~ le (1.7 mL, DMF) was added dicyclohexyl-carbodiimide (18 mg, 0.086 mmol) and N-hydroxysuccinimide (11 mg, 0.087 mmol), the reaction was covered with foil then stirred for 20 hours under N2. The reaction was filtered to afforded a solution of 6-carboxyfluorescein active ester (20) which was added to a solution of porphobilinogen (1) 20 mg, 0.082 mmol) in 0.10 M phosphate (pH=7.7, 2 0 4 mL). The reaction was covered with foil then stirred for 20 hours under N2.
and solvents removed in vaCuo. The crude material was purified twice by HPLC
(30% CH3CN/70% H2O + 0.1% formic acid) to give 8 mg (17%) of PBG-6-AMF (21).
H NMR (CD30D~: d 8.15-8.00 (m, 2H), 7.65 (s, lH), 6.70-6.40 (m, 7H), 4.45 (s, 2H), 3.45 (s, 2H), 2.275-2.40 (m, 4H); mass spec (M+H)+ 585; HPLC (30% CHICN/70%
2 5 H2O + 0.1% ~ormic acid): 8.14 min, >97%.

CA 02239969 l998-06-08 W O 97/24621 PCT~US96/19544 ExA~lpT~F 5 SYNTHESIS OF PORPHOBIL~NOGEN TRACER (27) 4-Dimethylaminopyridine (DMAP) (1.18 g, 9.7 mmol) was added to a room temperature solution of t-butyl 4-nitrobutyrate (2.74 g, 14.5 mmol) and 3-t(tetrahydro-2H-pyran-2-yl)oxy] propanal (1.53 g, 9.7 mmol) in dry CH2Cl2 and stirred for 96 h. The solvent was removed in vacuo and the crude mixture was purified by silica gel column chromatography (20-35% EtOAc in n-hexane) to l 0 afford a-hydroxynitro compound (22) 2.45 g (73%) as a colorless thick gum. 1H
NMR (CDC13): d 4.62-4.54 (m, 2H), 4.3û-3.45 (m, SH), 2.41-2.10 (m, 4H), 1.90-1.70 (m, 2H), 1.57-1.51 (m, 6H), 1.44 (s, 9H); mass spec (M+NH4)+ 365.
Acetic anhydride (4.83 mL, 51.3 mmol) was added dropwise to a 0~C solution of a-l~ydroxynitro compound (22) (4.45 g, 12.8 mmol), anl-ydrous pyridine (1.15 1 5 mL, 19.2 mmol) and dry CH2Cl2 (50 mL); after 2.0 h, tl~e mixture was allowed to warm to room temperature and stirred for 14 h. Workup consisted of pouring the reaction mixture into 10% aqueous NaHCO3 (50 mL), separated layers and extracted the aqueous layer with CH2Cl2 (2 X 50 m~). The combined organic layers were washed with 5% HCl (20 mL), water (30 mL), brine (15 mL), dried (MgSO4) and the solvents were removed in vacuo. Purification by silica gel column chromatography (20% EtOAc in n-l~exane) afforded 2.63 g of a-acetoxynitro compound (23) (51%) as a colorless thick liquid. 1H NMR (CDCl3):
d 5.50-5.40 (m, lH), 4.90-4.80 (m, lH), 4.60 (br s, lH), 4.51 (br s, lH), 3.90-3.72 (m, 2H), 3.58-3.32 (m, 2H), 2.45-2.15 (m, 4H), 2.08 (s, 3H), 2.04 (s, 3H), 2.04-1.50 (m, 8H), 2 5 1.44 (s, 9H); mass spec (M+NH4)+ 407.
1,8-Diazabicydol5.4.0]undec-7-ene (DBU) (0.76 mL, 5.08 mmol) was added to a 0~C mixture of a-acetoxynitro compound (23) (1.65 g, 4.24 mmol), benzylisocyanoacetate (0.890 g, 5.08 mmol) and TH~ (14 mL), stirred for 30 min at 0~C, warmed to room temperature and stirred for 24 h. The resulting orange-red CA 02239969 l998-06-08 W O 97/24621 PCT~US96/19544 color solution was quenched with water (20 mL) and extracted with F~OAc (3 X
50 mL). The combined organic layers were washed with water (20 mL), brine (15 mL), dried (MgS04) and the solvents were removed in vacuo. The crude compound was purified by silica gel column chromatography (20% EtOAc in n-S hexane) to afford 1.21 g of 24 (63%) as a colorless g~m. lH NMR (CDC13): d 8.86 (br s, lH), 7.45-7.26 (m, 5H), 6.70 (s, lH), 5.30 (m, 2~), 4.51 (br s, lH), 3.88-3.70 (m, 2H), 3.56-3.38 (m, 2H), 3.04 (t, J=7.2 Hz, 2H), 2.75 (t, J=7.8 Hz, 2H), 2.47 (t, J=7.5 Hz, 2H), 1.80-1.43 (m, 6H), 1.42 (s, 9H); mass spec (M+NH4)+ 475.
In a dry single necked round bottom flask equipped with magnetic stir bar, l 0 nitrogen inlet was placed 2-carbobenzyloxy pyrrole (24) (1.20 g, 2.18 mmol) in methanol (20 mL) and added pyridinium p-toluenesulfonate (PPTS, 0.58 g, 2.29 mmol) at room temperature and stirred for 48 h. The solvent was removed in vacuo and the mixture was diluted with water (30 mL)/EtOAc (75 mL). The aqueous layer was extracted with EtOAc (2 X 50 mL) and the combined organic 1 5 extracts were washed with brine (20 mL) and concentrated in vacuo. Purification by silica gel column chromatography (30-40% EtOAc in n-hexane) gave 1.66 g (92%) of pyrrole alcohol (25). 1H NMR (~DC13): d 8.90 (br s, lH), 7.42-7.27 (m, 5H), 6.71 (d, J=2.7 Hz, lH), 5.29 (s, 2H), 3.79-3.74 (m, 2H), 3.03 (t, J=6.3 Hz, 2H), 2.73 (t, J=7.8 Hz, 2H), Z.47 (t, J=7.8 Hz, 2H), 2.15-2.05 (m, lH), 1.41 (s, 9~I); mass spec 2 0 (M+H)~ 374.
Jones reagent (2.67 M, 0.64 mL, 1.69 mmol) was added in one portion to a 0~C
solution of pyrrole alcohol (25) (0.400 g, 1.13 mmol) in acetone (20 mL) and stirred for 1.5 h then quenched with isopropanol (3.0 mL~ and stirred for 30 minat rt. The mixture was diluted with acetone (22 mL), filtered and washed with 2 5 acetone (30 mL). After concentrating the filtrate in vacuo, the resulting crude acid was dissolved in DMF (0.5 mL), cooled to 0-5~C with ice bath and added a solution of O-~-butyl-N,N'-diisopropyl-isourea (0.313 g, 5.0 eq.) in DMF (1.0 mL).
The cooling bath was removed and the mixture was stirred for 43 h at room temperature before removing solvents in vacuo. Purification by silica gel .

