Immunofluorescence signal amplification by the enzyme-catalyzed deposition of a fluorescent reporter substrate (CARD)

Progress has been made in improving the immunohistochemical detection of antigens for imaging and flow cytometry. We report the synthesis of a novel fluorescent horseradish peroxidase substrate, Cy3.29-tyramide, and its application in an enzyme-based signal amplification system, catalyzed reporter deposition (CARD). The catalyzed deposition of Cy3.29-tyramide was used to detect cell surface markers such as CD8 and CD25 on tonsil tissue and human lymphocytes. We compared the fluorescence CARD method to standard indirect immunofluorescence detection methods and found that an amplification of up to 15-fold was possible with CARD. The detection of the intracellular protein myosin II in fibroblastic cells and rabbit serum proteins blotted onto nitrocellulose was also improved. Thus, fluorescent CARD is a simple modification that can be made to standard immunofluorescence staining protocols to enhance significantly the detection of antigens.     

Introduction of a novel HRP substrate-Nanogold probe for signal amplification in immunocytochemistry.

Amplification of immunological signals with catalyzed reporter deposition (CARD) allows improved detection of scarce tissue antigens in light and electron microscopy. The technique takes advantage of the oxidation ability of horseradish peroxidase (HRP), in the presence of hydrogen peroxide, to yield the accumulation of one of its specific reporter-tagged substrates. This immunocytochemical approach continues to be improved by the introduction of new reporter molecules tagged to tyramine or to other HRP substrates. In this study we introduced a novel HRP substrate tagged to Nanogold particles. The amplification protocol is based on the application of a specific primary antibody, a biotinylated secondary antibody, streptavidin-HRP, and an HRP substrate coupled to Nanogold, followed by silver intensification. In addition to amplification of immunological signals of high resolution, direct accumulation of Nanogold particles at target sites by enzymatic activity of HRP improves the efficiency of the technique compared to other amplification protocols. Moreover, this approach combines the CARD amplification potentials with the ultrasmall gold probe and the silver intensification method. Immunolabeling obtained by light and electron microscopy, as well as immunodot assay using this new amplification strategy, appear to be highly sensitive, specific, and of enhanced intensity.     

Immunogold signal amplification: Application of the CARD approach to electron microscopy.

Catalyzed reporter deposition (CARD) is a technique that allows amplification of routine immunolabeling in light microscopy. This procedure takes advantage of the horseradish peroxidase (HRP) from an HRP-avidin complex to catalyze the accumulation of reporter-conjugated tyramine (a phenolic compound) onto a surface displaying biotinylated antigen-antibody complexes. The large amount of labeled tyramine deposited allows the detection of an antigenic site with multiple reporter molecules. In this study we modified this amplification protocol to combine it with the immunogold technique for the ultrastructural localization of antigens in electron microscopy. We constructed various tyramide conjugates that permit the combination of this amplification method with a particulate colloidal gold marker. The new probes yield results of high specificity and enhanced intensity. Assessment of the level of resolution of the labeling has demonstrated that, in spite of the amplification, the resolution remains very good. Therefore, once associated, the immunogold and the CARD techniques lead to specific, high-resolution, sensitive and amplified signals that exhibit the advantages of both approaches.(J Histochem Cytochem 47:421-429, 1999)     

Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains.

A procedure is described for intensifying histochemical reactions by amplification of biotinylated sites. This is achieved by deposition of biotinylated tyramine on the tissue through the enzymatic action of horseradish peroxidase (HRP). The amplified biotin sites are subsequently visualized by binding them to avidin, to which a marker is attached. This amplification greatly increases the sensitivity of staining procedures that employ HRP (and/or biotin) in tissue. For neuroanatomical pathway tracing methods, the procedure greatly increases the detectability of the injected tracer. For lectin histochemistry and immunohistochemistry, the amplification requires that the lectin or primary antibody be greatly diluted. This dilution results in less background staining and yet strong signals are produced even when very dilute reagents are used. Alternatively, the amplification permits much shorter incubations in primary antibodies when dilutions are used that would ordinarily be used with conventional bridge techniques. The procedure is also useful for amplifying very weak signals, such as those of immunoreactions in glutaraldehyde-fixed tissue. The amplification procedure, together with the availability of avidin probes labeled with fluorochromes, colloidal gold, or enzyme systems other than HRP, provides a means of greatly increasing the versatility of a variety of histochemical reactions, including those for detecting in situ hybridization probes, in addition to increasing the sensitivity of the reactions.     

