Journal of Immunological Methods 336 (2008) 9–15

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Journal of Immunological Methods

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Research paper

Carbon–protein covalent conjugates in non-instrumentalimmunodiagnostic systems

Mikhail Rayev, Konstantin Shmagel⁎Institute of Ecology and Genetics of Microorganisms UB RAS, 13 Golev Street, Perm, 614081, Russia

a r t i c l e i n f o

Abbreviations: BSA, bovine serum albumin; Eimmunosorbent assay; FITC, fluorescein isothiocychorionic gonadotropin; PBS, phosphate-buffered salibuffered saline containing 0.05% Tween-20; PBS-T-Csaline containing 0.05% Tween-20 and 1% casein; RIA,⁎ Corresponding author. Tel.: +7 342 244 71 34; fax:

E-mail address: shmagel@iegm.ru (K. Shmagel).

0022-1759/$ – see front matter © 2008 Published bydoi:10.1016/j.jim.2008.03.005

a b s t r a c t

Article history:Received 15 October 2007Received in revised form 24 January 2008Accepted 5 March 2008Available online 7 April 2008

An original technique for obtaining stable conjugates using colloid carbon particles as indicatorlabels has been developed. The reliability and stability of the diagnostic reagents obtained isprovided by covalent binding of various affine compounds on the surface of carbon particles.The stability of the reagents has been studied under various storage conditions for 3–10 years. Itwas shown that even when storage conditions were outside the optimal range, someconjugates preserved their analytical characteristics for 10 years. A number of systems foranalytical detection of various ligands have been designed based on the carbon–proteinconjugates synthesized. Themajor characteristics of these systems are their high sensitivity andspecificity, reliability, and reproducibility, simplicity in operation, and quick results.

© 2008 Published by Elsevier B.V.

Keywords:Carbon conjugatesColloidal carbon particlesNon-instrumental immunoassayImmunochromatographyImmunofiltration

1. Introduction

The currentlyavailablemethods of immunodiagnostics canbe arbitrarily divided into two fundamentally different groupsaccording to the type of implementation procedure: instru-mental and non-instrumental systems. The former requiremeasuring equipment, the latter do not. Implementationprotocols for nearly all systems include three stages: forma-tion of a specific complex, labeling, and label reading with anyphysicalmethod or simple visualization. Unlike the first groupof methods, which includes radioimmunoassay (RIA), immu-noenzyme assay (ELISA), and bio- and chemiluminescentimmunoassays (Yallow and Berson, 1959; Engvall and Perl-mann, 1971; Rubinstein et al., 1972; Woodhead and Weeks,1985; Ullman et al., 1996), non-instrumental systems areusually taken outside the clinical laboratory, and are used forquick diagnostics without the need for measuring equipment,

LISA, enzyme-linkedanate; HCG, humanne; PBS-T, phosphate-, phosphate-bufferedradioimmunoassay.+7 342 244 67 11.

Elsevier B.V.

specific conditions, or professional training. Thus, it is easy tooutline the requirements for the success of non-instrumentalsystems on the biochemical market. These requirementsinclude: high sensitivity of detection, specificity that providesfor the diagnostic value of the results, simplicity in operation,and clarity, all of which will ensure wide availability of theassay and unambiguity in interpretation. Such test systemparameters are of increasing importance; stability andreliability are based on the qualities of the associated reagents.If these issues are not addressed, it is virtually impossible tocreate reliable, replicable and credible diagnostic tests. As arule, non-instrumental systems are utilized by untrainedusers, and consequently they should be able towithstandnon-optimal storage conditions, and retain their effectiveness for along time. This implies the use of non-instrumental diagnos-ticums at home, in emergency situations, including epidemicemergencies, in field expeditions, in the army, in veterinaryservices, and on farms.

In practice, the reliability of a test system depends pri-marily on the stability of the associated reagents. Mostcommonly, this implies diagnostic conjugates used duringthe specific complex visualization stage.

The design of diagnostic test systems in the non-instru-mental group is commonly based on direct labeling of affinecompounds (anti-ligands) that are specific to the substance

Fig.1. Immunochromatography. Strip state: (a) beginning of the assay; (b) endof the assay. The large gray arrow shows the direction of fluid movement. 1,Contact zone; 2, analytical zone; 3, absorbent element; K+, internal positivecontrol.

