A method of performing a diagnostic immunoassay utilizing colloidal non-metal particles having conjugated thereto a binding component capable of specifically recognizing an analyte to be determined. After reaction of the sample and colloidal non-metal particles, the presence or amount of analyte/colloidal non-metal particle complexes are determined by optical analysis as a measure of the amount of analyte in the sample. The method can be utilized for the specific detection of numerous analytes and is sensitive and has a wide detection range.

BACKGROUND OF THE INVENTION 
The present invention relates generally to diagnostic immunoassays and 
material useful therein More particularly, the present invention relates 
to employing colloidal non-metal particles, especially selenium in 
specific binding assays to determine the presence of small quantities of 
an analyte of interest 
Various analytical procedures are commonly employed in assays to determine 
the presence and/or concentration of substances of interest or clinical 
significance in fluids or other materials. Such clinical significant or 
interesting substances are commonly termed "analytes" and can include, for 
example, antibodies, antigens, and the broad category of substances 
commonly known by the term "ligands". Particularly, with respect to the 
diagnosis and treatment of disease or other conditions of the human body, 
the accurate determination, on a timely basis, of the presence or amount 
of certain analytes can have a profound influence on the ability of health 
care professionals to treat and manage pathological physical disorders or 
make an early and accurate determination of physiological conditions such 
as pregnancy. 
Many diagnostic immunoassays are known which generally employ the specific 
binding characteristics that exist between an analyte or protein and a 
specific antibody tagged with some traceable substituent. Assays of these 
type include, for example, radioimmunoassay (RIA), free radical assay 
technique (FRAT), enzyme immunoassay technique (EIA), and 
immunofluorescence techniques such as fluorometric analysis and 
fluorescence polarization. Analytes of interest have also been detected 
and/or quantified using metal sol particle immunoassay techniques, as 
described in U.S. Pat. No. 4,313,734 to Leurverinq. In Leurverinq, 
colloidal metal particles of at least 5 nm are coupled to binding protein 
and employed as an aqueous sol dispersion in immunochemical techniques 
such as "sandwich" assays and homogeneous agglutination assays. 
While the above techniques can be effective, they are subject to 
improvement. For example, although sensitive, a radioimmunoassay requires 
handling of radioactive compounds and use of sensitive instrumentation, 
such as a scintillation counter. Enzyme immunoassays are widely used; 
however, in general EIA's are not sensitive enough to detect small amounts 
of immunogens. Immunoassays employing metal sols are subject to limited 
detection range (i.e, wavelenqth range to monitor reaction) thereby 
requiring careful calibration of the instrument employed to monitor the 
change in optical properties of a sample. 
Accordingly, there is a need for an improved method for conducting an 
immunoassay exhibitinq both high sensitivity and a wide detection range 
for analytes and proteins of interest. 
SUMMARY OF THE INVENTION 
The present invention relates to a non-metal colloidal particle suitable 
for use in an immunoassay. The method of the present invention involves 
determining the presence or amount of an analyte in a sample by, 
contacting the sample with a non-metal labelled constituent comprising an 
aqueous dispersion of colloidal non-metal particles having conjuqated 
thereto a binding component capable of specifically recognizing the 
analyte of interest. After reaction of the sample and non-metal labelled 
constituent the presence or amount of analyte/colloidal non-metal particle 
complexes formed are analyzed as a measure of the amount of analyte in the 
sample. 
In a preferred embodiment of the present invention, selenium particles 
having an antibody adsorbed thereon serve as the non-metal labelled 
constituent. Preferably, selenium particles of from about 3 nm to about 
450 nm are used, with selenium particles of from about 11 nm to about 200 
nm most preferred. The selenium particles are employed in the preferred 
method as an aqueous dispersion of from about 0.005% to about 0.1% 
(weight/volume). Most preferably, an aqueous dispersion of from about 
0.02% to about 0.04% (weight/volume) selenium particles is used. 
The method of the present invention is suitable for solution type 
immunoassays and chromatographic assays employing solid carriers such as 
nitrocellulose or the like. In a preferred embodiment, the presence or 
amount of analyte is determined by measuring the change in optical 
properties in a reaction mixture of test sample and aqueous dispersion of 
colloidal non-metal labelled constituent resulting from agglutination of 
analyte and colloidal non-metal labelled constituent. While the change of 
optical properties can be detected either visually or by use of 
instrumentation, preferably the change in optical properties is analyzed 
using a spectrophotometer or fluorescence intensity measuring instrument.