Improved mRNA in situ hybridization on formaldehyde-fixed and paraffin-embedded tissue using signal amplification with different haptenized tyramides

 We report an optimized in situ hybridization (ISH) protocol with a rapid signal amplification procedure based on catalyzed reporter deposition (CARD) to increase the sensitivity of non-isotopic mRNA ISH on formaldehyde-fixed and paraffin-embedded tissue. The CARD method is based on the deposition of haptenized tyramide molecules in the vicinity of hybridized probes catalyzed by horseradish peroxidase. Commercially available and newly synthesized haptenized tyramides, including digoxigenin-, biotin-, di- and trinitrophenyl- as well as fluorescein-tyramide, were compared. The haptenized tyramides were visualized using peroxidase conjugated anti-hapten antibodies followed by the diaminobenzidine reaction. As a test system, we applied digoxigenin-labeled oligonucleotides to detect insulin and vasoactive intestinal polypeptide mRNA in pancreatic endocrine tumors and liver metastases. Our results indicate that specificity, sensitivity, and applicability of oligonucleotide mRNA ISH can be significantly improved by using chemically digoxigenin-labeled oligonucleotide probes and signal amplification by CARD. Furthermore, all tested tyramides provided approximately equal amplification efficiency. In conclusion, CARD signal amplification should further promote mRNA ISH studies on paraffin-embedded tissues and allow for multiple-target nucleic acid detection in situ. Accepted: 1 July 1998     

Enzymatic amplification staining for flow cytometric analysis of cell surface molecules

BACKGROUND: Flow cytometric analysis is a powerful technique for the single cell assessment of cell surface expression of selected molecules. The major deficiency of flow cytometry has been its relative insensitivity. Only molecules expressed in abundance have been readily observed. METHODS: We have developed an enzymatic amplification procedure for the analysis of cell surface molecules by flow cytometry. Transformed and nontransformed cells expressing MHC class I, CD5, CD3, CD4, CD6, CD7, CD34, CD45, MHC class II, Fas ligand, and phosphatidylserine were assessed. RESULTS: Our enzymatic amplification technology increased the fluorescence signal between 10 and 100-fold for all surface molecules tested. CONCLUSIONS: Enzymatic amplification staining produces a significant enhancement in the resolving power of flow cytometric analysis of cell surface molecules. Using this technique, we have been able to detect the presence of molecules that could not be observed by the standard procedure.     

Design and construction of novel molecular conjugates for signal amplification (II): use of multivalent polystyrene microparticles and lysine peptide chains to generate immunoglobulin-horseradish peroxidase conjugates

Spherical polystyrene microparticles expressing a large number of highly reactive functional groups were chemically engineered to generate antibody–enzyme conjugates as novel signal amplification systems. Chemically modified goat anti-human IgG and horseradish peroxidase (HRP) were combined in a 1:5 ratio and attached to 0.44 μm streptavidin microparticles or N-succinimidyl-S-acetylthioacetate (SATA)-activated 0.29 μm amino microparticles with highly reactive free sulfhydryl groups on their surface. The numbers of HRP molecules/microparticle were further increased by coupling HRP to primary amines on N-terminal biotinylated or bromoacetylated polypeptides containing 20 lysine residues prior to conjugation with streptavidin or sulfhydryl groups-containing microparticles. The antibody–poly-HRP immunoconjugates contained an estimated number of 10⁵ HRP/streptavidin microparticle and 10⁶ HRP/amino microparticle, respectively. These microparticle immunoconjugates efficiently bound to plasma anti-HIV-1 antibodies that had been captured by HIV antigens on 5 μm carboxyl magnetic microparticles and, upon reaction with orthophenyldiamine substrate, produced a detection signal with 5–8 times more sensitivity as compared to conventional HRP-conjugated goat anti-human IgG. The signal amplification technique by microparticle immunoconjugates may provide potentially novel tools for the development of highly sensitive diagnostic systems.     

