Immunoassay for aromatic ring containing compounds

The present invention provides methods, reagents and kits for determining the presence of aromatic ring-containing compounds by immunoassay techniques, including enzyme, fluorescent, chemiluminescent and biosensor immunoassay, as well as radioimmunoassay. Monoclonal and polyclonal antibodies may be used in the practice of the present invention.

FIELD OF THE INVENTION 
This invention relates to immunoassays for aromatic hydrocarbons, to 
reagents for and methods of carrying out such assays and to kits 
comprising reagents to practice such methods. 
BACKGROUND OF THE INVENTION 
The effects of chemicals found in the environment, both on man and animals, 
is of widespread concern. The history of chemical regulation in the United 
States is one of increasing stringency in terms of what is "acceptable" in 
the environment and the workplace. Fueled by an ever-expanding body of 
scientific knowledge, and supported by public outcry for a "clean 
environment," and labor and management's interest in a cleaner, safer 
workplace, the demand for ability to detect environmental contaminants, 
and aromatic hydrocarbons in particular, and to determine how much of such 
substances are present in the environment, is rapidly growing. 
Certain aromatic hydrocarbons, e.g., benzene, toluene, and xylene, have a 
broad basis of application and utilization as solvents and additives. 
Benzene is present in the workplace in the chemical manufacturing industry 
as well as industries involved in industrial manufacturing of motor fuels, 
inks, oils, paints, plastics, and rubber. Additionally, the materials are 
used in manufacturing detergents, explosives, pharmaceuticals, and dye 
stuffs. The National Institute of Occupational Safety and Health estimates 
that over two million workers in the U.S. are potentially exposed to 
benzene, a known cause of leukemia in humans. 
Furthermore, benzene is present and its uses are enhanced in unleaded fuels 
for automobiles equipped with catalytic converters. Thus, benzene is a 
persistent and notable constituent of gasoline. Gasoline is currently 
leaking from an existing half million or more underground storage tanks in 
the U.S. Hence, the ability to detect benzene in potable water by a rapid 
method becomes of increasing value in assessing the presence or absence of 
gasoline contaminants of water supplies. 
Toluene and xylene are also present in the workplace and in some petroleum 
fuels. Many other aromatic hydrocarbons, e.g. including chlorinated 
derivatives of benzene, toluene and xylene, naphthalene, styrenes, etc. 
are found in various environmental milieus. 
At present, systems for determining the presence of aromatic hydrocarbons 
in the workplace and the environment include gas chromatography, mass 
spectrophotometry and high performance liquid chromatography. 
In general, these current methods for analysis require a collection of 
samples of air, water, soil, etc., transportation of the sample to an 
analytical laboratory, and an analysis by professionals using costly 
equipment and reagents. The results obtained must then be transmitted back 
to the entity that ordered the testing. This process is expensive and 
time-consuming. 
Thus, alternative testing procedures for aromatic hydrocarbons and their 
derivatives which are simpler, less expensive, and adaptable to being 
rapidly carried out on-site are being needed. 
SUMMARY OF THE INVENTION 
The present invention provides methods, reagents, and kits for determining 
the presence of one or more aromatic hydrocarbons in a sample by 
immunoassay. 
It has been found that antigenic haptens can be made by conjugating serum 
protein antigens such as serum albumin or gamma globulin to an aromatic 
hydrocarbon, and that these conjugates can then be injected into animals 
where, in a known manner, antibodies to the aromatic ring will be produced 
and can be harvested. These antibodies may then be utilized in a variety 
of immunoassay procedures to detect environmental aromatic hydrocarbons, 
using various known markers to label either the aromatic hydrocarbon or 
antibody. In some embodiments of immunoassays either an aromatic 
hydrocarbon or an antibody is immobilized. Both polyclonal and mononclonal 
antibodies can be used in the practice of the present invention. The 
antibodies are heterospecific in that they will bind aromatic rings, 
whether or not they have attached functional groups. 
