WO1996013605A1 - Procede de dosage immunologique par hydrocarbures polyaromatiques (pah), composants correspondants, et kit d'immunodosage - Google Patents

Procede de dosage immunologique par hydrocarbures polyaromatiques (pah), composants correspondants, et kit d'immunodosage Download PDF

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Publication number
WO1996013605A1
WO1996013605A1 PCT/US1994/012218 US9412218W WO9613605A1 WO 1996013605 A1 WO1996013605 A1 WO 1996013605A1 US 9412218 W US9412218 W US 9412218W WO 9613605 A1 WO9613605 A1 WO 9613605A1
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WIPO (PCT)
Prior art keywords
monoclonal antibody
anthracene
sample
group
immunoassay
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PCT/US1994/012218
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English (en)
Inventor
Stephen B. Friedman
Randy L. Allen
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Ensys, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US08/097,223 priority Critical patent/US5449611A/en
Priority claimed from US08/097,223 external-priority patent/US5449611A/en
Application filed by Ensys, Inc. filed Critical Ensys, Inc.
Priority to PCT/US1994/012218 priority patent/WO1996013605A1/fr
Priority to AU10836/95A priority patent/AU1083695A/en
Publication of WO1996013605A1 publication Critical patent/WO1996013605A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids

Definitions

  • PAH Polyaromatic Hydrocarbon
  • the present invention relates to methods, compositions and kits for performing polyaromatic hydrocarbon (PAH) immunoassays.
  • PAH polyaromatic hydrocarbon
  • Testing is an essential, and integral, component of all environmental protection and restoration activities. It is the rate limiting element that influences the time, cost, and overall efficiency of project management.
  • Effective field screening methods can increase the efficiency of site management and improve overall data quality when used to supplement the services of regional laboratories.
  • the development of these methods requires a technology that will be compatible with numerous compounds and matrixes and yet be simple, effective and rugged enough to be incorporated into a protocol for use in the field.
  • Screening methods need to provide fast, simple, cost- effective and reliable information when operated under field conditions.
  • the reagents and equipment should be portable and stable at ambient conditions, and the claims relating to performance should accurately reflect anticipated field use.
  • the methods should be able to rapidly provide an ample quantity of data, and the protocol should be simple to perform and safe to use.
  • Performance characteristics relative to sensitivity, freedom from matrix interferences and cross- reacting compounds, and correlation to an acceptable reference method should be carefully evaluated. Developers must maintain high, and consistent, quality standards relative to the consistency of their manufacturing protocols, the adequacy of in-process and pre-release quality control methods, and the reliability of their product claims.
  • a necessary characteristic of particular significance for screening methods is that they exhibit a very low frequency of false negative results.
  • Screening methods detect contamination at specified concentrations.
  • concentration may relate to a hazardous threshold, a clean-up target, or a process-control parameter.
  • the potential implications of false negative data far outweigh those of false positive results.
  • False negative data provides an erroneous perception of a clean site, and may have serious environmental and legal consequences. Safeguards that minimize the incidence of false negative results are imperative. Appropriate control over the frequency of false positive data needs to be established and maintained.
  • Contamination of soil with polyaromatic hydrocarbons is a serious problem at manufactured gas plant sites, coking operations, wood preserving operations that have used creosote as a wood preservative, and petrochemical plant waste disposal sites.
  • the federal and state regulatory agencies are mandating the clean-up of many of these sites due to the carcinogenic nature of some of the PAHs.
  • PAHs Polyaromatic hydrocarbons
  • PNAs polynuclear aromatic compounds
  • PAH Compounds acenaphthene acenaphthylene anthracene benzo[a]anthracene benzo[a]pyrene benzo[b]fluoranthene benzo[g,h,i]perylene benzo[k]fluoranthene chrysene dibenzo[a,h]anthracene fluoranthene fluorene indeno[1,2,3-cd]pyrene naphthalene phenanthrene pyrene
  • the two and three ring PAHs are non- carcinogenic, while several of the four, five and six ring PAHs are carcinogenic.
  • the four ring PAHs, chrysene and benzo[a]anthracene, the five ring PAHs, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, and dibenzo[a,h]anthracene, and the six ring PAH, indeno[l,2,3- cd]pyrene, are listed as carcinogenic PAHs with benzo[a]pyrene being the most potent carcinogen among the PAHs. Regulatory concern is generally focused on benzo[a]pyrene, total carcinogenic PAHs, and total PAHs.
  • Immunoassay methods combine the specific binding characteristics of an antibody molecule with a read-out system that is used to detect and quantify compounds.