Linear amplification of catalyzed reporter deposition technology on nylon membrane microarray

The application of microarray analysis to gene expression from limited tissue samples has not been very successful because of the poor signal qualityfrom the genes expressed at low levels. Here we discussed the use of catalyzed reporter deposition (CARD) technology to amplify signals from limited RNA samples on nylon membrane cDNA microarray. When the input RNA level was greater than 10 microg, the genes expressed at high levels did not amplify in proportion to those expressed at low levels. Compared to conventional colorimetric detection, the CARD method requires less than 10% of the total RNA used for amplification of signal displayed onto a nylon membrane cDNA microarray. Total RNA (5-10 microg), as one can extract from a limited amount of specimen, was determined to produce a linear correlation between the colorimetric detection and CARD methods. Beyond this range, it can cause a nonlinear amplification of highly expressed and low-abundance genes. These results suggest that when amplification is needed for any applications using the CARD method, including DNA microarray experiments, precaution has to be taken in the amount of RNA used to avoid skew amplification and thus misleading conclusions.     

Recent advances in catalytic peroxidase histochemistry.

Immunoassays have developed to become an important analytical tool in life sciences for detection of endogenous and exogenous targets. Among the most important enzyme labels horseradish peroxidase (HRP), alkaline phosphatase (AP), and beta-D-galactosidase (GAL) is HRP the smallest enzyme and plays nowadays an outstanding role. The oldest substrates are chromogens widely applied for localization of sites of peroxidase (PO) activity in histochemistry as well as for colorimetric applications. They are represented by a diversity of aromatic amines and phenols. Encouraged by development of light excitation and measuring techniques and the commercial availability of highly sensitive equipment, luminescent labels represent the most sensitive and worthwhile detection tools to date. In contrast to chromogens fluorescent labels for detection PO activity are confined only to a few substrates developed more recently. These substrates are mostly applied in histochemistry at a short time scale due to their frequently high solubility. At the long time scale sole exception is so far the tyramine based fluorochome deposition technique (more general: catalytic reporter deposition, CARD). Despite quite different staining behavior both fluorometric and product deposition related principles are based on 4-hydroxy phenylalkyl substrates. The following article reviews basic principles of peroxidatic substrate degradation processes including chromogenic and fluorescent approaches with emphasis on recent advances in development of chromogens and fluorogens for application in histology. As a result of systematic efforts towards the design of substrates, the range of classical precipitating chromogens as well as fluorescent techniques could be complimented by novel highly sensitive substrates with superior staining capabilities: a) Metal chelating 2-hydroxy benzylamines are derived from classical aniline substrates (two steps) and utilize metal catalytic effects in an efficient intramolecular way. The enzymatically yielded dark colored polycondensation products are applicable in histochemistry, in colorimetry and especially as precipitating electron opaque labels with enhanced osmiophilic properties for light and electron microscopy. b) Fluorescent 4-hydroxy-styryl derivatives are capable of oxidative selfanchoring reactions at the cellular level close to sites of PO activity. In contrast to deposition of tyramine conjugated fluorochromes an altered fluorochrome with improved fluorescence properties is furnished during oxidative crosslinking of the substrate. This results in a highly specific and photostable fluorescence response and an outstanding low background staining. Histochemical and immunohistochemical applications are presented.     

Amplification methods to increase the sensitivity of in situ hybridization: play card(s).

In situ hybridization (ISH) has proved to be an invaluable molecular tool in research and diagnosis to visualize nucleic acids in their cellular environment. However, its applicability can be limited by its restricted detection sensitivity. During the past 10 years, several strategies have been developed to improve the threshold levels of nucleic acid detection in situ by amplification of either target nucleic acid sequences before ISH (e.g., in situ PCR) or the detection signals after the hybridization procedures. Here we outline the principles of tyramide signal amplification using the catalyzed reporter deposition (CARD) technique, present practical suggestions to efficiently enhance the sensitivity of ISH with CARD, and discuss some applications and possible future directions of in situ nucleic acid detection using such an amplification strategy.     