Immunoassay methods that can be used in the practice of the present 
invention to detect aromatic hydrocarbon rings in a sample include enzyme, 
fluorescent, chemiluminescent and biosensor immunoassay, as well as 
radioimmunoassay. Such assays may be heterogeneous or homogeneous. 
The assays of the present invention are used to determine the presence of 
compounds containing the aromatic ring, such as toluene, toluidine, 
2-(p-Tolyl) ethylamine, benzene, styrene, xylene, ethylbenzene, 
propylbenzene, or 2-methylnaphthalene, halogenated benzenes, biphenyl 
compounds, etc.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides immunoassays for testing a sample for the 
presence of aromatic hydrocarbons rings. In such immunoassays, the 
aromatic hydrocarbon ring (antigen)/antibody reaction can be detected by a 
variety of methods, using various markers to label either the antigen or 
antibody to permit detection of the reaction product. Furthermore, 
immobilization of either the antigen or antibody will facilitate detection 
in many cases. 
Antigen/antibody assays can be generally classed into two categories, 
heterogeneous and homogeneous. Heterogeneous assays require separation of 
the bound-labelled component from the free-labelled component prior to 
detection of the reaction product. Homogeneous assays do not require such 
a separation step. The assays can further be (1) competitive, for example, 
where antigen competes for labelled antibody with a solid-phase antigen or 
where antigen competes with labelled antigen for a solid-phase antibody or 
(2) noncompetitive where there is a direct relationship between label and 
antibody or antigen. 
In one embodiment of the present invention, the method of detecting the 
presence of at least one aromatic hydrocarbon ring in a sample comprises 
the steps: 
(a) Forming a conjugate between (i) an immunological equivalent of the 
aromatic hydrocarbon ring or the aromatic hydrocarbon ring itself, and 
(ii) a composition capable of detection; 
(b) Contacting the conjugate and the sample to be tested for the aromatic 
hydrocarbon ring with an antibody which reacts with the aromatic 
hydrocarbon ring, under conditions so that the conjugate and the aromatic 
hydrocarbon ring in the sample compete for binding sites on the antibody; 
and 
(c) Detecting the presence of the aromatic hydrocarbon ring by measuring 
the reaction of bound conjugate with a composition responsive to the 
composition capable of detection. 
In some embodiments, the antibody is immobilized to facilitate detection. 
In yet another embodiment of the present invention, the method of detecting 
the presence of at least one aromatic hydrocarbon ring in a sample 
comprises the steps of: 
(a) Immobilizing the aromatic hydrocarbon ring or its immunological 
equivalent; 
(b) Forming a conjugate between (i) an antibody which binds the aromatic 
hydrocarbon ring or an immunological equivalent thereof, and (ii) a 
composition capable of detection; 
(c) Contacting the conjugate and the sample to be tested for the 
immobilized aromatic hydrocarbon ring with the aromatic hydrocarbon ring 
or immunological equivalent thereof of step (a) under conditions so that 
the immobilized hydrocarbon and the aromatic hydrocarbon ring in the 
sample compete for binding sites on the conjugate; and 
(d) Detecting the presence of the aromatic hydrocarbon ring by measuring 
the reaction of bound conjugate with a composition to which it is 
responsive. 
Immunoassay methods that can be used in the practice of the present 
invention to detect the presence or absence of aromatic hydrocarbons in a 
sample include enzyme, fluorescent chemiluminescent and biosensor 
immunoassay, as well as radioimmunoassay. In enzyme-lined immunoassays 
(ELISA), in accordance with the present invention, the aromatic 
hydrocarbon ring(s) or interest can be labelled directly with an enzyme or 
indirectly by use of enzyme-labelled antibodies which under appropriate 
conditions catalyze a reaction with a substrate. The enzyme activity is 
typically detected by formation of a colored reaction product i.e., a 
colored end point that may be easily detected by eye or measured by 
spectroscopic or reflectance means. Several enzymes, including alkaline 
phosphatase, horseradish peroxidase (HRP) and glucose oxidase have been 
coupled to both antigen and antibody. HRP is commonly used and several 
substrates are available for it. For visual detection, the substrate will 
usually comprise a solution of a peroxide such as hydrogen peroxide and a 
chromogenic material such as ophenylenediamine or tetramethylbenzidine 
which manifests a color upon oxidation. 