DETAILED DESCRIPTION OF THE INVENTION 
The term "analyte" as used herein is defined as a compound or composition 
to be measured, which may be an antibody, antigen or ligand, which is 
mono- or polyepitopic, antigenic or haptenic, single or plurality of 
compounds which share at least one common epitopic site or a receptor. 
Ligand, as used herein, refers to a molecule, to which a binding protein, 
such as a receptor or an antibody, can be obtained or formed. Liqands are 
generally protein-free compounds, generally of low molecular weight, which 
do not induce antibody formation when injected into an animal but which 
are reactive to antibodies. Liqands which are chemically modified for 
configuration to a carrier material, such as a protein or tracer compound 
are termed haptens. Antibodies to haptens are generally raised by first 
conjugatinq the haptens to a protein and injecting the conjugate product 
into an animal. The resulting antibodies are isolated by conventional 
antibody isolation techniques. 
Generally, in the present invention, the immunological reaction is based on 
the reaction between a specific binding protein (i.e. antigen or antibody) 
and the analyte which is a corresponding bindable substance in an aqueous 
test sample. In the present invention, the analyte of interest in the test 
sample can be any one of a large number of antigens, antibodies, or 
ligands of interest. The corresponding binding protein is an antigen or 
antibody capable of specifically recognizing the corresponding analyte. 
The analytes to be determined using the method of the present invention are 
limited primarily by their ability to be attached to the specific binding 
component. Generally, the molecular weight will be between 100 and about 
100,000; however, the present method can be applied to detect 
macro-molecules of a much higher molecular weight. Antigens, haptens and 
their antibodies, hormones, vitamins, drugs, metabolites and their 
receptors and binding materials may be determined using the present 
method. Representatives of analytes determinable by the method of the 
present invention include steroids such as estrone, estradiol, cortisol, 
testosterone, progesterone, chenodeoxycholic acid, digoxin, cholic acid, 
digitoxin, deoxycholic acid, lithocholic acids and the ester and amide 
derivatives thereof; vitamins such as B-12, folic acid, thyroxine, 
triiodothyronine, histamine, serotorin, prostaqlandins such as PGE, PGF, 
PGA; antiasthmatic drugs such as theophylline, antineoplastic drugs such 
as doxorubicin and methotrexate; antiarrhythmic drugs such as 
disopyramide, lidocaine, procainamide, propranolol, quinidine, 
N-acetylprocainamide; anticonvulsant drugs such as phenobarbital, 
phenytoin, primidone, valproic acid, carbamazepine and ethosuximide; 
antibiotics such as penicillins, cephalosporins, erythromycin, vancomycin, 
gentamicin, amikacin, chloramphenicol, streptomycin and tobramycin; 
antiarthritic drugs such as salicylate; antidepressant drugs including 
tricyclics such as nortriptyline, amitriptyline, imipramine and 
desipramine; and the like as well as the metabolites thereof. 
Additional liqands that may be determined by the methods of the present 
invention include drugs of abuse such as morphine, heroin, hydromophone, 
oxymorphone, metapon, codeine, hydrocodone, dihydrocodiene, dihydrohydroxy 
codeinone, pholcodine, dextromethorphan, phenazocine and deonin and their 
metabolities. Higher molecular weight analytes such as aminoacids, 
polypeptides, and proteins such as hCG, TSH, LH, ferritin, CEA and 
C-reactive protein may also be determined by the methods of the present 
invention. 
According to the present invention a method for performing immunoassays 
employing a non-metal labelled constituent in the form of a aqueous 
dispersion of colloidal non-metal particles having conjuqated thereto a 
specific binding component (i.e. antibody or antigen) has been found to be 
surprisingly useful. Preferred non-metals which can be used in the present 
invention include elements within Group VI A. of the Periodic Table, 
sulfur, selenium, and tellurium. Most preferably the present invention 
utilizes an aqueous dispersion of colloidal selenium particles. 
The aqueous dispersion of colloidal non-metal particles can include from 
about 0.005g to about 0.1g, on a weight/volume basis, of the colloidal 
non-metal particles. Most preferably, aqueous dispersions having from 
about 0.02% to about 0.04% of the non-metal particles on a weight/volume 
basis are used. The non-metal particles themselves preferably have a 
particle size from about 3 nm to about 450 nm, most preferably from about 
11 nm to about 200 nm. In the case of the most preferred selenium 
particles the overall shape is globular and exhibits a ratio of length to 
width of between 0.7 and 0.95. Surprisingly, as evidenced by the examples 
hereto it has been observed that use of colloidal non-metal particles 
conjugated to a binding protein provides for an immunoassay exhibiting 
high sensitivity and wide detection range for an analyte of interest. 