Amplification methods for the immunolocalization of rare molecules in cells and tissues

The needs to precisely assign macromolecules to specific locations and domains within tissues and cells and to reveal antigens which are present in low or even in trace amounts, led to the elaboration of a wide spectrum of immunocytochemical amplification procedures. These arise from the successive improvements of tissue preparation techniques, of antigen retrieval procedures and of immunological or non-immunological detection systems. Improvement of detection systems may be the most active in the development of amplification techniques. Since the early work of Coons, in which by the introduction of the indirect technique has started amplifying the signal, different systems have succeeded in increasing the sensitivity of antigens detection. Indeed, amplification techniques such as the multiple antibody layers, the multiple bridges, the enzyme complexes, the avidin-biotin, the silver intensification, and the numerous variations and combinations among these have increased the sensitivity for the detection of scarce tissue antigens. However, as shown by the recent progress carried out with new approaches such as the catalyzed reporter deposition (CARD) and the enhanced polymer one-step staining (EPOS), more efficient methods are still needed. In electron microscopy, few techniques have reached the resolution afforded by the post-embedding immunogold approach. In spite of this and in order to further increase its sensitivity, new probes and novel approaches are allowing combination of the gold marker with the amplification capacity of enzymes afforded by the CARD technique. Immunogold amplification strategies, such as the multiple incubations with the primary antibody and the use of an anti-protein A antibody have also led to enhanced signals displaying the advantages in terms of resolution and possibilities of quantification inherent to the colloidal gold marker.     

The enhanced reactivity of endogenous biotin-like molecules by antigen retrieval procedures and signal amplification with tyramine

In diagnostic pathology and immunocytochemical research, immunohistochemical techniques using the streptavidin–biotin–peroxidase system have played an extremely valuable role. This system, based on the high affinity of streptavidin for biotin, may, however, provoke false positive results because of endogenous streptavidin-binding sites in human tissues. With the advent of the antigen retrieval procedure and signal amplification method, this problem can be serious enough to cause mistakes in interpreting immunohistochemical staining results. Therefore, we examined the distribution of endogenous biotin-like molecules in various human tissues and the influence of various antigen retrieval procedures with or without signal amplification using biotinylated tyramine to reveal these biotin-like activities. We observed that endogenous biotin-like molecules were present in a wide range of tissues, and their activity was markedly enhanced by employing antigen retrieval procedures or signal amplification. Furthermore, the extent to which the activity of endogenous biotin-like activities was enhanced depended on the kinds of antigen retrieval procedures and signal amplification employed. Pressure cooking and tyramine amplification with microwave heating showed the highest activities. These results show that the antigen retrieval procedures and signal amplification with tyramine can enhance the activity of endogenous biotin or biotin-like molecules as well as antigenicity, which can be a pitfall in the interpretation of immunohistochemical data.     

Dual enhancement of double immunofluorescent signals by CARD: participation of ubiquitin during formation of neurofibrillary tangles

Amplification with catalyzed reporter deposition (CARD) greatly enhances peroxidase signals, which has been utilized to amplify immunohistochemical labelings including fluorochromes. Here we describe a strategy to amplify each of two immunofluorescent signals without crosstalk on double-stained histological sections from human autopsied brains with Alzheimer's disease (AD). One of the two primary antibodies (anti-Abeta or anti-PHF-tau) was probed by a species-specific secondary antibody conjugated with horseradish peroxidase (HRP), which was visualized by FITC-labeled tyramide. After inactivation of HRP, the other primary antibody was probed by another species-specific secondary antibody conjugated with HRP. Amplification with biotinylated tyramide was followed by streptavidin-conjugated Cy-5, which specifically labeled the latter epitope. It was found that Abeta and PHF-tau were localized to senile plaques and neurofibrillary tangles (NFTs), respectively, which verified lack of crosstalk on the double-stained section. Localization of ubiquitin and PHF-tau was looked for at higher magnification in NFT-bearing neurons. Although these two epitopes were colocalized in some neurons, ubiquitin was not always present in PHF-tau positive NFTs. Discrepancy between PFH-tau and ubiquitin, verified inter- and intracellularly, may represent different stages of NFT formation. This is the first report of successful CARD amplification of two different fluorescent signals on double-labeling immunohistochemistry, which is now proved to be powerful in detecting epitopes in relation to AD-related lesions. Improved intensity over tenfold of the two fluorescent signals without crosstalk will expand the application of the multilabeling method with fluorochromes.     