In fluorescent immunoassay techniques for use in the present invention, the 
aromatic hydrocarbon ring(s) of interest can be labelled directly with 
fluorochromes, or indirectly with fluorochrome-labelled antibodies. 
Fluorochromes are dyes that absorb radiation (e.g., ultraviolet light), 
are excited by it, and emit light (e.g., visible light). 
The assays of the present invention are applicable to any compound 
containing an aromatic hydrocarbon ring that is free of steric hindrance 
or blockage to reaction with an antibody. Exemplary aromatic hydrocarbon 
rings containing compounds include toluene, toluidene, 2-(p-Tolyl) 
ethylamine, benzene, styrene, xylene, ethylbenzene, propylbenzene, or 
2-methyl-napthalene, halogenated aromatic hydrocarbons, etc. 
The amount of aromatic hydrocarbon ring detected can vary over a wide 
range. For example, immunoassays in accordance with the present invention 
can be designed by one skilled in the art to detect from about 1 part per 
trillion (10.sup.-12 g) to about 1000 parts per million (10.sup.-3 g) of 
aromatic hydrocarbon ring. 
Monoclonal antibodies to aromatic hydrocarbon rings for use in the practice 
of the present invention are made using immunization and hybridoma 
culturing techniques well known to those in the art. Polyclonal antibodies 
to aromatic hydrocarbons for use in the practice of the present invention 
are also made using techniques known to those skilled in the art. 
Heterospecific antibodies are particularly useful in conducting tests of 
the present invention for screening purposes such as, e.g., detecting the 
presence of gasoline in a soil sample, because a variety of aromatic 
hydrocarbons are found in gasoline. 
The present invention also provides a polyclonal IgG antibody preparation 
which binds at least one aromatic hydrocarbon ring, the antibody 
preparation produced by a method which comprises the steps of: (a) 
administering to a host a predetermined quantity of a composition 
comprising a hydrocarbon containing the ring or an immunological 
equivalent coupled to a biologically acceptable carrier protein, (b) 
collecting sera from the host, and (c) purifying IgG antibody from the 
sera. An immunological equivalent of an antigen has the ability, when 
introduced into a host, to cause the production of antibodies to the 
antigen. A heterospecific polyclonal antibody preparation, preferred in 
some embodiments, e.g., aromatic hydrocarbon screening tests, of the 
subject invention was produced by developing antibodies in rabbits against 
the toluene derivative, tolylacetic acid. The immunogen used consisted of 
tolylacetic acid molecules conjugated to Bovine Serum Albumin molecules. 
The resulting antibody binds toluene and also binds a number of other 
aromatic hydrocarbons containing the benzene ring, including, but not 
limited to, benzene, toluidine, 2-(p-Tolyl)ethylamine, styrene, xylene, 
ethylbenzene, propylbenzene and 2-methylnaphthalene. 
In accordance with the present invention, the antibody or the aromatic 
hydrocarbon may be labelled with radioactivity, enzymes, fluorochromes or 
luminogens. At present, enzymes are a preferred label. Although any enzyme 
which can be conjugated to an antibody or an aromatic hydrocarbon can be 
used in assays according to the present invention, peroxidases are a 
preferred class of enzymes, and horseradish peroxidase is particularly 
preferred. The chromogen for use in enzyme immunoassays, according to the 
present invention, can be any chromogen that is capable of changing or 
producing a color in the presence of an enzyme and its substrate. 
3,3',4,5'-tetramethylbenzidine (TMB) is a preferred chromogen when the 
enzyme used is horseradish peroxidase. 