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under investigation (ligand). These labels are optically denseparticles or colored polymers of various origin and size. Forexample, latexes, including colored latex (Remington et al.,1983; Bangs, 1988, 1990, 1996; Tarcha et al., 1990), erythro-cytes (Guesdon and Avrameas, 1980), non-metal colloids ofsulfur, selenium, tellurium (Spallholz, 1982; Yost et al., 1989),polymerized colloid dyes (Snowden and Hommel, 1991), andcolloid gold particles (Moeremans et al., 1984; Roth and Heitz,1989; Chakraborty et al., 1990). The use of these reagentsallows for immediate visual detection of the ligand on thesolid phase surface without an additional label reading stage,which favorably distinguishes them from well-known enzy-matic diagnostic systems. These methods, while sufficientlysensitive and reliable under sophisticated conditions (specifictemperature, environment and duration of storage), may beuseless in less civilized settings. During military action,natural and technology-induced disasters, expeditions andtraveling, when the storage regimen cannot be controlled andusers lack the required qualifications, the demand forreliability of test systems increases drastically.

The main goal of this study was to design stable carbon–protein conjugates based on covalent sheathing of colloidcarbon particles with various affine compounds, and there-fore, to increase the reliability and sensitivity of analyticalsystems for awide range ofmethods used in non-instrumentaltesting.

2. Materials and methods

2.1. Preparation of carbon particles

Carbon particles used as indicator labels were obtained byburning toluene. The soot was condensed on a glass surfaceand then collected. Carbon was suspended in tenfold theamount (by weight) of toluene, and boiled in a flask with abackflow condenser for 30 min, then cooled and filteredthrough a Shott's glass filter. The same procedure but withoutheating and cooling was repeated five times and resulted in acompletely discolored solvent after the last filtration. Inaddition, the carbon was washed at room temperature withdimethyl formamide and hexane five more times. The finalproduct was dried under a vacuum to ensure removal oftraces of organic solvent. The carbon was then annealed at250 °C for 12 h to achieve a fixed weight. A matt-black dust-like hydrophobic powder was obtained.

2.2. Carbon conjugate preparation

For introduction of carbon particles into water, the pow-der was instilled into a 2% protein solution (e.g. streptavidin,antibodies or G-protein, etc.) in standard phosphate-bufferedsaline (PBS: 0.15 M NaCl, 0.015 M Na2HPO4, 0.015 MNaH2PO4; pH 7.2) at room temperature with vigorous mixingfor 36 h. A 5% concentration of carbon in the suspension wasobtained. The peptized particles were treated with 25%glutaraldehyde; 25% glutaraldehyde (pH 7.2) was addedgradually to an equal volume of the 5% carbon suspensionwhile constantly and actively mixing. The total reaction timewas 100 min. Then the suspension was centrifuged for 5 minat 6000 × g in order to remove large, aggregated particles.Exclusion of glutaraldehyde and unbound protein molecules

was achieved via gel filtration on Sepharose 6B-CL (Pharma-cia, Sweden). The chromatographic process was controlledvisually by observation of the carbon particle flow. Fractionswith the maximal carbon content were combined. Glycerol,bovine serum albumin (BSA), and Na3N were added toconcentrations of 20%, 1% and 0.1%, respectively.

This procedure resulted in covalent cross-linking betweenthe sorbed polymer molecules. It allowed the introduction ofreactive groups of the bifunctional reagent onto the surface ofthe complex, and provided for binding of the active complexto free anti-ligand molecules. Thus, a rigid covalent structurewas formed, comprised of a carbon particle coated with across-linked anti-ligand. Particle sizes were evaluated using aCoulter N4M counter.

2.3. Carbon conjugate stability testing

The stability of the reagents obtained was studied usingthe streptavidin–carbon conjugate model synthesized usinga technique developed in 1997. The reagent was stored ina hermetically sealed glass flask at the temperature of ahousehold refrigerator: +4 °C to +8 °C. During storage, aportion of the conjugate was placed at the room temperature(+22 °C) for 2 weeks every 2 months. The analytical efficiencyof this portion of the reagent was assessed in comparisonwiththe conjugate that was stored in the refrigerator (+4 °C to+8 °C) for the entire time, and with the streptavidin–carbonconjugate that was prepared ex tempore. BSA biotinylationwas performed according to Guesdon et al. (1979).