  • Current immunoassay technology benefits from the diversity of detection systems developed that use enzyme-catalyzed chromogenic reactions, radionuclides, chemiluminescence, fluorescence, fluorescence polarization and a variety of potentiometric and optical biosensor techniques. Improvements in the sensitivity achieved has necessitated the generation of new descriptive nomenclature for methods that can now detect "zeptomolar" (10 ⁇ 21 , 600 molecules) concentrations.
  • an immunoassay method is needed which will provide reliable accurate and fast results in the field for PAH contaminants, regardless of origin or exact composition.
  • Such an assay would increase the efficiency of environmental site management activities such as characterization (mapping) , remediation monitoring, and regulatory compliance.
  • one object of the present invention is to provide a PAH immunoassay method which is field compatible, fast and accurate.
  • a further object of the present invention is to provide a PAH immunoassay method which gives minimal false negative results.
  • Another object of the present invention is to provide a PAH immunoassay method which detects PAH contamination derived from a wide variety of unknown sources about equivalently, where such method is independent of the detection of a single compound in the sample, since concentrations of single compounds vary between samples and locations.
  • Another object of the present invention is to provide a PAH immunoassay which is simple to perform and easy to interpret in a field or laboratory setting by operators inexperienced in the art.
  • Another object of the present invention is to provide an anti-PAH monoclonal (MAb) antibody which is useful in the above-mentioned PAH immunoassay.
  • MAb monoclonal
  • Another object of the present invention is to provide an anti-PAH monoclonal antibody with specific reactivity towards a characteristic plurality of compounds found in PAH contaminated samples, namely the 3-4 ring PAHs and at least some of the 5-6 ring PAHs, with preferential recognition for the more prevalent 3 ring phenanthrene.
  • Another object of the present invention is to provide a reagent (e.g., an enzyme conjugate reagent) for use with the monoclonal antibody in competitive binding type immunoassays.
  • a reagent e.g., an enzyme conjugate reagent
  • Another object of the present invention is to provide immunogens which elicit an anti-PAH monoclonal antibody response.
  • Another object of the present invention is to provide a kit for performing a PAH immunoassay on various sample matrices.
  • Another object of the present invention is to provide a sample processing procedure for preparing samples from various matrices for use in the immunoassay of the present invention.
  • PAH contaminated samples of different sources differ from each other with regard to the nature of the PAH compounds present. However, most (>85%) contain phenanthrene in addition to a plurality of other PAH compounds, although the relative amounts of each individual compound in this plurality may vary from one PAH contaminated site to another.
  • the antibody of the present invention recognizes this plurality of compounds, primarily phenanthrene, which is a common and prevalent PAH contaminant, and others with relative equivalency, and can therefore be used to detect the presence of PAH contamination in a sample.
  • the present invention accordingly provides, among its varied embodiments, both an antibody and a test kit which incorporates the antibody.
  • the kit is useful to detect the presence of PAH contamination in a sample because the antibody exhibits approximately equivalent detection of PAH compounds and preferentially detects phenanthrene, regardless of origin or diversity of chemical composition, and thereby provides an easy-to-use and highly reliable kit for environmental PAH testing applications.
  • Figure 1 displays the sensitivity profiles of 201 observations assayed at room temperature across four validation lots, twelve matrices and 3 operators, using the PAH immunoassay of the present invention.
  • the present invention provides an immunoassay for determining the presence of PAH contamination in a sample (suspected to contain PAH) .
  • the immunoassay comprises:
  • the invention provides an immunoassay kit which comprises three basic components: (l) a monoclonal antibody with specific reactivity towards a characteristic plurality of PAH compounds (2) a reagent cross reactive with the monoclonal antibody and susceptible to detection, and optionally (3) a signal-generating reagent.
  • an immunogen which contains a derivative moiety which mimics the structural features of the prevalent phenanthrene and a characteristic plurality of other PAH compounds.
  • the immunogen is synthesized, by standard methods, by coupling a derivative moiety to an immunologic carrier molecule.
  • Suitable immunological carrier moieties can be used, including albumin (e.g., bovine serum albumin), thyroglobulin (e.g., bovine thyroglobulin), hemocyanin (e.g., keyhole limpet hemocyanin) , polyamino acids and other molecules having a minimum size, complexity, and foreignness to the host animal.
  • Carriers are usually of the class of proteins, polypeptides or peptides having a molecular weight of at least 1,000 daltons and preferably >10,000 daltons.
  • Carrier molecules may have a reactive group(s) available for coveilent conjugation of the derivative.
  • R-groups e.g. , COOH, NH 2
  • amino acids or sugar moieties of glycoproteins are often used for this purpose in the synthesis of immunogens.