column chromatography (20% EtOAc in n-hexane) afforded 0.190 g of tl2%) pyrrole di t-butyl ester (26). 1H NMR (CDC13): d 8.88 (br s, lH), 7.26-7.41 (m, 5H), 6.73 (d, J=3.0 Hz, lH), 5.29 (s, 2H), 3.77 (s, 2H), 2.70 (t, J=7.2 Hz, 2H), 2.48 (t, J=6.6 Hz, 2H), 1.42 (s, 9H), 1.40 (s, 9H); mass spec calcd for C25H34NO6: 444.2386; found:
444.2383; HPLC: 0.1% formic acid in water:MeCN (75:25), 4.15 min, 97%.
Palladium on carbon (10% Pd/C) (0.029 g) was added to a solution of pyrrole di-t-butyl ester (26) (0Ø94 g, 0.212 mmol) in absolute ethanol (4 mL) and stirred under hydrogen (1 atrn) for 2.0 h at room temperature. The mixture was diluted with ethanol (10 mL), filtered and solvent removed in vacuo to afford 0.065 g 1 0 (89%) of 2-acyl analogue of PBG (27). 1H NMR (acetone-d6): d 10.52 (br s, lH), 6.88 (d, J=3.3 Hz, lH), 3.84 (s, 2H), 2.71 (t, J=8.4 Hz, 2H), 2.49 (t, ~=8.4 Hz, 2H), 1.47 (s, 9H), 1.45 (s, 9H); mass spec calcd for C18H28NO6: 354.1917; found: 354.1914; HPLC:
0.1% formic acid in water:MeCN (25:75), 2.57 min, 96%.
EDAC (0.033 g, 0.175 mmol) was added to a room temperature solution of l 5 acid (27) (0.017 g, 0.05 mmol) and HOSu (0.015 g, 0.125 mmol) in dry DME (0.5 mL) and stirred for 36 h. The mixture was diluted with potassium phosphate pH
6.0 buffer solution (3 mL) and extracted with Et2O (3 X 20 mL). The combined ethereal extracts were washed with water (5 mL), dried (MgSO4) and concentrated in vaCuo to afford 0.021 g of (89%) 2-carboxypyrrole active ester (28).
2 0 1H NMR (CDC13): d 10.32 (br s, lH), 7.12 (s, lH), 3.88 (s, 2H), 2.97 (s, 4H), 2.83 (t, J=8.1 Hz, 2H), 2.59 (t, J=7.8 Hz, 2H), 1.57 (s, 9H), 1.55 (s, 9H); mass spec (M+NH4)+
468; HPLC: 0.1% formic acid in water:MeCN (25:75), 2.68 min, 89%.
In a dry single-necked round bottom flask equipped with magnetic stir bar was placed active ester (28) (0.008 g, 0.017 mmol) in dry DMF (0.5 mL); added 6-2 5 (aminomethyl)-fluorescein hydrochloride (6-AMF) (0.010 g, 0.025 mmol) followed by triethylamine (Et3N) (0.018 mL, 0.13 mmol) to the reaction mixture and stirred at room temperature for 18 h. The mixture was pllrified by preparative reversed phase C18 HPLC, eluting with 0.1% formic acid in water:MeCN/45:55 (v/v), to afford 9.6 mg (77%) of 2-acyl analogue of PBG tracer W O 97/24621 PCTrUS96/19544 di-t-~utyl ester (29~. I H NMR (CD30D): d 7.96 (d, J=7.8 Hz, lH), 7.68 (d, J=7.8 Hz, lH), 7.21 (s, lH), 6.69-6.48 (m, 7H), 4.59 (s, 2H), 3.52 (s, 2H), 2.67 (t, J=7.5 Hz, 2H), 2.43 (t, J=7.2 Hz, 2H), 1.38 (s, 9H), 1.34 (s, 9H); mass spec (M~H)+ 697; HPLC: 0.1%
formic acid in water:MeCN (45:55), 6.43 min, >99%.
Trifluoroacetic acid (1.0 mL) was added to a mixture of di-t-butyl ester (29) (0.010 g, 0.14 mmol) in dry CH2Clz (1.0 mL), and stirred at room temperature for1.5 h. The mixture was purified by preparative reversed phase Cl8 HPLC, eluting witl 0.1% formic acid in water: MeCN (70:30) to afford 2.4 mg (34%) of PBG 2-acyl analogue tracer (30). 1H NMR (CD30D): d 7.95 (d, J=7.8 Hz, lH), 7.72-1 0 7.68 (m, lH), 7.21 (s, lH), 6.67-6.51 (m, 7H), 4.61 (s, 2H), 3.37 (s, 2H), 2.77 (t, J=7.8 Hz, 2H), 2.50 (t, J=7.2 Hz, 2H); mass spec (M)+ 584; HPLC: 0.1% formic acid in water:MeCN (70:30), 7.08 min, >99%.