免疫检测信号放大抗体偶联物的制备及应用*

目的: 以聚赖氨酸(polylysine,PL)为骨架提高辣根过氧化物酶(horseradish peroxidase,HRP)与羊抗兔IgG的连接数量,比较几种化学偶联剂的偶联效果,通过免疫检测技术对其灵敏度进行检测和比较。方法: 对HRP与PL、聚合物HRP-PL与N-琥珀酰-S-乙酰乙酸(N-succinimidyl-S-acetylthioacetate,SATA)和2-亚氨基硫烷(Traut’s)两种试剂、羊抗兔IgG与琥珀酰亚胺基4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯[succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate,Sulfo-SMCC]、活化后羊抗兔IgG及HRP-PL进行摩尔比的优化;对偶联物IgG-PL-HRP及商品化二抗分别进行斑点免疫印迹、ELISA和免疫组化,并计算偶联物IgG-PL-HRP的检测放大倍数。结果: 当PL与HRP摩尔比为1∶5,HRP-PL与Traut’s摩尔比为1∶15,羊抗兔IgG与Sulfo-SMCC摩尔比为1∶30,羊抗兔IgG与HRP-PL摩尔比为1∶10时,反应效率较高;商品化二抗及偶联物IgG-PL-HRP在斑点免疫印迹实验中的最低检测限分别为2.5 μg和312.5 ng,最大稀释倍数分别为50和100倍;在ELISA实验中的最大稀释倍数分别为5 000和20 000倍;在免疫组化实验中偶联物IgG-PL-HRP的检测特异性及强度均大于商品化二抗。结论: 成功合成抗体偶联物IgG-PL-HRP,且免疫检测信号放大倍数为商品化二抗的3~7倍,这对后续免疫诊断及生物学的研究具有重要意义。     

Signal amplification of microarray-based immunoassay by optimization of nanoliposome formulations

The use of microarray-based immunoassay is often limited by its sensitivity. To increase the sensitivities of such an immunoassay, liposome encapsulation was explored. Two different liposome formations and several preparation methods were examined to optimize encapsulation and signal-enhancing efficacy for enzyme-linked immunosorbent assay (ELISA) and antibody array. The signal amplification by liposome encapsulation was demonstrated through a detection for foodborne pathogenic Listeria. In plate-trapped antigen (PTA) ELISA, horseradish peroxidase (HRP)-loaded liposome increased signal 9-fold more than the control. Limits of detection (LODs) of HRP-encapsulated liposome were 6.4×10(5) and 5.5×10(6)CFU/ml in sandwich ELISA and antibody array, respectively. Furthermore, when chromogenic 4-chloro-1-naphthol (4-CN) substrate was used for signal development in the antibody array, the signal could be detected with the naked eye. These results suggest that the liposome encapsulation technique can have great potential for signal amplification and, therefore, for increasing assay sensitivity for various formats of immunoassay, especially microarray-based format.     

Ferrocenemonocarboxylic-HRP@Pt nanoparticles labeled RCA for multiple amplification of electro-immunosensing

A multiple amplification immunoassay was proposed to detect alpha-fetoprotein (AFP), which was based on ferrocenemonocarboxylic-HRP conjugated on Pt nanoparticles as labels for rolling circle amplification (RCA). Firstly, the capture antibody (anti-AFP) was immobilized on glass carbon electrode (GCE) deposited nano-sized gold particles. After a typical immuno-sandwich protocol, primary DNA was immobilized by labeling secondary antibody, which acted as a precursor to initiate RCA. The products of RCA provide large amount of sites to link detection DNAs, which were labeled by signal probes (ferrocenemonocarboxylic) and horseradish peroxidase (HRP). Moreover, the enzymatic amplification signals could be produced by the catalysis of HRP and Pt nanoparticles with the addition of H?O?. These lead to multiple amplification signals monitoring by electrochemical instrument and further resulted in high sensitivity of the immunoassay with the detection limit of 1.7 pg/mL.     

基于免疫性PTR的血小板膜糖蛋白及T/B淋巴细胞表达水平研究

摘要 目的：探究血小板膜糖蛋白及T/B淋巴细胞免疫在免疫性血小板输注无效发生机制中的重要作用。方法：采用血小板特异性抗体检测试剂盒及流式细胞技术检测免疫性PTR患者血小板特异性抗体（抗GPIIb/IIIa、抗GPIa/IIa、抗GPIb/IX、抗GPIV）、血小板膜糖蛋白（CD36、CD61、CD41a）表达情况及外周血T/B淋巴细胞数量,运用SPSS19.0软件及R软件分析免疫性PTR与血小板膜糖蛋白、外周血T/B淋巴细胞的相关性。结果：免疫性PTR与血小板输注有效者比较,血小板特异性抗体的产生无显著差异,血小板膜糖蛋白CD36和CD61的表达具有显著差异,CD36对免疫性PTR发生风险具有极大的预测价值,外周血CD8⁺T细胞比例增高,而CD4⁺T细胞比例减低。结论：免疫性PTR患者产生血小板抗体具体机制不明确,了解患者细胞免疫状态有助于明确免疫性PTR的发生机制,为患者提供更优质的诊疗策略。     