  • the immunogen is a compound of the formula
  • Ar is an aromatic benzenoid hydrocarbon group having from 2 to 4 benzenoid rings, each ring of which may be substituted by one or more substituents selected from the group consisting of H, CH 3 and CH 2 CH 3 ;
  • X and Y are each, independently
  • X is -CH 2 —( )— ' ⁇ co0 "/ or a single bond
  • R 1 is selected from the group consisting of
  • n is 0 or an integer from 1 to 4 where Z is an immunologic carrier molecule and the rest of the molecule is the derivative moiety of the immunogen.
  • one or two of the substituents on the Ar group is methyl.
  • a most preferred immunogen is
  • R 2 to R 8 are each independently a member selected from the group consisting of H, CH 3 , and CH 2 CH 3 and Z is an immunologic carrier molecule.
  • naphthalene and phenanthrene by themselves are too small to stimulate an immune response and elicit an antibody response.
  • derivatives of small molecules such as naphthalene
  • the small molecules and accompanying bridge structures of the derivative are designed so that they mimic immunologically at least one common feature present in the defined molecular pool of interest.
  • a number of modifications to the bridge structure (X-(CH 2 ) n -Y) are possible and produce similar results.
  • Any vertebrate is a suitable host for the immunization procedure, such as mice, rats, dogs, pigs and other domestic animals, with mice being preferred.
  • in vitro immunization may be used.
  • the immunoassay method of the present invention uses a hybridoma-derived monoclonal antibody.
  • the monoclonal antibody is selected using a multi-parameter screening process.
  • a reactive panel profiling system is developed to identify appropriate cell lines and monoclonal antibodies on the basis of sensitivity, PAH recognition characteristics, and freedom of interference from confirmed negative soil samples or cross-reacting compounds.
  • the monoclonal antibody binds selectively to a defined pool of molecules present in PAH contaminants and preferably to phenanthrene and anthracene.
  • the antibody allows the method to rapidly detect PAH contamination, regardless of origin, at, or above, a concentration of 1 ppm in soil at a 95% confidence level, with lower concentrations detectable at lower confidence levels, because most samples contain phenanthrene and because of the cumulative concentration of other PAH compounds.
  • the antibody operates through the selective recognition of specific polycyclic aromatic compounds.
  • the reporter molecule reagent of the present invention which in a preferred embodiment is an enzyme conjugate reagent, is cross reactive with the monoclonal antibody and capable of providing a detectable signal, either alone or in combination with one or more co-reagents.
  • the reporter molecule reagent comprises a compound of formula
  • Ar is an aromatic benzenoid hydrocarbon group having from 2 to 6 benzenoid rings, each ring of which may be substituted by one or more substituents selected from the group consisting of H, CH 3 and CH 2 CH 3 ;
  • X and Y are each, independently
  • X is -CH 2 —(O)— ' "COO-, or a single bond
  • R 9 is selected from the group consisting of
  • the reporter moiety used in the present invention may include an enzyme, a fluorescent compound, a chemiluminescent compound, a bioluminescent compound, a dyed latex particle or a radioactive atom.
  • a preferred reporter moiety for use in the enzyme-conjugate reagent of the present invention includes an enzyme selected from the group consisting of horseradish peroxidase, alkaline phosphatase, glucose oxidase, ⁇ - galactosidase, glucose oxidase, G6-PDH (glucose-6-phosphate dehydrogenase) or urease, with horseradish peroxidase being most preferred.
  • a most preferred enzyme conjugate reagent is a compound of formula
  • R 10 to R 17 are each independently selected from the group consisting of H, CH 3 , and CH 2 CH 3 , and HRP represents horseradish peroxidase.
  • the enzyme conjugate is prepared using conventional methods such as those described in U.S. Applications Serial Nos. 07/984,098 and 08/068,093 to Friedman et al.
  • the enzyme conjugate of the present invention must be cross-reactive with the anti-PAH antibody of the present invention and capable of inducing a detectable change in the sample by reaction with a signal producing substrate or reagent used in the immunoassay.
  • the immunoassay-based system of the present invention uses a signal producing reagent which, upon reaction with the bound enzyme conjugate/antibody complex after incubation, provides a detectable signal indicating the presence or absence of PAH contamination in the sample being tested.
  • Suitable signal producing reagents include a hydrogen peroxide/tetramethyl benzidine pair, or a phenylene diamino, in combination with an enzyme conjugate reagent containing horseradish peroxidase, o-nitrophenyl- ⁇ -D-galactopyranoside (ONPG) in combination with an enzyme conjugate reagent containing ⁇ -galactosidase, o-nitrophenylphosphate in combination with an enzyme conjugate reagent containing alkaline phosphatase, or glucose in combination with glucose oxidase or G6-PDH.