Fx~ yIp~F 6 l 5 SYNTHESIS OF PORPHOBILINOGEN TRACER (32) In a dry single-necked round bottom flask equipped with magnetic stir bar was placed ~-t-BOC-6-amino hexanoic acid (1.15 g, 5.0 mmol), HOSu (0.69 g, 6.0 mmol) and dry DMF (15 mL); added EDAC (1.24 g, 6.5 mmol) at room 2 0 temperature and stirred for 24 h. The solvent was removed in vacuo and diluted with water (25 mL). Extracted the aqueous mixture with Et2O (50 mL, 2 X
30 mL), combined Et2O layers were washed with water (2 X 20 mL), brine (15 mL) and dried (MgSO4). Removal of the solvent in vacuo afforded 1.52 g of (93%) of N-t-BOC-6-amino hexanoic acid active ester as a colorless solid. 1 H NMR
2 5 (CDC13): d 4.60 (br s, lH), 3.14-3.05 (m, 2H), 2.97 (s, 4H), 2.83 (s, 4H), 2.61 (t, J=7.2 Hz, 2H), 1.71-1.82 (m, 2H), 1.54-1.40 (m, 4H), 1.43 (s, 9H); HPLC: 0.1% formic acid in water:MeCN (50:50), 8.98 min.
Triethylamine (0.060 mL, 0.45 mmol) was added to a room temperature solution of N-t-BOC-6-amino hexanoic acid active ester (û.014 g, 0.045 mmol), 6-W O 97/24621 27 PCTrUS96/19544 (aminomethyl)fluorescein hydrochloride (6-AMF) (0.020 g, 0.05 mmol) and dry DMF (0.4 mL); the mixture was stirred for 24 h at room temperature.
Purification by preparative HPLC, eluting with 0.1% formic acid in water:MeCN
(32:68) afforded 0.026 g (95%) of N-t-BOC-6-aminohexanoic-(6-aminomethyl)-~ 5 fluorescein. lH NMR (CD30D): d 8.05 (d, J=8.1 Hz, lH), 7.69 (d, J=6.9 Hz, lH), 7.19 (s, lH), 6.87-6.70 (m, 6H), 4.51 (d, J=6.0 Hz, 2H), 3.12-3.05 (m, 2H), 2.30-2.20 (t, J---7.8 Hz, 2H), 1.65-1.46 (m, 4H), 1.~6 (s, 9H), 1.35-1.25 (m, 2Hj; mass spec (M+H)+ 575;
HPLC: 0.1% formic acid in water:MeCN (60:40), 7.98 min, 96%.
In a dry single-necked round bottom flask equipped with magnetic stir bar 1 0 was placed the above prepared N-t-BOC-6-aminohexanoic-(6-aminomethyl)-fluorescein (0.024 g, 0.069 mmol) in dry CH2C12 (1.0 mL); added trifluoroacetic acid (0.5 mL~ and stirred at room temperature for 10 min. The mixture was purified by preparative reversed phase C18 HPLC, eluting with 0.1% formic acid in water:MeCN (60:40) to give 20 mg (>95%) of aminofluorescein derivative. 1H
1 5 ~MR (CD30D): d 7.96 (d, J=8.1 Hz, IH), 7.55 (d, J=8.7 Hz, lH), 7.10 (s, lH), 6.79-6.53 (m, 6H), 4.86 (s, 2H), 2.83 (t, J=8.1 Hz, 2H), 2.21 (t, J=7.5 Hz, 2H), 1.62-1.50 (m, 4H), 1.38-1.29 (m, 2H); mass spec (M+H)+ 475; HPLC: 0.1% formic acid in H20:MeCN
(60:40), 2.54 min, >99%.
Triethyl~rnine (0.020 mL, 0.15 mmol) was added to a solution of active ester 2 0 (28) (6 mg, 0.013 mmol), aminofluorescein compound (7 mg, 0.014 mmol) and dry DMF (0.5 mL); the reaction mixture was stirred at room temperature for 20 h.Purification by preparative reversed phase C18 HPLC, eluting with 0.1% formic acid in water:MeCN (45:55) afforded 4.5 mg (45%) of di-t-butyl ester tracer (31).
1H NMR (CD30D): d 7.97-7.94 (m, lH), 7.80-7.78 (m, lH), 7.08 (s, lH), 6.68-6.53 (m, 2 S 7H), 4.40 (s, 2H), 3.62 (s, 2H), 2.70 (t, J=7.5 Hz, 2H), 2.47 (t, J=8.1 Hz, 2H), 2.20-2.16 (m, 2H3, 1.60-1.20 (m, 8H), 1.42 (s, 9H), 1.40 (s, 9H); mass spec (M+H)+ 810; HPLC:
0.1% formic acid in water:MeCN (45:55), 6.27 min, 98%.
Trifluoroacetic acid (1.0 mL) was added to a 5~C mixture of di-t-butyl ester (31) (4 mg, 0.005 mmol) in dry CH2Cl2 (1.0 mL) and stirred at room temperature W O 97/24621 PCTrUS96/19544 for 1.5 h. The mixture was purified by preparative reversed phase Clg HPLC, eluting with 0.1% formic acid in water:MeCN (60:40) to afford 2 mg (59%) of 2-acyl analogue of PBG tracer (32). 1H NMR (CD30D): d 7.90 (d, J=7.5 Hz, lH), 7.60-7.55 (m, lH), 7.07 (s, lH), 6.69-6.50 (m, 7H), 4.42 (s, lH), 4.39 (s, lH), 3.53 (s, 2H), 2.78 (t, J=7.5 Hz, 2H), 2.53 (t, J=7.8 Hz, 2H), 2.17 (t, J=6.9 Hz, 2H), 1.56-1.24 (m, 8H);
mass spec (M+H)~ 698; HPLC: 0.1% formic acid in water: MeCN (60:40), 3.46 min, 99%.