2.4. Solid phase material

Nitrocellulose membranes with a pore diameter of0.45 μm (BioRad, USA), and 5.0 μm AE-98 (Schleicher &

Fig. 3. Probable scheme of the modified carbon particle. Apparently, theprimary carbon–protein interaction is determined by an absorption process.In our case, it takes place when the particles are introduced into the anti-ligand solution. When glutaraldehyde is added, one part of it is absorbed onthe carbon and another part is bound covalently to the immobilized proteins(anti-ligand). As the density of the absorbed proteins approaches a sufficientlevel after 36 h of solubilization, the glutaraldehyde links them to each otherand forms a rigid shell around a particle. Unaltered sites of the anti-ligand areexpressed outside the particle and are able to interact with the ligand.

Fig. 2. Device (cell) for the immunofiltration assay. 1, Ready-mounted cell; 2, bottom section; 3, macroporous absorptive element; 4, polypropylene support;5, solid-phase immunosorbent; 6, cell cover with the hole for introducing reagent.

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Schuell, Germany) were used as the porous solid phase. Anti-ligand solutions in PBS were used to sensitize their surface.PBS with Tween-20 added to a final concentration of 0.05%(PBS-T) was used as a buffer for sample dilution and as awashing buffer.

A stable covalent carbon–protein G conjugate was used inthe detection of antibodies to HIV antigens. We applied ourcarbon–protein G conjugate for HIV diagnostics by detectionof human antibodies to a set of viral proteins. Nitrocellulosestrips imprinted with transverse lines of env I, gag I, pol I, andenv II recombinant proteins were used as solid-phase reagent(BTC Bioservice, Russia). According to precept recombinantproteins that did not contain antigenic HIV-1, two determi-nants served as the internal negative control, and human IgGanti-serum as the internal positive control.

A mixture of sera from AIDS patients with verified HIV-1and HIV-2 infection (Regional AIDS Controlling Center, Perm,Russia) was used as a test sample. Pooled serum wasrepeatedly diluted in PBS-T using the double dilutiontechnique starting with a 1/250 dilution. Ready-made strips(2 series, 11 strips each) were placed in the test samples andafter incubation for 30 minwere washed three times in PBS-Tfor 2 min. Detection was performed with two reagents:carbon-G protein conjugate and enzyme conjugate of murinemonoclonal antibodies to human IgG with horseradishperoxidase (BTC Bioservice, Russia).

2.5. Detection of human chorionic gonadotropin (HCG)

Immunochromatographic detection of HCG was per-formed using carbon particle conjugate with monoclonalantibodies to the β-subunit of the hormone (Sigma) as adetection reagent. The monoclonal antibodies to the α-subunit of the hormone (1.0 mg/ml; Sigma) and HCG(Sigma) alone (internal positive control; 50 IU/ml) weresorbed as transverse lines onto nitrocellulose strips (4-mmwide). After the applied reagents had dried completely, theresultant immunosorbent was blocked by 1% casein solutionin PBS-T (PBS-T-C) for 30 min, then dried at 37 °C, washedwith PBS-T, and dried again. The prepared immunosorbentswere glued onto transparent polyvinyl chloride Scotch tape.The same Scotch tape was used to firmly attach the uppermargin of the sensitized membrane to a strip of thickmacroporous cellulose filter paper that served as an absorbingelement. The lowermarginwas attached to a similar strip (thelower contact element). Standard HCG diluted in PBS-T wasused in the study. Urine from a healthy man mixed withTween-20 to a final concentration of 0.05% served as thenegative control. Equal volumes of conjugate at double

concentration (relative to the working dilution) were addedto prepared samples. Immediately after mixing, the lowercontact elements of the chromatographic strips were placedinto the mixture for 5–7 s, and then transferred into the PBS-Tsolution for the same period. The scheme for this analyticalsystem is shown in Fig. 1.

To carry out the immunofiltration (also known as the flow-adsorptive method) for the detection of HCG, we used thesame immunoreagents as in immunochromatography. Aplastic cylinder with an embedded outlet in the upper partwas used to perform this method. The cylinder was filled withmacroporous chromatographic paper as an absorbing ele-ment, topped with a supporting disc made of macroporouspolypropylene, and a nitrocellulose membrane as an immu-nosorbent. The device for the assay (a cell) is presented inFig. 2.

3. Results

3.1. Some physical properties of carbon–protein conjugates

The original technique of conjugate synthesis includingpreparation of the particles, their peptization, standardizationby size, and finally the conjugation itself, creates an unusualaggregate state for similar heterogeneous systems. On the onehand, our system involves particles with real surface, and thusmanifests the features of a suspension. On the other hand, the

Fig. 4. Results of carbon particle size testing (diagram ribbon fragment).