Although any suitable immunoassay technique, such as RIA, EIA, or ELISA, 
may be used for the detection of aromatic hydrocarbon rings, according to 
the present invention, a preferred immunoassay technique is a colorimetric 
ELISA competitive immunoassay, where the antibody is immobilized and the 
aromatic hydrocarbon to be detected is conjugated with an enzyme. In this 
type of competitive enzyme immunoassay, a predetermined number of 
conjugate molecules compete with an unknown number of aromatic hydrocarbon 
molecules in the sample for a predetermined number of binding sites on the 
antibody. In such colorimetric enzyme immunoassays, sufficient conjugate 
must be bound so that when the color formation or change occurs, the color 
or color change is capable of detection visually or by an appropriate 
instrument. Concentrations of aromatic hydrocarbons from about one part 
per million (10.sup.-6 g) to about 1000 parts per million (10.sup.-3 g) 
have been found useful in ELISA's of aromatic hydrocarbons according to 
the present invention. However, the method is suitable for any amount of 
aromatic hydrocarbon present as long as the color can be detected. 
A programmable differential rate spectrophotometer is used in the Examples 
set forth below. However, the methods of the present invention can be 
adapted by one skilled in the art to use in other types of 
spectrophotometers. Similarly, the detectable reaction product is not 
limited to a chromophore but includes other labels as described above. 
In one embodiment of the present invention, the assay matrix is a triple 
antibody layer. Rabbit IgG is immobilized on the inside of a polystyrene 
tube using techniques known to those skilled in the art, e.g., via 
adsorption or the glutaraldehyde method (See, e.g., T. Boenisch, Protides 
of Biological Fluids, 24th Colloq. (1976), p. 743, Ed. H. Peeters.). Then, 
using conventional techniques, goat anti-rabbit IgG is immunologically 
bound to the rabbit IgG, followed by the immunological binding of a rabbit 
antibody prepared in accordance with the present invention which reacts 
with the aromatic hydrocarbon ring in question. 
The invention also provides kits for performing the assay of aromatic 
hydrocarbons. One embodiment of such a kit comprises in combination: (a) 
an antibody which reacts with at least one aromatic hydrocarbon ring; and 
(b) a conjugate of (i) an immunological equivalent of the hydrocarbon or 
the hydrocarbon ring itself, and (ii) a label substance capable of 
detection. 
In such kits, wherein the detectable reaction product comprises a substance 
which emits radiation or produces a conductivity change, a luminogen, a 
fluorogen or an enzyme responsive to a chromogen, there may also be 
provided a composition which can react in the presence of the conjugate to 
produce a color, a color change, emission of light, fluorescence or a 
conductivity difference. 
The present invention also includes a kit for carrying out an immunoassay 
to detect the presence of at least one aromatic hydrocarbon ring 
comprising in combination: 
(a) an immobilized aromatic hydrocarbon or an immunological equivalent 
thereof; and 
(b) A conjugate between (i) an antibody which binds the aromatic 
hydrocarbon ring or which binds the immunological equivalent thereof, and 
(ii) a composition capable of detection. 
In the kits described above, either the conjugate or antibody may be 
immobilized to facilitate detection. The kits may also include items such 
as standards, buffers and so forth. 
Samples which can be tested for the presence of aromatic hydrocarbons by 
use of immunoassays in accordance with the present invention include, but 
are not limited to, particulate samples, e.g., activated charcoal, air, 
water and soils. 
Immunoassays in accordance with the present invention can be applied to 
several areas where the detection and/or quantitative or semi-quantitative 
analysis of aromatic hydrocarbons is required. The test can be carried out 
on site in the industrial workplace for individual monitoring of workers 
exposed to hazardous substances. In this case, the aromatic hydrocarbons 
present in the air are adsorbed on the activated charcoal contained in a 
dosimeter badge. The charcoal is extracted with methanol, as described 
below, and assayed by an immunoassay of the present invention. Groundwater 
can also be assayed, and in many instances, requires no extraction step; 
in such cases, an aliquot of the water to be tested is simply added to the 
assay instead of the extraction solution. Soil or gravel samples, e.g., 
those known or suspected to be contaminated with gasoline or kerosene, can 
be extracted in the same manner as is described above for the activated 
charcoal. 