FX ~P~.F.7 1 0 SYNTHESIS OF PORPHOBILINOGEN TRACER (33) EDAC (19.2 mg, 0.10 mmol) was added to a solution of N1-sulfonyl compound (11) (20 mg, 0.034 mmol), HOSu (19.6 mg, 0.17 mmol) and 1 mL DMF
then stirred under nitrogen for 14 hours. The reaction mixture was poured into l 5 50 mL Et2O, washed 1 x 30 mL phosphate buffer (500 mM, pH=6.0) and removed solvents in v~cuo to afford 22.8 mg (99%) of desired active ester (12). lH NMR
(CDCl3) d 6.88 (s, lH), 5.32 (br s, lH), 4.38 (d, J=5.9 Hz, 2H), 3.53 (s, 2 H), 3.40 (t, J=7.2 Hz, 2H), 2.83 (s, 4H), 2.74 (t, J=7.2 Hz, 2H), 2.63 (t, J=7.2 Hz, 2H), 2.20-2.08 (m, 4H), i.43 (sl br s, 27 H); mass spec (M+H)+ 686.
2 0 5-Aminomethylfluorescein (25 mg, 0.048 mmol) was added to a solution of active ester ~12) (22.7 mg, 0.033 mmol), diiopropylethylamine (44 mL, 0.25 mmol) and 1 mL DMF, the reaction was stirred for 18 hours and solvents removed in vaCuo. Preparative HPLC purification gave 14.3 mg (46%) of desired protected tracer (33). lH NMR (CDCl3) d 7.88-7.80 (m, lH), 7.52-7.40 (m,2 5 lH), 7.05-6.92 (m, lH), 6.87 (s, 2H), 6.70-6.30 (m, 4H), 5.40 (br s, lH), 4.52-4.28 (m, 4H), 4.11~.02 (m, 2H), 3.65-3.52 (m, 2H), 3.35-3.20 (m, 2H), 2.65-2.52 (m, 2H), 2.51-2.42 (m, 4H), 2.10-1.95 (m, 2H), 1.43 (sl br s, 27~I); mass spec (M)+ 932.
Trifluoroacetic acid (1 mL) was added to a suspension of protected tracer (33) (14 mg, 0.015 mmol) in 1 mL methylene chloride, stirred 30 minutes at room W O 97/24621 PCT~US96/19544 temperature and solvents removed in vacuo to afford an orange residue.
Preparative HPLC purification gave 3.1 mg (28%) of desired protected tracer (34).
lH NMR (CD30D + 5 drops CDC13) d 8.03 (s, lH), 7.76 (d, J=8.Q Hz, lH), 7.26 (d, J=8.0 Hz, 2H), 7.14 (s, lH), 6.97 (s, 2H), 6.95 (d, J=8.9 Hz, 2H), 6.80 (d, J=8.9 Hz, 2H), 4.55 (s, 2H), 4.36 (s, 2H), 3.63 (s, 2H), 3.60-3.46 (m, 2H), 2.71 (t, J=7.1 Hz, 2H), 2.55 (t, J=7.1 Hz, 2H), 2.01 (t, J=7.2 Hz, 2H); mass spec (M+H)+ 720.