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stabilized phase state in this technique does not allowparticles to settle down in a quiescent state, i.e. spontaneousphase separation typical of suspensions does not occur.Hence, the reagent obtained was named a “suspensoid”. Thesuggested scheme of a modified carbon particle is shown inFig. 3. According to the results (Fig. 4), the average particlesize was 162±52 nm (mean±SD).

The analytical properties of the conjugates stored undervarious conditions were evaluated by their ability to bindwith biotinylated BSA that was dot-sorbed on a nitrocellulosemembrane (Fig. 5).

Observations showed that 10-year storage of diagnosticreagents at the temperature of a household refrigerator doesnot have a significant effect on the efficiency of the analyticalsystem in the detection of the biotinylated BSA. This was trueeven in the case of periodic discontinuation of refrigeratedstorage conditions. Two similar investigations studied altera-tions in the analytical characteristics of carbon conjugates:one testing a G protein–carbon conjugate in the detection ofhuman immunoglobulins (refrigerator storage at +4 to +8 °Cfor 5 years), and the other investigating a carbon–antibodyconjugate to human chorionic gonadotropin (HCG) in thedetection of HCG (refrigerator storage at +4 to +8 °C for3 years). In both cases, no decline in sensitivity or non-specificsignaling was observed.

These results show that the synthesized diagnosticums aresufficiently stable, and the systems for analytical testing

Fig. 5. Detection efficacy of biotinylated BSA by carbon–streptavidin conjugates(B) Conjugate stored at 4−8 °C for 10 years. (C) Conjugate stored at 4−8 °C for 10 ye

created based on their use are reliable and reproducible. Webelieve that the primary determining factor in the stability ofthe reagents is the covalent sheathing of the carbon particlesby conjugate components and the chemical inertness of thecarbon itself. Our conjugate synthesis technique has allowedus to design and implement a number of analytical systems.

3.2. Model of dot-analytical system for HIV diagnostics

The majority of test systems used in the diagnosis of HIVinfection at the stage of confirmatory analysis are immu-noenzyme kits based on immune blotting. They are con-venient to use, have the necessary controls, and containdetailed recommendations for the analytical procedure andinterpretation of the results. However, they require quitestringent storage conditions, and their storage time is usuallylimited to 9–12 months.

Instability of immunoenzyme conjugates also posesserious problems in relation to transportation of the diag-nostic systems, particularly to distant regions and to regionswith hot climates. The test systems are often rejected due tonon-specific background levels, manifesting as both falseexposure of the negative control and in background coloringof the immunosorbent surface. In these cases, the risk ofnegative control failure results in hyperdiagnostics. False-positive and false-negative results occur mainly for tworeasons: the first is immunosorbent instability caused by

stored under various conditions. (A) Ex tempore synthesized conjugate.ars with periodic 2-week exposure at 22 °C.

Fig. 6. Comparative assessment of anti-HIV-1 and anti-HIV-2 antibodies by carbon and enzyme conjugates. (A) Detection by carbon–protein G conjugate;(B) detection by enzyme-antibody conjugate. 1, Internal positive control; 2, internal negative control. K−; serum without anti-HIV antibodies.

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ineffective stabilizing protection of sorbed anti-ligands, andthe second is relatively low stability of enzyme conjugates.

Results of comparative detection of anti-HIV-1,2 antibo-dies by immunoenzyme and immunocarbon conjugatesdemonstrate the advantages of non-enzyme detection(Fig. 6), where the sensitivity is 8–16 times higher than thatof enzyme detection. Enzyme detection involves a reactioncatalyzed by horseradish peroxidase during which theprecipitating substrate, 4-Cl-1-naphthol, turns blue. Thecolor intensity (chromaticity) of the product of this reactionis considerably weaker than that of colloid carbon particles,

Fig. 7. HCG determination by immunochromatographic analysis. 1, Samplewithout hormone; 2, HCG standard (10 IU/l); 3, HCG standard (1000 IU/l);4, HCG standard (10,000 IU/l).

and this inevitably influences the sensitivity of detection. Thespecificity of the detection system is shown by the lack ofstaining in the negative control.