Cell surface receptor-antibody association constants and enumeration of receptor sites for monoclonal antibodies

BACKGROUND: Fluorescent markers (labeled antibodies) and flow cytometry are used to enumerate the average number of receptors (antigens) on formed bodies (cells) in whole blood by using a new method that avoids the extra steps of separating bound from unbound fluorescent markers or the use of external standards. METHODS: Mean channel fluorescence intensities of equilibrated marker-cell suspension mixtures, total concentrations of marker, and targeted cell counts obtained by standard cytometry procedures are used to complete the analyses for receptors per cell. Also, flow cytometric assays using competitive binding between fluorescent marker (CD4-RD1, CD8-FITC, CD3-FITC, CD3-RD1) and unlabeled antibody (CD4, CD8, CD3, CD3-dextran) for receptors on white blood cells in whole blood are described for determination of relative and specific binding constants of unlabeled/labeled antibody for targeted receptors. RESULTS: Ranges that were obtained for receptors per cell (lymphocytes) in normal blood donors were as follows: CD4, 4.9 x 10(4)-1.5 x 10(5); CD8, 5.0 x 10(5)-2.1 x 10(6); CD3, 6.6-7.8 x 10(5). Binding constants were highest for unlabeled CD4 antibody, 2. 7 x 10(10)-2.1 x 10(12) M(-1), and then unlabeled CD3 antibody, 1.1 x 10(10)-1.9 x 10(11) M(-1). FITC- and RD1-labeled antibodies typically had binding constants that were 10-to 100-fold lower than the native antibodies. CONCLUSIONS: Values of receptors per cell and binding constants obtained by the new method from flow cytometric analyses of mixtures of whole blood with FITC- or RD1-labeled CD4, CD8, and CD3 antibodies compare well with literature values determined by other methods.     

Enzymatic amplification detection of DNA based on "molecular beacon" biosensors

We described a novel electrochemical DNA biosensor based on molecular beacon (MB) probe and enzymatic amplification protocol. The MB modified with a thiol at its 5' end and a biotin at its 3' end was immobilized on the gold electrode through mixed self-assembly process. Hybridization events between MB and target DNA cause the conformational change of the MB, triggering the attached biotin group on the electrode surface. Following the specific interaction between the conformation-triggered biotin and streptavidin-horseradish peroxidase (HRP), subsequent quantification of DNA was realized by electrochemical detection of enzymatic product in the presence of substrate. The detection limit is obtained as low as 0.1nM. The presented DNA biosensor has good selectivity, being able to differentiate between a complementary target DNA sequence and one containing G-G single-base mismatches.     

Preparation and Characterization of Ligand-Modified Labelled Liposomes for Solid Phase Immunoassays

Abstract

Small unilamellar vesicles conjugated with an enzyme label and with specific ligands for biological molecules may prove to be useful as signal enhancement vehicles in the development of enzyme-linked immunoadsorbent assays and other detection applications. Bifunctional vesicles have been prepared by covalently attaching horseradish peroxidase (HRP) and monoclonal antibodies to the outside of the lipid bilayer. The reaction conditions were optimized to obtain 7-12 antibody molecules and 100-200 HRP molecules per vesicle. The enzyme retained 70-80% of its specific activity after immobilization with no apparent change in vesicle stability. These bifunctional vesicles were used in a noncompetitive immunoassay for D-Dimer, a fibrin dimer formed at the early stages of thrombogenesis. The assay results using vesicles led to a detection limit for D-Dimer in human plasma which was five times lower than what was achieved using a conventional enzyme-antibody conjugate assay. HRP labelled (bifunctional) liposomes can also be used in competitive assays for the detection of small ligands in bulk solution. HRP and biotin-conjugated vesicles were prepared and used in competitive assays for biotin in free solution. The lowest detection limit for biotin using vesicles as the signal generation mechanism was found to be a factor of 10 lower than what could be observed with a traditional biotin-HRP conjugate. A model has been developed for the competition between a small ligand in solution and a large ligand-conjugated vesicle for binding sites on a solid surface.