SYNTHESIS OF PORPHOBILINOGEN TRACER (36) l O

5-Aminomethylfluorescein (50 mg, 0.085 mmol) was added to a solution of active ester (17) (24.3 mg, 0.042 mmol), diiopropylethylamine (73 mL, 0.42 mmol) and 0.80 mL DMF, the reaction was stirred for 3.5 days at 50~C and solvents removed in vacuo. Preparative HPLC purification gave 23 mg (66%~ of 1 5 desired protected tracer (35) 1H NMR (CDCl3 + 2 drops CD30D) d 7.99 (s, lH),7.71 (d, J=6.1 Hz, 2H), 7.12 (d, J=6.1 Hz, 2H), 6.68 (s, 1~), 4.74 (br s, 2H), 4.23 (br s, 2H), 3.35 (s, 2H), 3.00-2.92 (m, 2H), 2.62 (t, J=7.1 Hz, 2H), 1.47 (sl br s, 18 H), 1.38 (s, 9H); mass spec (M)+ 826.
Trifluoroacetic acid (3.5 mL) was added to a suspension of protected tracer (35) (23 mg, 0.028 mmol) in 3.5 mL methylene chloride, stirred 30 minutes at room temperature, added 5 mT toluene and solvents removed in ~acuo to afford an orange residue. Preparative HPLC purification afforded 7.8 mg (45%) of desired protected tracer (36); mass spec (M)+ 613.

2 5 FXAMPI.F g A~ll~i~.l~A PRODUCTION

Imrnunogens of Formulas II, III and IV were used for antisera production.
Each of the immunogens was used for immuni2:ation in a separate group of W O 97/24621 rcTrusg6/lg544 rabbits and a separate group of sheep. The rabbits were initially immunized with 0.5 mg of immunogen and subsequently boosted with 0.25 mg of the immunogen every 6 weeks while the sheep were initially immunized with 1 mg of immunogen and subsequently boosted with 0.5 mg of the immunogen 5 every 6 weeks. l~nimAl~ were bled at 2 weeks and the bleeds were titrated to select antisera collections demonstrating adequate binding and displacement at a reasonable dilution. Each bleed obtained from all ~nimAl~ were tested for binding of tracer using all tracers synthesized. Testing was performed on the Abbott IMx(g) analyzer, different dilution's of the antisera in FPIA buffer werel 0 combined with the tracers in a 1 mL final volume for 5-30 minutes. Only antibodies derived from immunogens (7) and (14) showed significant binding with selected tracers. A typical pool for measuring the levels of lead via the quantification of porphobilinogen is diluted 1 to 100, has a binding of about 180 millipolarization units (mP) and a displacement of about 65 mP's with a lead l 5 solution containing 800 mg lead per milliliter. Figure 10 shows a bar graph of polarization vs. selected antibodies (derived from immunogens of present invention)/selected tracers. Each bar represents the polarization obtained at a 1/100 antisera dilution when combined with the matching tracer. A working calibration curve is demonstrated using tracer (21) and antibodies derived from 2 0 immunogen (7) and using tracer (34) and antibodies derived from immlmogen (14) as shown in Figure 9.