3.3. Immunochromatographic detection of HCG

In application of our immunochromatographic strips, dueto the forces of capillary absorption, the hormone solutionand the carbon diagnosticum moved along the strips at a rateof about 1.5 cm/min. In 2–3 min the results of the test wereevaluated visually by the presence (positive result) or absence(negative result) of suspensoid conjugate binding in the zoneof immobilized antibodies on the strip (dark bands against alight grey background). A dark band in the immobilizationzone of the internal positive control served as evidence thatthe assay was performed correctly (Fig. 7).

The results of immunochromatographic detection of HCGshowed a high level of sensitivity due to the use of colloidcarbon particles conjugated with antibodies to HCG as thedetecting reagent. Thiswas confirmed by staining in the zone ofimmobilized antibodies specific to the HCG α-subunit afteranalysis of the samplewith a hormone concentration of 10 IU/l.

Fig. 8. HCG immunofiltration assay. +, positive reaction; −;, negative reaction.

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High specificity of the test was indicated by the lack of stainingon the immunosorbent surface in the negative control zone.Detection of 10 IU/l in itself makes the analytical characteristicsof this diagnostic system extremely attractive.

3.4. Detection of HCG by immunofiltration

The immunosorbent was prepared by sorption of mono-clonal antibodies to the α-subunit of HCG (1.0 mg/ml) onto anitrocellulose membrane filter (pore diameter 5 μm) in avertical line. The hormone itself (internal positive control;50 IU/ml) was sorbed in a horizontally intersecting line. Afterapplication of the reagent and drying at +37 °C for 30min, themembranes were blocked by soaking in PBS-T-C for 1 h atroom temperature followed by washing in PBS-T, and dryingas before.

Equal volumes of the sample under test (urine frompregnant or non-pregnant women) and carbon particleconjugate with monoclonal antibodies to the β-subunit ofHCG at double concentration (relative to the workingdilution) were mixed. At the beginning of the assay, 0.5 mlof PBS-T was introduced into the top opening to wet theimmunosorbent. Then 0.5 ml of the previously preparedmixture was added. After absorption of the mixture, 1.0 ml ofPBS-T was introduced to wash the surface of the membrane.Assay time was 2–3 min (Fig. 8).

4. Discussion

The use of carbon particles as labels for detecting reagentsis not novel. In the late 1960s, studies by A.K. Adamov wereterminated for reasons that were unclear, although for anumber of years he had been developing a concept fordesigning immunological reagents: suspension antigens andantibodies (Adamov and Agafonov, 1969).

The concept was based on the use of so-called suspensionantigens and antibodies as diagnostic reagents. Antigens andantibodies were fixed by absorption on the surface of variousparticles including carbon. The resulting reagents were usedto detect matching antibodies and antigens in reactions of so-called agglomeration (agglutination per se). There wereprobably at least three reasons for the termination of thesestudies: (a) difficulties in obtaining solution-stable suspen-sions; (b) instability of the reagents produced after storage;(c) a high rate of non-specific interactions.

We have succeeded in solving many of these problems bymaking a relatively homogeneous carbon suspension and bycarrying out covalent sheathing of colloid carbon particleswith protein compounds. As a result, the stability of theconjugates has increased. Storage of streptavidin-carbonconjugate for 10 years at the temperature of a householdrefrigerator does not have a significant effect on the efficiencyof the analytical system for detection of biotinylated BSA. Afurther interesting characteristic of our carbon–protein watersuspension is that it did not vary with time, in contrast tostandard suspensions. The harmless property of the reagentshould also be kept in mind. The universality of carbonconjugates has been demonstrated in various analyticalsystems with different anti-ligands.

The high degree of optical sharpness (chromogenicity) ofthe carbon label gives it an advantage among all color

materials used in diagnostic systems and provides signifi-cant analytical sensitivity. At the same time, the naturalproperties of carbon create serious obstacles towards itschemical modification. Our studies have demonstrated thepossibility of producing reliable diagnosticums that utilizethe unique natural properties of carbon particles. Chemicallyinert material can be involved in physical–chemical mod-ification that results in the creation of a fundamentally newproduct.

5. Conclusions

The carbon–protein conjugates designed here presentone approach in the creation of artificial constructionsintended for use in simplified non-instrumental assays.These conjugates possess some attractive properties. Theirblack color allows maximum contrast, and makes them verysuitable for visual registration. Diagnostic reagents based oncovalent binding are able to withstand unfavorable storageconditions without losing their analytical and operationalproperties. We have succeeded in including an inert carbonparticle in a protein pouch and then fastened it by covalentbonds.