While the immunocomponent of the non-metal labelled constituent can be 
conjugated to the colloidal non-metal particles using a number of methods 
known in the art including physical immobilization, covalent bonding 
hydrophilic bonding, hydrophobic bonding and ionic interaction preferably 
antibody capable of specifically recognizing the analyte of interest is 
adsorbed onto the colloidal non-metal particles. 
The method of the present invention involves contacting a liquid sample, 
having or suspected of having an analyte of interest, with a labelled 
substance and binding component capable of recognizing the analyte sought 
to be quantified. In a preferred embodiment, a sample of biological fluid 
such as urine, blood, serum, saliva or the like is mixed with an aqueous 
dispersion of non-metal labelled constituent in the form of colloidal 
selenium particles having conjugated thereto a binding compound capable of 
recognizing the analyte of interest. After an appropriate incubation time, 
(i.e. sufficient time to allow the labeling substance to complex with all 
the analyte of interest in the sample) the presence or amount of 
analyte/selenium labelled constituent complexes is determined as a measure 
of the amount of analyte in the sample. In the preferred embodiment the 
change in optical properties resulting from formation of the 
analyte/colloidal selenium labelled constituent complexes is measured. 
The measurement of the change in optical properties can be performed using 
a number of known techniques. The particular manner in which the change in 
optical properties is determined depends on the immunoassay techniques 
employed. For example, the present method can be used in liquid chemistry 
immunoassay techniques such as homogeneous agglutination in which the 
immunoloqical component of interest in the test sample complexes with the 
labelled immunochemical component resulting in a change of optical 
properties. Also, the present method can be employed in solid carrier type 
immunoassays which involve "sandwich" techniques known to those skilled in 
the art. The typical sandwich assay of this type includes an immunological 
component, such as a non-metal labelled antibody, if an antigen is to be 
determined, rendered insoluble by coupling to the solid carrier such as 
the surface of a reaction vessel or fiber matrix. After an initial 
incubation period which can be followed by a washing step, a second 
incubation takes place with a reagent capable of participating in a 
specific binding reaction with the analyte/non-metal labelled antibody 
complex formed during the initial incubation period. 
In the case of homoqeneous liquid agglutination techniques employing the 
method of the present invention, optical changes can be detected in a 
number of known ways. In one preferred method the change in optical 
properties resulting from formation of analyte/colloidal non-metal 
labelled constituent complexes are measured using a visual 
spectrophotometer, such as the ABBOTT SPECTRUM.RTM. available from Abbott 
Laboratories, Abbott Park, Ill. 60064. Alternatively, known fluorescence 
quenching techniques can be employed to determine the change in optical 
properties based on the fluorescence intensity of analyte/colloidal 
non-metal labelled complexes formed in the reaction mixture. Fluorescence 
intensity can be detected and/or quantified using commercially available 
clinical instruments such as the Abbott TDx.RTM., available from Abbott 
Laboratories, Abbott Park, Ill. 60064. 
It is contemplated that the method of the present invention can be employed 
not only in the above-described immunoassay techniques but can be modified 
by one of skill in the art for carrying out numerous heteroqeneous and, 
homogeneous specific binding assays without departing from the inventive 
concepts embodied herein. For example the method of the present invention 
can be modified to employed non-metal labelled constituent in nephelometry 
or histochemistry. 
When the method of the present invention is employed using colloidal 
selenium particles immunoassays with sensitivity detection as low as 10 
mIU/ml and at least up to 5,000 mIU/ml can be conducted. Further, not only 
does use of colloidal selenium having binding component conjugated thereto 
make possible high detection sensitivity when employed in the present 
method, but, the wavelength range for accurately detecting and quantifying 
an immunoreactive response is surprisingly wide. For example as presented 
in the Examples hereto, formation of analyte/colloidal selenium binding 
component complexes may be accurately measured over the range of from 
about 300 nm to about 600 nm. 
EXAMPLE 1 
Preparation of colloidal selenium 
To 200 ml of boiling distilled water, 2 ml of 3% SeO and 4.5 ml of freshly 
dissolved 1% ascorbic acid were added. The solution was refluxed for 10 
minutes and cooled to room temperature. The resulting brownish red 
solution was then centrifuged for 20 minutes at 2500 g and 4.degree. C. 
The pellet was resuspended in 200 ml of distilled water. 