Immunoassays, as taught therein, have several advantages over the 
traditional gas chromatography methods currently used. The advantages 
include reduced cost to assay a sample; the capability of performing on 
site evaluations; and shorter turn around times. Moreover, such 
immunoassays can be designed to be rapid (the immunoassays described below 
take, on the average, 10 minutes to perform). 
The following examples are provided to further illustrate the invention. 
EXAMPLE I--ANTIBODY PREATION 
Materials, Methods, and Techniques 
Tolylacetic Acid was covalently linked to a number of proteins including 
Bovine Serum Albumin (BSA) and Bovine Gamma Globulin (BGG). 
Hapten BGG conjugates were used to immunize rabbits and mice. 
Preparation of the N-Hydroxy Succinimide (NHS) Ester p-Tolylacetic Acid 
The preparation of hapten protein conjugates requires the activation of 
p-Tolylacetic Acid, and the subsequent coupling of the activated hapten to 
the desired protein. 
Tolylacetic Acid was purchased from Aldrich and used without modification 
or purification. 
Experimental Protocol: NHS-ester of p-Tolylacetic Acid 
1. The hapten (p-Tolylacetic Acid), hydroxethyl succinimide and 1-ethyl 
(3-dimethylaminopropyl) carbodiimide (1:1.1:1.1 molar ratio, respectively) 
were weighed into a flask equipped with a stir bar and vacuum dried 
overnight at room temperature. 
2. Dimethylformamide (DMF) (dried over 4A molecular sieves) was added to 
the flask to obtain a 0.2 M hapten solution. The flask was stoppered and 
allowed to stir overnight at ambient temperature. 
3. The crude NHS-ester was used in conjugations without further 
purification. 
Conjugation of P-Tolylacetic-NHS ester to BGG and BSA 
1. The activated hapten (p-Tolylacetic-NHS ester) in aliquots of 5-20 
microliters (depending on reaction size) was added to a cold (ice bath) 
stirring solution of protein (BGG or BSA) in 0.1 M Carbonate, pH 9.5, 
Buffer. The protein concentration was approximately 60-100 mg/ml. The 
activated hapten was added slowly over a period of 1-1.5 hours to an 
approximate end point of 50:1 hapten: protein ratio. 
2. The conjugation was monitored by withdrawing aliquots of the protein 
conjugation solution and determining the amine content by TNBS Titration. 
It is assumed that the moles of amines lost is equal to the moles of 
hapten bound. Reactions were usually allowed to proceed 30 minutes past 
the last hapten addition. 
3. The crude conjugates were purified by chromatography on a SEPHADEX G-25 
column in 0.1 M Carbonate (pH 8.5) Buffer, dialyzed against four changes 
of distilled water, and lyophilized. A small aliquot of the 
chromatographed protein is saved to determine the hapten number. 
4. The hapten number was determined by amine content by Trinitrobenezine 
Sulfonate (TNBS) Titration of the protein sample before and after 
conjugation. The protein concentrations are determined by UV and/or the 
Lowry Protein Method. 
The hapten numbers of the conjugates prepared are given below: 
______________________________________ 
HAPTEN PROTEIN 
CONJUGATE NUMBER YIELD 
______________________________________ 
p-Tolylacetic BGG 
44.5 340 mg 
p-Tolylacetic BSA 
32 190 mg 
______________________________________ 
Animal Programs 
Animal immunization programs with three rabbits and ten mice were initiated 
with p-Tolylacetic BGG conjugates. The initial injections were prepared 
with Complete Freunds' Adjuvant while subsequent injections (boost) were 
prepared with Incomplete Freunds' Adjuvant. 
Immunization and Bleeding 
1. New Zealand White Rabbits, with approximate weights of 7-8 pounds per 
animal, were used in the immunization program. 
2. 2.5 mg/ml of the antigen was injected into each animal per month (3-4 
weeks). 
3. Test bleeds of 7-10 milliliters of blood were obtained each month or 
7-10 days following immunization. 
4. The serum was separated from the blood using the following procedure: 
blood was drawn from the ear vein and allowed to stand at room temperature 
for one hour. 