The sufficiently high sensitivity of carbon–protein con-jugates may become the basis for a wide range of efficientsystems designed for simplified diagnostic testing in biology,medicine, criminology, agriculture, etc. The attractive char-acteristics of such test systems could be successfully realizedin various “one patient–one test” kits for outpatient, pre-clinical, and clinical tests, including urgent clinical andepidemic situations, field expeditions, military assignments,and also popular “home” systems suitable for individual use.

The remaining tasks for these future applications are towork out the format and procedural arrangements ofanalytical systems based on the use of carbon conjugates,and to design appropriate devices convenient for implemen-tation of non-instrumental assays.

References

Adamov, A.K., Agafonov, V.I., 1969. Suspension antigens, antibodies andimmunosorbents. Meditsina, Moscow (in Russian).

Bangs, L.B., 1988. Latex agglutination tests. Am. Clin. Lab. News 7 (4A), 20.Bangs, L.B., 1990. Latex immunoassays. J. Clin. Immunoass. 13, 127.Bangs, L.B., 1996. Immuno-latex chromatographic procedure for detection

of cignatoxin, and related polyether marine toxins. US patent, serialno. 5,525,525.

Chakraborty, U.R., Black, N., Brooks, H.G., Campbell, C., Gluck, K., Harmon, F.,Hollenbeck, L., Lawler, S., Levison, S., Mochnal, D., 1990. An immunogoldassay system for the detection of antigen or antibody. Ann. Biol. Clin. 48,403.

Engvall, E., Perlmann, P., 1971. Enzyme-linked immunosorbent assay (ELISA).Quantitative assay of immunoglobulin G. Immunochemistry 8, 871.

Guesdon, J.L., Avrameas, S., 1980. Sensitive titration of antibodies andantigens using erythro-immunoassay. Ann. Immunol. 131, 389.

Guesdon, J.L., Ternynck, T., Avrameas, S., 1979. The use of avidin-biotininteraction in immunoenzymatic techniques. J. Histochem. Cytochem. 27(8), 1131.

Moeremans, M., Daneels, G., Van Dijck, A., Langanger, G., Mey, J.De., 1984.Sensitive visualization of antigen–antibody reactions in dot and blotimmune overlay assay with immunogold and immunogold/silverstaining. J. Immunol. Methods 74, 353.

Remington, J.S., Eimstad, W.M., Aranjo, F.G., 1983. Detection of immunoglo-bulin M antibodies with antigen-tagged latex particles in an immuno-sorbent assay. J. Clin. Microbiol. 17 (5), 939.

Roth, J., Heitz, P.U., 1989. Immunolabeling with the Protein A–gold technique:an overview. Ultrastruct. Pathol. 13, 467.

15M. Rayev, K. Shmagel / Journal of Immunological Methods 336 (2008) 9–15

Rubinstein, K.E., Schneider, R.S., Ullman, E.F., 1972. “Homogeneous” enzymeimmunoassay. A new immunochemical technique. Biochem. Biophys.Res. Commun. 47, 846.

Snowden, K., Hommel, M., 1991. Antigen detection immunoassay usingdipsticks and colloidal dyes. J. Immunol. Methods 140, 57.

Spallholz, J.E., 1982. Stable isotopic immunoassay method employingnonradioactive selenium label. US patent, serial no. 4,341,757.

Tarcha, P.J., Wong, M., Donovan, J.J., 1990. Indicator reagents, diagnosticassays and kits employing organic polymer latex particles. Eur. patent,serial no. 89116594.6.

Ullman, E.F., Kirakossian, H., Switchenko, A.C., Ishkanian, J., Ericson, M.,Wartchow, C.A., Pirio, M., Pease, J., Irvin, B.R., Singh, S., Singh, R., Patel, R.,

Dafforn, A., Davalian, D., Skold, C., Kurn, N., Wagner, D.B., 1996.Luminescent oxygen channeling assay (LOCI): sensitive, broadly applic-able homogeneous immunoassay method. Clin. Chem. 42, 1518.

Woodhead, J.S., Weeks, I., 1985. Chemiluminescence immunoassay. PureAppl. Chem. 57, 523.

Yallow, R.S., Berson, S.A., 1959. Assay of plasma insulin in human subjects byimmunological method. Nature 184, 1648.

Yost, D.A., Russel, J.C., Yang, H., 1989. Non-metal colloidal particle immu-noassay. Eur. patent, serial no. 0298368 A2.