  • the method of the present invention preferably uses the combination of a hydrogen peroxide (H 2 0 2 ) substrate solution with a tetramethylbenzidine (TMB) chromogen, with the combination producing a blue chromophore upon oxidation of TMB by H 2 0 2 .
  • H 2 0 2 hydrogen peroxide
  • TMB tetramethylbenzidine
  • the standard concentration should not be set at the concentration of interest, but below it. Setting the standard concentration to coincide with the regulatory concentration would result in a 50% false negative incidence.
  • standards used in the immunoassay of the present invention should be offset in concentration to produce a >95% confidence level of detecting contamination at, or above, the desired level. For example, to obtain a method which gives results having a >95% confidence level for detection of PAHs at 1 ppm, a 0.65 ppm standard could be used. The standard is set below the target level. The actual concentration of standard used is a function of the binding characteristics of the assay and the overall precision of the method.
  • Immunoassay methods have conventionally been used to test liquid matrixes such as blood, urine, and water. This method is designed to test also solid matrices. However, the testing of solid matrices requires that the issues of sample collection, dispersion, extraction and clarification be addressed and integrated with the immunoassay component. While the assay of the present invention may be performed on samples of a wide range of qualities, a reproducible, particulate-free, leachate is preferred for optimum results. The extraction and recovery of a compound from soil requires the selection of an appropriate solvent system, adequate sample dispersion, sufficient time for partitioning, non-invasive clarification and compatibility with the subsequent immunochemistry. Current sample processing for analytical detection methods limits the efficiency of these methods and is slow and expensive.
  • One suitable method for sample preparation involves the gravimetric collection of a 10 g sample using a small battery-operated balance.
  • the sample is then transferred into a dispersion vial containing methanol and conventional dispersing pellets, and is subjected to a one minute manual agitation for adequate dispersion and partitioning of the analyte into the solvent.
  • Filtration of the sample suspension to produce a particulate-free extract can be accomplished using a fingertip-operated filter unit fitted with non-adsorbing filters.
  • the clarified leachate is then ready for testing with the immunoassay of the present invention following dilution or otherwise removal of the solvent.
  • Solvent-resistant antibody reagents could be used to eliminate or limit the need for the solvent dilution step.
  • Buffers, detergents (e.g. Tweens, Tritons, etc.) or solvents, used alone, or in combination, have proven to be effective for extraction.
  • Analytical methods for the analysis of solid waste rely upon gravimetrically collected samples, and results are reported in gravimetric units. Volumetric sampling for solid waste should preferably be avoided because of the potential bias that may be caused by the specific gravity of the sample.
  • the present immunoassay method permits the simultaneous testing of soil samples to detect contamination at multiple concentrations and to facilitate the construction of contamination contour profiles.
  • the method is preferably for screening purposes and several safeguards have been incorporated to minimize the incidence of false negative results.
  • the basic characteristics of specificity and equivalent detection of the assay of the present invention are a result of the monoclonal antibody (MAb) and the reagent.
  • the assay provides approximately equal recognition of PAH contaminants, with preference for the more prevalent phenanthrene, from different origins having different chemical compositions, low cross-reactivity with unrelated compounds and is not significantly affected by normally found constituents of negative soil extracts, such as humic acid and fulvic acid.
  • MAb monoclonal antibody
  • the method for producing MAbs is extremely powerful and allows for the preparation of a defined and reproducible Ab reagent.
  • Hybridoma technology permits one to explore the entire b-cell repertoire of the immune system and to select unique antibody producing cells that produce antibodies having unique binding characteristics.
  • the production of polyclonal antisera is much less controlled since polyclonal antisera contain numerous Ab populations each having varying specificity and sensitivity characteristics that are the products of numerous responding b-cell clones.
  • MAb reagents are also homogeneous with a defined specificity, unlike polyclonal antisera which contain a mixed population of antibodies. Use and selection of hybridoma cell lines provides access MAb reagents that offer unique performance characteristics to the test system and consistency of the methods that utilize them.
  • a unique strategy is used for antibody production. This strategy involves developing an antibody to one or more specific structural elements that are present on a defined set of PAH compounds.
  • the antibody is designed to recognize a defined pool of PAH compounds, having preference for phenanthrene.
  • Recognition of a panel of compounds, instead of a single compound, allows the assay of the present invention to average out the concentration difference of any single component present in the differing PAH contaminants, while focusing on a prevalent indicator of PAH contamination, such as phenanthrene. Additionally, recognition of a collection of compounds rather than a single constituent contributes significantly to the sensitivity of the assay.