EXAMPLE 2 
Conjugation of anti-.beta.hCG polyclonal antibody with colloidal selenium 
Polyclonal antibody against .beta.hCG was adsorbed onto colloidal selenium 
by mixing 25 ul of goat anti-ghCG polyclonal antibody (4.6 mg/ml) in 2 ml 
of phosphate buffer (20 mM, pH 7.3) containing 30 mM NaCI with 25 ml of 
the above selenium colloid. After stirrinq for 10 minutes at 26.degree. C, 
1 ml of 1% polyethylene glycol (avg. MW 8000) was added. The solution was 
then centrifuged for 5 minutes at 5000 q and 4.degree. C to obtain a dark 
red loose pellet. The pellet was resuspended in 1 ml of 0.1 g sodium 
phosphate buffer, pH 7.3, containing 0.15 g NaCI and 2% (w/v) BSA. 
EXAMPLE 3 
Assay of serum samples containing various amounts of hCGg 
A. Spectrophotometric Analysis 
In a series of test tubes were put 0.6 ml of 0.1 M sodium phosphate buffer, 
pH 7.3, containing 0.15 M NaCI and 30 ul of the above selenium-antibody 
conjuqate. To each tube, 70 ul of serum containing a specific amount of 
hCG standard were added. The tubes were briefly vortexed and incubated for 
3 minutes at 26.degree. C. before a spectrum was taken for each test tube 
on a spectrophotometer. Results of spectrophotometer measurements at 
A.sub.356, A.sub.420 and Ahd 510 are shown in FIG.1. As seen in FIG. 1, 
the absorbance units associated with the hCG standards were essentially 
the same at various wavelengths, demonstrating the broad range at which 
the change of optical properties resulting from analyte/collidal non-metal 
labelled constituent can be monitored. 
B. Radiative Energy Attenuation Analysis 
In a separate experiment, g60 ul of the above conjugate of selenium and 
antibody were mixed with 1.2 ml of 0.1 g sodium phosphate buffer, pH 7.3, 
containing 0.15 M NaCl. To this solution 140 ul of serum sample containing 
various amount of hCG were added. Each solution was mixed and incubated at 
26.degree. C. for 3 minutes. To this solution 25 ul of fluorescein 
solution were added. The concentration of the fluorescein was to give 
16,000 vertical intensity units on a Abbott TDx.RTM. fluorometer when the 
25 ul solution was mixed with 1.4 mL of the sodium phosphate buffer. The 
amount of fluorescence quenching due to the absorbance by selenium 
conjugate at the excitation and emission wavelengths of fluorescence (set 
at 485 and 525 nm on TDx.RTM.) was then measured employing a technique 
described in U.S. Pat. No. 4,495,293 (Radiative Energy Attenuation, REA, 
assay). The vertical intensity of fluorescence for each sample was 
collected. The fluorescence change due to the presence of different 
amounts of hCG in serum was then compared to the change in absorbance 
units measured by a spectrophotometer as described above and the results 
are shown in FIG. 2. 
EXAMPLE 4 
Solid phase immunoassay technique 
A mix and run sandwich-type immunoassay device for the detection of swine 
antitrichina antibodies were produced. Nitrocellulose assay strips were 
prepared and were treated with trichina antigen immobilized at a detection 
zone. 
Collidal selenium particles of Example I were utilized with various volumes 
(40, 80 and 150 ul aliquots) of concentrated selenium sol pipetted into 
individual vials containing 4 ml of water each and the pH of each, 
solution was adjusted to 7.2 by addition of 0.01 M potassium carbonate. To 
each of the vials was then added 150 ul of goat anti-swine antibody to 
IgG(H+L) (1 mg/ml concentration). The solutions were mixed and allowed to 
incubate for 10 minutes. A 0.5 ml aliquot of a 0.5% solution of alkaline 
treated casein was added to each solution and mixed well. Three ml 
aliquots of each selenium conjugate solution were centrifuged in 1 ml 
portions on a TDx.RTM. table centrifuqe, and the pellets were combined for 
each conjugate after the supernatent was decanted off. The combined 
pellets of each conjugate were resuspended with a solution of 4% casein in 
20 ul of TBS. 0.5 ul aliquots of the selenium particle labelled antibody 
solutions were then mixed with sera samples comprising varying 
concentrations of swine antitrichina antibodies. 
The sample mixtures were contacted to the nitrocellulose strips, and 
specific binding of the labelled antibody was detected at the detection 
zone. All conjugates gave a visible red color which indicates a positive 
signal, with the conjuqate utilizing 80 nm selenium particles providing 
the best results. All the conjugate solutions were tested against a 
negative control which indicated no specific binding. 
Although this invention has been described with respect to specific 
modifications, the details are not to be construed as limitations, for it 
will be apparent that various equivalents, changes and modifications may 
be resorted to without departing from the spirit and scope thereof and it 
is understood that such equivalent embodiments are intended to be included 
therein.