The sample was cooled to 4.degree. C. (refrigerated) for four hours to 
clot, and centrifuged to separate the cells from the serum. 
The serum was separated from the clot and centrifuged again at high speed 
to remove lipids. 
5. The animal sera was screened for presence of specific antibodies by 
ouchterlony immunodiffusion analysis using the BSA hapten conjugate as the 
antigen. 
6. Antisera which gave a positive response to ouchterlony immunodiffusion 
analysis were evaluated for inhibition in competitive solid phase EIA, 
using conventional techniques. 
7. Production bleeds of 40-50 milliliters of animal blood (18-23 ml serum) 
were obtained in the same manner as test bleeds. 
Injections 
The injections containing the immunogens are prepared one to three days 
prior to use. The lyophilized immunogen is dissolved in saline. An equal 
volume of adjuvant (Freunds' Complete for the initial immunization and 
Freunds' Incomplete for subsequent boost) and immunogen-saline mixture is 
combined and emulsified. One milliliter of the emulsion containing 2.5 mg 
of the immunogen is injected beneath the skin of the rabbit. The animals 
were bled 7-10 days following immunization. 
EXAMPLE 2--COMPETITIVE ENZYME IMMUNOASSAY REAGENT AND PROCEDURE 
Assay Principle 
The test described below is a competitive enzyme immunoassay in which a 
fixed number of enzyme-labelled antigen molecules compete with an unknown 
number of antigen molecules in the sample for a fixed number of binding 
sites on antibodies directed against the antigen. As the number of antigen 
molecules in the sample increases, the number of bound labelled antigen 
molecules decreases due to competition. Ergo, after the unbound antigen 
(labelled and unlabelled) is removed and substrate and chromogen are 
added, the degree of color development will depend on how much labelled 
antigen remains immunologically bound. The greater the concentration of 
antigen in the sample, the less labelled antigen bound after separation 
and the lower the rate of color development. A programmable, differential 
rate spectrophotometer which references the rate of color development in 
the sample against a calibrator (designed reference) containing a fixed 
mass of antigen was used in carrying out the assays described below. 
Preparation of Assay Matrix 
The assay matrix is a triple antibody layer comprising rabbit IgG 
(Pel-Freeze), anti-rabbit IgG (Pel-Freeze) and primary antibody (prepared 
in accordance with Example 1 above) in a 12.times.75 mm polystyrene tube. 
Rabbit IgG was bound to the inside of the polystyrene tube via the 
glutaraldehyde method (See, e.g., Boenisch, supra.). 
The following steps immunologically bind the second and the primary 
antibody resulting in a triple layer. A 1:500 dilution of Goat anti-Rabbit 
serum (Pel-Freeze) was made with 10 mM phosphate buffer, 0.15 M 
phosphate-buffered saline (PBS) of pH 7.5 to which 1 mg/ml Bovine Serum 
Albumin (BSA) is added (PBS-BSA). Six hundred microliters of this solution 
was pipetted into tubes and incubated overnight at room temperature. Then 
the tubes were rinsed 1.times. with deionized water. A 1:20,000 dilution 
of the primary antibody was prepared in PBS-BSA and six hundred microliter 
aliquots were added to the tubes. The tubes were incubated, aspirated, 
rinsed, and placed in a drying room for about 24 hours. The tubes are 
subsequently stored at 4.degree. C. 
The primary antibody was developed in rabbits against the toluene 
derivative para-tolylacetic acid conjugated to BSA or BGG as described in 
Example 1 above. The resulting antibodies react with several compounds all 
containing the aromatic ring; toluene, toluidine, 2-(p-Tolyl)ethylamine, 
benzene, styrene, xylene, ethylbenzene, propylbenzene, 
2-methyl-naphthalene, for example. 