  • the pool of compounds selected for detection by the assay are predominantly the 3 and 4 ring polynuclear aromatic hydrocarbon compounds and a portion of the 5 and 6 ring polynuclear aromatic hydrocarbons. More preferably the compounds include the compounds acenaphthene, naphthalene, phenanthrene, anthracene, fluorene, benzo[a]anthracene, chrysene, fluoranthene, pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene and indeno(l,2,3- cd)pyrene, with a cross-reactivity with the monoclonal antibody of at least 9% for each of the PAH compounds.
  • the monoclonal antibody has cross-reactivity of at least 60% with the compounds phenanthrene, anthracene, fluorene, benzo[a]anthracene, chrysene and fluoranthene. Most preferably the monoclonal antibody has cross-reactivity of at least 100% with phenanthrene and anthracene.
  • the immunogen of the present invention is used to immunize suitable hosts, such as mice.
  • Hosts responding to the immunization protocol are selected as splenocyte (or lymphoid cell) donors for hybridoma production.
  • tissue culture medium such as HAT (hypoxanthine, aminopterine, thymidine)
  • myeloma cells capable of providing a hybridoma
  • s-s fusion product normally has a short lifetime and dies within days.
  • the m-m fusion product has a very short lifetime in the tissue culture medium used, lacking the metabolic components needed for DNA synthesis.
  • the s-m fusion product (or hybridoma) survives in tissue culture and retains the Ab- producing characteristics of the splenocyte parent, and the high rate of growth and relative immortality of the myeloma cell parent.
  • This hybridoma cell line replicates itself readily in culture producing daughter cells that provide reproducible, homogeneous, and a consistent supply of the monoclonal anti-PAH antibody of the present invention.
  • the structure of the reporter molecule reagent can have a significant influence on immunoassay performance. Both the small molecule portion and the bridge structure of the derivative play an important role in antibody binding. Antibody and reagent pairs should be able to satisfy the following criteria:
  • Antibody and reagent pairs which best satisfy the above criteria are most suitable for the assay of the present invention.
  • the immunoassay of the present invention is performed according to the following procedure.
  • Monoclonal anti-PAH antibody is provided in an assay vessel, preferably immobilized in the assay vessel or other solid support, preferably to the bottom of a polystyrene tube, at a concentration, of from 0.005 to 20 ⁇ g/tube, preferably 0.1 to 0.4 ⁇ g/tube.
  • concentration and affinity of the antibody for the sample molecules and reporter reagent molecules directly influences the overall sensitivity of the final method.
  • High, equivalent, affinity, and minimal non-specific signal generation produces assays having superior sensitivity, with affinity binding constants, Ka, from 10 4 -10 12 L/mol, preferably 10 4 -10 9 L/mol.
  • the assay of the present invention compares a sample to be tested with a standard solution containing a compound cross-reactive with the antibody of the present invention, preferably phenanthrene, which is present in the standard solution in an amount sufficient to provide a >95% confidence level for detection at the desired level.
  • the following describes a simultaneous test of a negative sample, a sample containing >100 ppm of PAHs, and a standard solution containing the equivalent of approximately 65 ppm of phenanthrene.
  • a negative sample a sample containing >100 ppm of PAHs
  • a standard solution containing the equivalent of approximately 65 ppm of phenanthrene.
  • sample molecules and reporter reagent molecules compete for the limited number of antibody binding sites that are available on the bottom of each of the tubes.
  • the antibody concentration present is insufficient to permit the binding of all of the sample and reagent molecules simultaneously, and a situation somewhat analogous to the game of musical chairs exists, with the limited antibody binding capacity of the antibody molecules serving as the chairs in this example.
  • the concentration of reporter reagent immobilized in each tube after incubation is inversely proportional to the concentration of PAHs in the sample or standard.
  • the standard in the Standard tube limits the binding of the reporter reagent, the Negative sample permits more reporter reagent to bind (relative to the standard) , and the positive sample permits less of the reporter reagent to bind (relative to the standard) .
  • the tubes are washed leaving only the reagent that was retained by the immobilized antibody on the bottom of each tube.
  • the bound reagent/antibody complex remaining in each vessel or tube is next used to produce a detectable signal.
  • a signal forming reagent(s) preferably a substrate/chromogen reagent pair
  • the enzyme molecule catalyzes the formation of a detectable signal.
  • the signal that is generated is directly proportional to the concentration of reporter reagent present.
  • a preferred substrate/chromogen reagent pair is H 2 0 2 and tetramethyl- benzidine (TMB) which, when used with the preferred reporter reagent wherein the reporter molecule is the enzyme HRP (horseradish peroxidase) , reacts with the enzyme portion of the reporter reagent, which facilitates the oxidation of TMB by H 2 0 2 and generates a blue color.