~AMPLE 10 SAMPLE PRETREATMENT FOR QUA~TIFICATION OF PORPHOBILINOGEN

A pretreatment solution (200 mL), consisting of 9% trichloroacetic acid, 0.6 N HNO3 and 5 mM periodic acid, was added to 200 mL of test sample in a 1.5 mL microfuge tube which was capped and vortexed for 30 seconds.
Centrifugation at 10,000 rpm for 2 minutes afforded a translucent supernatant W O 97/24621 31 PCTrUS96tl9544 which was used without further modification in the assay described in Example 11.

F~Al~PT.R 11 S LEAD STANDARD CUl~V~

A gravimetric solution of lead (Pb) traceable to NIST purchased from GFS
Chemicals was diluted to 80, 60, 40 and 20 mg/dl in water that had been adjustedto pH 0.5 with HNO3. This was neutralized to p~I 7.1 with a pH 7.85 l 0 neutralizing buffer consisting of 1.25 M MOPS, 1.25 M HEPES, 30 mM
hydroxyquinoline-5-sulfonic acid and 0.1% Neomycin Sulfate. The enzyme aminolevulinic acid dehydratase and rabbit antisera solution were diluted into a buffer consisting of 0.25 M MOPS, 0.9 M ammonium sulfate, 0.5%
polyethylene glycol 8K and 0.1% Neomycin Sulfate pH 7Ø The substrate l 5 consisted of 50 mM aminolevulinic acid, 25 mM tris(2-carboxyethyl)-phosphinehydrochloride and 250 rnM zinc chloride. The fluorescent tracer was diluted in Abbott IMx~ FPIA buffer to approximately 13 nM.
The ass~y was run in the Abbott IMx(~ analyzer using the following steps.
A blank read was taken on the empty cuvette. The lead (Pb) standard (150 ml) 2 0 was neutralized with 9Q ml of neutralizing buffer and 50 ml of Abbott IMx~) FPIA buffer. A portion (140 ml) of this mixture was combined with 90 ml of the enzyme/antisera solution and 95 ml of Abbott IMx@~ FPIA buffer then incubated for 6.25 minutes in the cuvette. The substrate (90 ml) and 150 m~ of Abbott IMx(g) FPIA buffer were added to the cuvette and incubated for 20 minutes 2 5 followed by the addition of fluorescent tracer (220 ml) and 375 ml of FPIA buffer and a final incubation of 6.25 minutes. ~ final polarization read was measured on the cuvette solution. Lead standards of 0, 20, 40, 60 and 80 mg/dl were run in replicates of 2 or 4 (See Figure 9).

W O 97/24621 32 PCTnUS96/19544 It is expected that the compounds and methods described herein will be readily adapated to all sorts of automated immunoassay formats, where speed, cost, convenience and ease of use, as well as accuracy, precision and reliability are important.
While the invention has been described in each of its various embodiments, it is expected that certain modifications thereto may be made by those skilled in the art without departing from the true spirit and scope of theinvention as described in the specification and further set forth in the 10 accompanying claims.

Claims (30)

WE CLAIM:

1. An immunoassay method for the direct detection and quantification of porphobilinogen without measurement of porphobilinogen complexes, said method comprising the steps of:
(a) contacting a test sample with a labeled reagent and an antibody reagent to form a reaction solution therewith, said antibody reagent comprising antibodies which are capable of binding to porphobilinogen, wherein (i) said antibodies are produced with an immunogen prepared from a porphobilinogen derivative of the formula:

wherein for [(Y-P)=H] X is a linker group conjugated to an immunogenic carrier material P. The linking group consists of 0 to 8 carbon atoms and 0 to 2 heteroatoms linked together where the linking group is linked to the immunogenic carrier material P and wherein for [(X-P)=H] Y is a linker group conjugated to an immunogenic carrier material P. The linking group consists of 0 to 8 carbon atoms and 0 to 2 heteroatoms linked together where the linking group is linked to the immunogenic carrier material P; and wherein (ii) said labeled reagent for the specific quantification of porphobilinogen is prepared from a derivative of the formula:

wherein for [(C-Q)=H] A is a linking group consisting of 0 to 2 heteroatoms and 0 to 8 carbon atoms linked a detectable moiety Q and for [(A-Q)=H] C is a linking group consisting of 0 to 2 heteroatoms and 0 to 8 carbon atoms linked a detectable moiety Q; and b) measuring the amount of said labeled reagent in said reaction solution which either has or has not participated in a binding reaction with said antibodies as a function of the amount of porphobilinogen.