Aromatic Hydrocarbon - HRP Conjugate 
Reference and Sample 
Tolylacetic acid molecules were conjugated to the enzyme Horseradish 
Peroxidase using methods known to those skilled in the art. See, e.g., 
Enzyme Labelling of Antibodies, J. of Immunoassay, Vol. 4, No. 3, 1983, 
First Edition, E. Ishikawa. The resultant conjugate was diluted in a 
protein medium consisting of 50% fetal calf serum, 0.5 M Trizma Base, with 
conventional antimicrobial agents, and 0.05% surfactant, about pH 7.0. 
Two vials of conjugate are required for conducting a single test when using 
a differential rate spectrophotometer, as described above. In one such 
vial, a predetermined quantity of the conjugate and of tolylacetic acid is 
present which gives the same test response as a certain mass of the 
aromatic ring-containing compound(s) to be detected (or combined benzene, 
toluene, and xylene-referred to as BTX). 35 mg of p-tolylacetic acid which 
has been determined to give the same response (absorbance after a set 
time) as 21 mg of Toluene (or combined BTX). This vial is designated as 
the Reference or `R` vial. The other vial containing the predetermined 
quantity of conjugate but no tolylacetic acid is labelled `S` for Sample. 
These materials are then lyophilized and stoppered under vacuum. 
RUNNING THE TEST 
One `R` vial and one `S` vial are placed in a tray. A predetermined 
quantity of a dissolving solution comprising 0.09% TRIS buffer and 0.025% 
detergent, e.g., Brij-35 is used to dissolve the freeze-dried materials. 
If the material to be tested for the presence of aromatic ring containing 
compound(s) requires extraction as from, e.g., activated charcoal, it is 
extracted by shaking it in a small container with a predetermined volume 
of methanol. A measured quantity of the methanol extract is placed in the 
`S` vial. The same volume of pure methanol is placed in the `R` vial to 
compensate for any effects of methanol in the `S` vial. The entire 
contents of the `R` and `S` vials are transferred to the `R` and `S` 
antibody-coated tubes, respectively. 
The reaction mixture is incubated for about 5 minutes followed by 5 
forceful rinses with water. Six drops, about 250 ml, of chromogen 
(3,3',5,5'-Tetramethylbenzidine; 40% methanol; 20% glycerol) are added to 
each tube. Then quickly and accurately the volume in each tube is brought 
up to a volume of between 500-600 microliters with the substrate solution, 
a 0.04% urea peroxide, 0.09% M sodium acetate, 0.1 M citric acid, pH 5.0. 
After swirling and tapping the color tube development is monitored by means 
of a computer-linked Differential Rate Spectrophotometer, as described 
above. 
An intermittent mixing device was built into the spectrophotometer using 
conventional techniques in order to assure accurate absorbance readings 
(color development is more rapid on the bottom of the tube where the 
surface area is greater). Absorbance readings are taken every 20 seconds 
(a few seconds after the mixing stops) and the readings are stored in the 
computer linked to the spectrophotometer. The first six readings are 
ignored. Calculations are the performed based on the equation 
(100+K)(r)-K=ppm (g per ml), where K is a constant determined from 
experimental data and r is the ratio: 
##EQU1## 
A mean of about nine such calculations was computed and shown on the 
liquid crystal display of the linked computer. A printer may also be 
attached to the spectrophotometer for a hard copy of data. Since 
calculations are based on the ratio of the rate of color development of 
the reference against the rate of color development of the sample, many 
factors which affect the assay, such as temperature and increased 
incubation time do not affect the result of the assay; both the reference 
and the sample should be affected equally by these factors. 
EXAMPLE 3--ANTIBODY/AROMATIC HYDROCARBON BINDING 
The reactivity of an antibody prepared in accordance with Example 1, above, 
with various aromatic hydrocarbons was determined using the general 
procedure outlined in Example 2, above. 
Preparation of Standards 
All standards were made with methanol (reagent grade) by making serial 
dilutions of the pure chemical to be tested. If the substance was in 
crystalline form, a weight/volume method was used. If the compound was a 
liquid, the relationship--density of the compound X 10.sup.6 parts per 
million (microgram per ml) was used to determine the actual mass. 
Subsequent serial dilutions were then made until a desired concentration 
(microgram per ml or ppm) was achieved. 