  • TMB tetramethyl- benzidine
  • the negative sample tube rapidly produces a solution that is visibly darker (i.e. greater absorbance) than the standard tube.
  • the positive test sample produces a solution having less color (i.e. lower absorbance) than the standard tube.
  • the assay method of the present invention can process multiple samples in less than 30 minutes.
  • the method is self-contained, field-compatible and does not require refrigeration or use hazardous components.
  • the detection level can be set at the users discretion, for example by dilution of the sample extract, with 1 ppm as the maximum obtainable sensitivity obtainable using solid matrix samples and 30 ppb for aqueous samples.
  • the kit of the present invention contains four basic components: (1) a sample processing component for preparing the sample for testing by the assay method (described above) , (2) at least one assay vessel, preferably a plurality, each containing an equivalent amount of an anti-PAH monoclonal antibody, preferably immobilized, (3) a standard solution containing the equivalent of 0.5-1.0 ppm (for soil) of a PAH compound in a suitable carrier, (4) a reporter reagent which is reconstituted to form a solution containing a reporter reagent which is cross-reactive with the monoclonal anti-PAH antibody, in a suitable carrier, where the reporter reagent is preferably a lyophilized enzyme-conjugate reagent, and (5) means for forming and detecting a signal indicating the presence or absence of PAH contamination in the sample being tested.
  • the kit contains one or more of the following additional components: (6) wash solutions for performing the wash step of the assay method, (7) weighing boats into which the sample is weighed using (8) a battery- operated scale, (9) one or more filters for preparing a clarified leachate sample, (10) one or more dilution vials, (11) substrate solution and (12) stop solution to halt signal formation by the signal-forming reagent.
  • the assay is provided in a coated-tube format in which the means for forming and detecting a signal comprises the use of a substrate/chromogen reagent pair which forms a detectable colored signal with the level of PAH contamination determined by comparison of the color developed by the test samples to the color of the kit standard.
  • the standard is preferably run in parallel with the samples. Therefore, any assay variations will equally affect both the standard and the samples. Using this format with the standard run in parallel, the assay has the following characteristics:
  • the sensitivity in solid samples is 1 ppm of PAH at >95% confidence level.
  • the test is reproducible from lot-to-lot, day-to-day, and person-to-person.
  • the assay is operable over a temperature range of 4- 37°C, preferably 15-37°C.
  • the immunoassay of the present invention has many advantages, including field screening compatibility, speed in obtaining a result, and a lack of false negative results. Specific characteristics had to be integrated into the method in order to achieve these advantages. The first was to minimize the incidence of false negative results. A false negative test does not detect a contaminated sample at the concentration of interest and therefore, can be potentially dangerous both to the customer and the environment. A false positive, on the other hand, might be inconvenient but not dangerous. Therefore, the immunoassay of the present invention was designed to detect positive solid matrix samples containing >1 ppm of PAHs with a minimum of false negative results.
  • the immunoassay has also been simplified by making it a semiquantitative test using a standard at a single concentration.
  • the use of one standard concentration greatly simplifies the test protocol and eliminates the data manipulation and expensive instrumentation that are normally necessary for quantitative tests.
  • the test becomes a semiquantitative "yes/no" test that determines whether a soil sample contains greater or less than a target level of PAH contamination.
  • the use of multiple standards at varying concentrations could be used to allow one to perform a quantitative assay by interpolation of sample signal from the signal of a standard curve.
  • the immunoassay of the present invention makes possible the rapid screening of numerous samples in the field for PAHs.
  • the rapid, on-site screening of multiple samples is a significant advantage compared to the standard analytical GC or HPLC methods.
  • the quick screening of samples allows quick answers to important questions concerning the project, such as, has the plume of contaminated soil been located, has the area been remediated to acceptable levels, and does additional soil need to be removed?.
  • personnel, time and equipment can be used more cost-effectively.
  • mice received a primary subcutaneous immunization with 50 ug of /3-methylnaphthalene derivatized to bovine serum albumin in adjuvant (MPL + TDM Emulsion, RIBI Immunochem Research, Inc. , Hamilton, MT) . Subcutaneous secondary injections (25 ug) were given at day 21, 35, and then monthly. The production of monoclonal antibodies was performed according to conventional procedures (see Milstein, Cancer, _9_:1953-1957 (1982)). Hybridoma cultures producing monoclonal antibodies recognizing free hapten by EIA were cloned by limiting dilution. Clone F35-2Z11 was selected based upon specificity, and interference criteria. Horseradish peroxidase conjugate reagent
  • Horseradish peroxidase (HRP) conjugate reagent was prepared with a hydrazide derivative of naphthalene using the conventional periodate method (see Nakane et al J. Histochem. Cytochem. 22.:1084 (1971)).