2. The method of claim 1 wherein said test sample is selected from biological fluids such as blood, plasma, urine and the like, food, aqueous mixtures, soil, sludge, sediment, paint and dust.

3. The method of claim 1 wherein said immunogenic carrier material is selected from the group consisting of bovine serum albumin, keyhole limpet hemocyanin, and thyroglobulin.

4. The method of claim 1 wherein said detectable moiety is selected from the group consisting of enzymes, fluorescent molecules, chemiluminescent molecules, phosphorescent molecules, and luminescent molecules .

5. The method of claim 1 wherein said immunoassay method is a fluorescent polarization immunoassay wherein said detectable moiety of said labeled reagent is a fluorescent molecule which is capable of producing a detectable fluorescence polarization response to the presence of said antibodiesfor the quantification of porphobilinogen.

6. The method of claim 5 wherein the amount of said labeled reagent is measured by (a) passing a plane of polarized light through said reaction solution to obtain a fluorescence polarization response and (b) detecting said fluorescence polarization response to said reaction solution as a function of porphobilinogen present.

7. The method of claim 5 wherein said fluorescent molecule is selected from the group consisting of aminomethylfluorescein, amino-fluorescein, 5-fluoresceinyl, 6-fluoresceinyl, 5-carboxyfluorescein, 6-carboxyfluorescein, thioureafluorescein, and methoxytriazinolyl-aminofluorescein.

8. A fluorescence polarization immunoassay method for the direct detection and quantification of porphobilinogen without measurement of porphobilinogen complexes, said method comprising the steps of:
a) contacting a test sample with a labeled reagent and an antibody reagent to form a reaction solution therewith, said antibody reagent comprising antibodies which are capable of binding to porphobilinogen, wherein (i) said antibodies are produced with an immunogen prepared from a derivative of the formula:

wherein for [(Y-P)=H; X is a linker group conjugated to an immunogenic carrier material P. The linking group consists of 0 to 8 carbon atoms and 0 to 2 heteroatoms linked together where the linking group is linked to the immunogenic carrier material P and wherein for [(X-P)=H] Y is a linker group conjugated to an immunogenic carrier material P. The linking group consists of 0 to 8 carbon atoms and 0 to 2 heteroatoms linked together where the linking group is linked to the immunogenic carrier material P; and wherein (ii) said labeled reagent for the specific quantification of porphobilinogen is prepared from a derivative of the formula:

wherein for [(C-Q)=H] A is a linking group consisting of 0 to 2 heteroatoms and 0 to 8 carbon atoms linked a detectable moiety Q and for [(A-Q)=H3 C is a linking group consisting of 0 to 2 heteroatoms and 0 to 8 carbon atoms linked a detectable moiety Q; and b) measuring the amount of said labeled reagent in said reaction solution which either has or has not participated in a binding reaction with said antibodies as a function of the amount of porphobilinogen.

9. The method of claim 8 wherein said test sample is selected from biological fluids such as blood, plasma, urine and the like, food, aqueous mixtures, soil, sludge, sediment, paint and dust.

10. The method of claim 8 wherein said immunogenic carrier material is selected from the group consisting of bovine serum albumin, keyhole limpet hemocyanin, and thyroglobulin.

11. The method of claim 8 wherein the amount of said labeled reagent is measured by (a) passing a plane of polarized light through said reaction solution to obtain a fluorescence polarization response and (b) detecting said fluorescence polarization response to said reaction solution as a function of porphobilinogen present.

12. The method of claim 8 wherein said fluorescent molecule is selected from the group consisting of aminomethylfluorescein, amino-fluorescein, 5-fluoresceinyl, 6-fluoresceinyl, 5-carboxyfluorescein, 6-carboxyfluorescein, thioureafluorescein, and methoxytriazinolyl-aminofluorescein.

13. The method of claim 8 wherein said antibodies used in the fluorescence polarization immunoassay are derived from the immunogen where [(Y-P)=H] are paired with the labeled reagent where [(C-Q)=H].

14. The method of claim 8 wherein said antibodies used in the fluorescence polarization immunoassay are derived from the immunogen where [(X-P)=H] are paired with the labeled reagent where [(A-Q)=H].