Assay Steps 
a) 250 ml of the Universal diluent is pipetted into each of from about 6 to 
about 10 triple-layer anti-tolyl coated polystyrene tubes and one uncoated 
polystyrene tube (to check for nonspecific binding-NSB). 
b) 150 ml of dissolving solution as described in Example 2 above was added 
to each tube. 
c) 50 ml of a working dilution of tolylacetic-HRP conjugate was added to 
the tubes. The working dilution of the conjugate for these particular 
tests varied, but the dilution is noted in the tabulation provided below. 
d) 100 ml of 100% methanol was pipetted into one-half of the reference 
tubes and the NSB tubes. This represented the zero dose or the maximum 
binding (i.e., maximum color development). Into the other tubes, 100 l was 
pipetted of a standard solution containing the aromatic hydrocarbon to be 
tested for cross-reactivity. 
e) The tubes are capped (a precaution since the aromatics are hydrophobic 
and the assay mixture is aqueous based) and incubated for 5 minutes. 
f) After the 5 minute incubation, the solutions were decanted, and the 
tubes were rinsed 5.times. forcefully with a squeeze bottle. 
g) The water was shaken out of the tubes thoroughly. Equal volumes of 
substrate and chromogen were added to a glass vial, vortexed, and 500 1 of 
the mixture was added to each tube quickly. 
h) Color was allowed to develop from about 5 to about 10 minutes until the 
reaction was terminated with 1 ml of 1NH.sub.2 SO.sub.4, switching the 
color from blue to yellow. The tubes were then read at 450 nm. 
The aromatic hydrocarbons listed below were tested for reactivity with an 
antibody preparation made in accordance with Example 1 above. The test 
procedures and volumes were essentially as described above. 
The following aromatic hydrocarbons were determined to react with the 
antibody: Benzene, Xylene, Toluene, Styrene, Toluidine, 
2-(p-Tolyl)ethylamine, Ethylbenzene, 2-Methyl-naphthalene, and 
Propyl-benzene. 
EXAMPLE 4 
Correlation with Gas Chromatography analysis 
Samples comprising a mixture of aromatic hydrocarbons were analyzed using 
the general procedures outline in Example 2, above, as well as by 
conventional gas chromatography techniques, to determine the presence of 
such aromatic hydrocarbons. The analysis using the immunoassay procedures 
of Example 2 has been determined by regression analysis, a technique 
well-known to those skilled in the art, to correlate well with the 
analysis using conventional gas chromatography techniques. The correlation 
coefficient was determined to be 0.989. 
The samples used in the regression analysis consisted of (i) serial 
dilutions of gasoline and (ii) soil samples, including gravel and clay, 
which were spiked with gasoline and then extracted with methanol as 
described in the above examples. 
The antibody preparation used in the regression analysis was prepared in 
accordance with Example 1 above. 
EXAMPLE 5--MONOCLONAL ANTIBODY PREATION 
Monoclonal antibodies are prepared according to the classical method of 
Kohler & Milstein by injecting an immunogen such as tolylacetic acid bound 
to BSA into a mouse spleen. After two to three weeks, the mouse is 
sacrificed, the spleen is removed and digested and the lymphocyte cells 
are extracted. A selection process involving the screening of the 
lymphocytes for antibody activity on microtiter plates is carried out as 
taught by Kohler & Milstein. The lymphocytes selected in the screening 
process are then each fused with a mouse myeloma cell to form a hybridoma. 
Each hybridoma is then injected into the stomach of a mouse. Each injected 
mouse is sacrificed approximately 2 weeks later and the ascites fluid 
produced by its stomach in the interim is recovered. From each separate 
batch of ascites fluid a different monoclonal antibody capable of bind 
aromatic ring-containing compounds is recovered. 
The examples, using solid-phase, enzyme-linked competitive immunoassay, are 
merely one example of the use of the present invention. Variations in the 
actual process described in the examples will be apparent to those skilled 
in the art. 
Therefore, the present invention is to be considered as limited only by the 
appended claims.