  • Antibody coated tubes were prepared by passively coating polystyrene (12X75 mm, NUNC) tubes with the monoclonal F12-3C8 antibody diluted in PBS and incubating overnight.
  • the NUNC polystyrene tubes were rinsed with hexane prior to use and were all from the same lot.
  • the immunoassay method of the present invention has been demonstrated as follows:
  • the sample and the two standards were then poured into three identical antibody coated tubes, respectively, and the resulting solution mixed.
  • the resulting solution was incubated for 10 minutes at ambient temperature and then washed 4X with a wash buffer (detergent-saline solution) .
  • 250 uL of tetramethylbenzidene solution KP Laboratories, Cat. No. 50- 76-02 was added along with 250 uL of hydrogen peroxide solution (KPL, Cat. No. 50-65-02) and the resultant solution mixed. After incubating for 2.5 minutes the color development reaction was stopped with 250 uL of stop solution (1 M sulfuric acid) .
  • the optical density (OD) of the test sample was compared to the OD of the phenanthrene standard (0.65 ppm soil equivalents) which was tested simultaneously with the sample. If the sample OD is less than the standard OD, the sample contains more than 1 ppm PAH. If the sample OD is greater than the standard OD, the sample contains less than 1 ppm PAH. Other concentration ranges can be assessed by diluting the test samples with suitable diluents, as long as they are non- reactive with the components of the assay.
  • the concentration of the phenanthrene standard was determined by using a statistical approach.
  • the standard was set at a point where the mean of the standard absorbance minus 2 standard deviations (SD) does not overlap the mean of the signal at 1 ppm.
  • SD standard standard deviations
  • the standard was established to be 0.65 ppm (soil equivalents) (Table II) .
  • a dilution of the sample is used to reduce interference from methanol and to test at varying target concentrations. A 1 ppm phenanthrene sample thus yields a positive interpretation, relative to the 0.65 ppm kit standard, with 95% confidence.
  • Sensitivity profiles were prepared as a measurement of assay performance using phenanthrene spikes compared to the kit standard. These profiles serve three purposes in the validation process. First, they indicate whether the kit standard is set appropriately. Second, the profiles help establish and support the sensitivity claims with 95% confidence. Third, they are used to evaluate matrix effects and assay variability due to lot, operator and temperature differences.
  • Figure 1 displays the sensitivity profiles of 201 observations assayed at room temperature across four validation lots, twelve matrices and 3 operators. This figure demonstrates that the standard (0.65 ppm soil equivalents) is set appropriately since it gave correct results for each phenanthrene sample having concentration at 0.4 and 0.8 ppm. Furthermore, Figure 1 illustrates a >95% confidence level in negative results at 0 ppm and in positive results at > 1 ppm.
  • the most interesting carcinogenic PAH from a market perspective is benzo(a)pyrene. If this compound is present in soils at concentrations of > 8.3 ppm the PAH immunoassay of the present invention will provide a positive result.
  • Chlorinated and methylated naphthalenes are frequently found at high levels at gas manufacturing plants. A high contamination of the naphthalene derivatives would give a positive interpretation of total PAHs.
  • the PAH assay of the present invention can be operated at temperatures between 4°C and 32°C with >95% confidence in results at the 0 ppm and 1 ppm phenanthrene levels. An increase in the frequency of false positive and false negative results was observed at 36°C. The differences between the standard pairs was not affected by the operating temperature (compare to Table V) which demonstrates that the temperature extremes did not affect the assay precision. Interestingly, the Bo signal was suppressed at either high or low temperatures without affecting the assay interpretation. In conclusion, the preferred operating range for the PAH test of the present invention is 4°C to 32°C.
  • test kit of the present invention containing the immunoassay components of the present invention, has been found to be stable for extended periods of time. Projections indicate a shelf life at 37°C of > 1 year.
  • This method offers significant versatility and performance advantages. It is a convenient and effective new tool that can enhance the efficiency of site management activities and the utilization of our national laboratory system.

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Abstract

La présente invention concerne un procédé de dosage immunologique intégrant un composant de traitement de l'échantillon qui permette de tester une variété de matrices d'environnement. L'invention concenre un également les composants nécessaires à la mise en ÷uvre de ce procédé de dosage immunologique caractérisé par l'utilisation d'un anticorps monoclonal anti-PAH pour détecter la présence ou l'absence de contamination PAH dans un échantillon lorsque l'examen se fait in situ ou en laboratoire.