15. An antibody reagent comprising antibodies which are capable of binding to porphobilinogen, wherein said antibodies are produced with an immunogen prepared from a porphobilinogen derivative of the formula wherein for [(Y-P)=H] X is a linker group conjugated to an immunogenic carrier material P The linking group consists of 0 to 8 carbon atoms and 0 to 2 heteroatoms linked together where the linking group is linked to the immunogenic carrier material P and wherein for [(X-P)=H] Y is a linker group conjugated to an immunogenic carrier material P The linking group consists of 0 to 8 carbon atoms and 0 to 2 heteroatoms linked together where the linking group is linked to the immunogenic carrier material P

16. The antibody reagent of claim 15 for the detection and quantification of porphobilinogen wherein said test sample is selected from biological fluids such as blood, plasma, urine and the like, food, aqueous mixtures, soil, sludge, sediment, paint and dust

17. The antibody reagent of claim 15 wherein said immunogenic carrier material is selected from the group consisting of bovine serum albumin, keyhole limpet hemocyanin, and thyroglobulin

18. A labeled reagent which is recognizable by antibodies capable of binding porphobilinogen, wherein said labeled reagent is prepared from a derivative of the formula:

wherein for [(C-Q)=H] A is a linking group consisting of 0 to 2 heteroatoms and 0 to 8 carbon atoms linked a detectable moiety Q and for [(A-Q)=H] C is a linking group consisting of 0 to 2 heteroatoms and 0 to 8 carbon atoms linked a detectable moiety Q.

19. The labeled reagent of claim 18 wherein said detectable moiety is selected from the group consisting of enzymes, fluorescent molecules, chemiluminescent molecules, phosphorescent molecules, and luminescent molecules.

20. The labeled reagent of claim 19 wherein said fluorescent molecule is selected from the group consisting of aminomethylfluorescein, amino-fluorescein, 5-fluoresceinyl, 6-fluoresceinyl, 5-carboxyfluorescein, 6-carboxyfluorescein, thioureafluorescein, and methoxytriazinolyl-aminofluorescein.

21. An immunogen of the formula:

wherein for [(Y-P)=H] X is a linker group conjugated to an immunogenic carrier material P. The linking group consists of 0 to 8 carbon atoms and 0 to 2 heteroatoms linked together where the linking group is linked to the immunogenic carrier material P and wherein for [(X-P)=H] Y is a linker group conjugated to an immunogenic carrier material P. The linking group consists of 0 to 8 carbon atoms and 0 to 2 heteroatoms linked together where the linking group is linked to the immunogenic carrier material P.

22. The immunogen of claim 21 said immunogenic carrier material is selected from the group consisting of bovine serum albumin, keyhole limpet hemocyanin, and thyroglobulin.

23. A compound of the formula:

wherein for [(Y-P)=H] X is a linker group conjugated to an immunogenic carrier material P. The linking group consists of 0 to 8 carbon atoms and 0 to 2 heteroatoms linked together where the linking group is linked to the immunogenic carrier material P and wherein for [(X-P)=H] Y is a linker group conjugated to an immunogenic carrier material P. The linking group consists of 0 to 8 carbon atoms and 0 to 2 heteroatoms linked together where the linking group is linked to the immunogenic carrier material P.

24. The immunogen of claim 23 said immunogenic carrier material is selected from the group consisting of bovine serum albumin, keyhole limpet hemocyanin, and thyroglobulin.

25. The compound of claim 23 wherein for [(Y-P)=H] X is -CO-(CH2)n-CO-P, n=1-6 and P is bovine serum albumin.

26. The compound of claim 23 wherein for [(X-P)=H] Y is -SO2-(CH2)n-(CO)-P, n=1-6 and P is bovine serum albumin.

27. The compound of the formula wherein for [(C-Q)=H] A is a linking group consisting of 0 to 2 heteroatoms and 0 to 8 carbon atoms linked a detectable moiety Q and for [(A-Q)=H] C is a linking group consisting of 0 to 2 heteroatoms and 0 to 8 carbon atoms linked a detectable moiety Q.

28. The compound of claim 27 wherein said fluorescein or a fluorescein derivative is selected from the group consisting of aminomethylfluorescein, aminofluorescein, 5-fluoresceinyl, 6-fluoresceinyl, 5-carboxyfluorescein, 6-carboxyfluorescein, thioureafluorescein, methoxytriazinolyl-aminofluorescein.

29. The compound of claim 27 wherein for [(C-Q)-H] A is -CO-Q and Q
is 6-fluoresceinyl.

30. The compound of claim 27 wherein for [(A-Q)=H] C is -SO2-(CH2)3-CO-NH-CH2-Q and Q is 5-fluoresceinyl.