PCT/US1994/012218 1993-07-27 1994-11-01 Procede de dosage immunologique par hydrocarbures polyaromatiques (pah), composants correspondants, et kit d'immunodosage WO1996013605A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US08/097,223 US5449611A (en) 1993-07-27 1993-07-27 Polyaromatic hydrocarbon (PAH) immunoassay method, its components and a kit for use in performing the same
PCT/US1994/012218 WO1996013605A1 (fr) 1993-07-27 1994-11-01 Procede de dosage immunologique par hydrocarbures polyaromatiques (pah), composants correspondants, et kit d'immunodosage
AU10836/95A AU1083695A (en) 1993-07-27 1994-11-01 A polyaromatic hydrocarbon (pah) immunoassay method, its components and a kit for use in performing the same

Applications Claiming Priority (2)

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US08/097,223 US5449611A (en) 1993-07-27 1993-07-27 Polyaromatic hydrocarbon (PAH) immunoassay method, its components and a kit for use in performing the same
PCT/US1994/012218 WO1996013605A1 (fr) 1993-07-27 1994-11-01 Procede de dosage immunologique par hydrocarbures polyaromatiques (pah), composants correspondants, et kit d'immunodosage

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6277964B1 (en) 1998-03-12 2001-08-21 Osaka Gas Company Limited Anti-PAH monoclonal antibodies and cell lines producing the same
CN102087280A (zh) * 2010-11-26 2011-06-08 无锡市金坤生物工程有限公司 食品中苯并芘含量的检测方法
CN111471110A (zh) * 2020-04-16 2020-07-31 江西省农业科学院农产品质量安全与标准研究所 一种采用CdTe标记抗体检测菲的方法
CN115028733A (zh) * 2022-07-11 2022-09-09 华中农业大学 用于检测芘和苯并[a]芘的单抗及酶联免疫方法与试剂盒
CN115028732A (zh) * 2022-07-11 2022-09-09 华中农业大学 抗苯并[a]芘的单克隆抗体及其应用

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WO1988009798A1 (fr) * 1987-06-09 1988-12-15 Peck Dana P Immunoanalyse pour composes contenant un cycle aromatique
US5145790A (en) * 1990-05-04 1992-09-08 Abbott Laboratories Reagents and method for detecting polychlorinated biphenyls

Patent Citations (2)

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WO1988009798A1 (fr) * 1987-06-09 1988-12-15 Peck Dana P Immunoanalyse pour composes contenant un cycle aromatique
US5145790A (en) * 1990-05-04 1992-09-08 Abbott Laboratories Reagents and method for detecting polychlorinated biphenyls

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Title
CHEMICAL ABSTRACTS, Volume 120, Number 23, issued 06 June 1994, P.P. McDONALD et al., "PAH RIS Soil Test-a Rapid, On-Site Screening Test for Polynuclear Aromatic Hydrocarbons in Soil", page 873, Abstract Number 297406f. *
CHEMICAL RESEARCH IN TOXICOLOGY, Volume 3, Number 4, issued 1990, M. GOMES et al., "Immunologic Methods for the Detection of BenzoÄaÜPyrene Metabolites in Urine", pages 307-310. *
ENVIRONMENTAL TECHNOLOGY, Volume 12, issued 1991, R.A. BACIGALUPO et al., "Development and Applications of an Ultrasensitive Quantitative Enzyme Immuno-Assay for Benzo(a)Pyrene in Environmental Samples", pages 1027-1035. *
JOURNAL OF SOIL CONTAMINATION, Volume 1, Number 3, issued 1992, R.L. ALLEN et al., "A Rapid and Sensitive Immunoassay for the Detection of Gasoline and Diesel Fuel in Contaminated Soil", pages 227-237. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6277964B1 (en) 1998-03-12 2001-08-21 Osaka Gas Company Limited Anti-PAH monoclonal antibodies and cell lines producing the same
CN102087280A (zh) * 2010-11-26 2011-06-08 无锡市金坤生物工程有限公司 食品中苯并芘含量的检测方法
CN111471110A (zh) * 2020-04-16 2020-07-31 江西省农业科学院农产品质量安全与标准研究所 一种采用CdTe标记抗体检测菲的方法
CN115028733A (zh) * 2022-07-11 2022-09-09 华中农业大学 用于检测芘和苯并[a]芘的单抗及酶联免疫方法与试剂盒
CN115028732A (zh) * 2022-07-11 2022-09-09 华中农业大学 抗苯并[a]芘的单克隆抗体及其应用
CN115028732B (zh) * 2022-07-11 2023-09-08 华中农业大学 抗苯并[a]芘的单克隆抗体及其应用

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