Use of a biomimetic peptide in the design of a competitive binding assay for biotin and biotin analogues

A competitive binding assay for biotin, biocytin, and desthiobiotin utilizing a genetically engineered enzyme-ligand conjugate is described herein. This assay is unique in that the enzyme-ligand conjugate consists of the streptavidin binding peptide Strep-tag II, which mimics the binding of biotin to streptavidin, rather than biotin itself. This allows for the construction of a well-defined, oligosubstituted enzyme-ligand conjugate for which the site of attachment of the ligand on the enzyme is known precisely. The assay has detection limits of 5 x 10(-8) M for biotin, 1 x 10(-7) M for biocytin, and 2 x 10(-6) M for desthiobiotin, and it serves as a model system in that it demonstrates the feasibility of using enzyme-ligand conjugates in which a peptide mimic of the analyte ligand is genetically fused to the enzyme. This avoids the problems associated with covalent attachment of the ligand to the enzyme, such as multiple substitution of the ligand and variability of the site of attachment. To our knowledge, this is the first example of using an enzyme-peptide mimic conjugate to detect a nonpeptide analyte.     

New approach to heterogeneous enzyme immunoassays using tagged enzyme-ligand conjugates

A new approach to heterogeneous enzyme immunoassays has been developed that uses a tag molecule linked to an enzyme-ligand conjugate. The insoluble phase is an insolubilized receptor to that tag. The antibody to the ligand, in addition to complexing either the free ligand or the one covalently linked to the tagged enzyme, also serves to mask the tag on the tagged enzyme-ligand conjugate so that it can no longer bind to the insolubilized receptor. Accordingly, the proportion of enzyme conjugate associated with the insoluble fraction is proportional to the amount of analyte ligand being assayed. This heterogeneous EIA based on the “antibody masking the tag” is called AMETIA. In the model system we use DNP-lysine as the ligand, β-galactosidase as the enzyme, biotin as the tag, and avidin immobilized to Sepharose as the insoluble receptor.     

Influence of hydrophobic and hydrophilic spacer-containing enzyme conjugates on functional parameters of steroid immunoassay

Introduction of spacers in coating steroid antigen or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced hydrophobic and hydrophilic spacers between enzyme and steroid moieties and studied their effects on functional parameters of enzyme immunoassays, using cortisol as a model steroid. Cortisol-3-O-carboxymethyloxime-bovine serum albumin (F-3-O-CMO-BSA) was used as immunogen to raise the antiserum in New Zealand white rabbits. Three enzyme conjugates were prepared using cortisol-21-hemisuccinate (F-21-HS) as carboxylic derivative of cortisol and horseradish peroxidase (HRP) as an enzyme label. These were F-21-HS-HRP (without spacer), F-21-HS-adipic acid dihydrazide-HRP (adipic acid dihydrazide as hydrophobic spacer), and F-21-HS-urea-HRP (urea as hydrophilic spacer). The influence of hydrophobic and hydrophilic spacers on the functional parameters of assays such as lower detection limit, ED50, and specificity was studied with reference to enzyme conjugate without spacer. The results of the present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate decreases the lower detection limit, decreases the ED50, and marginally improves the specificity of assays. These improvements in functional parameters of assays may be due to the decreased magnitude of the overall hydrophobic interactions existing between the spacer in enzyme conjugate and the antigen binding site of the antibody.     

Chitosan-pentaglycine-phenylboronic acid conjugate: a potential colon-specific platform for calcitonin

Novel drug delivery vehicles based on the biodegradable, mucoadhesive polysaccharide chitosan covalently linked to a boronic acid protease inhibitor have been prepared and characterized. It was anticipated that these conjugates could protect a proteinaceous drug, such as salmon calcitonin, against proteolysis by serine proteases, an obstacle that prevents its oral administration. Specifically, 4-formylphenylboronic acid was linked to chitosan. Three types of conjugates were prepared. In the first, 4-formylphenylboronic acid was directly linked to chitosan. The other two conjugates employed glycylglycine and pentaglycine spacers. Enzyme-inhibition assays toward trypsin and elastase, in the presence of the enzyme chitosanase, demonstrated a strong inhibitory effect for the chitosan-pentaglycine-phenylboronic acid conjugates, while no inhibitory effect could be detected without chitosanase. The chitosan-pentaglycine-phenylboronic acid conjugate with the highest degree of substitution of 4-formylphenylboronic acid was able to decrease the salmon calcitonin degradation rate by trypsin. It is concluded that chitosan-pentaglycine-phenylboronic acid conjugates are a potential multifunctional, colon-specific vehicle for orally administered salmon calcitonin.     

Evaluation of different chemical strategies for conjugation of oligonucleotides to peptides

We developed novel assays for high-throughput detection of one or many kinases or proteases. The assays use hundreds of different peptide substrates, each covalently linked to an oligonucleotide tag. After incubation with sample, the pool of substrates is hybridized to a microarray containing oligonucleotides complementary to the tag sequences. We screened several specific chemistries for the conjugation based on the following criteria: easy derivatization of oligonucleotides and peptides; high efficiency of the conjugation reaction; good stability of the conjugates; and satisfactory conjugate performance in our assays. We have validated selected method during the successful generation of thousands oligonucleotide-peptide conjugates.     

Tandem conjugation of enzyme and antibody on silica nanoparticle for enzyme immunoassay

We present a new type of enzyme-antibody conjugate that simplifies the labeling procedure and increases the sensitivity of enzyme-linked immunosorbent assay (ELISA). The conjugates were prepared through layer-by-layer immobilization of enzyme and antibody on a silica nanoparticle scaffold. A maximal amount of enzyme was immobilized on the nanoparticle, followed by antibody linkage through Dextran 500. The conjugate could be easily purified from unreacted reagents by simple centrifugations. In comparison with the conventional antibody-enzyme conjugate used in ELISA, which often has one or two enzyme molecules per antibody, the new type of conjugate contained more enzyme molecules per antibody and provided a much higher signal and increased sensitivity. When used in an ELISA detection of the hepatitis B surface antigen (HBsAg), the detection limit was three times lower than that of the commercially available ELISA kit.     

Immunoenzyme method of sequential saturation for determining antigens using the model of insulin

The method for the determination of insulin by means of the enzyme immunoassay, based on the use of insulin-peroxidase conjugates, has been developed. In this assay the scheme of the successive saturation of the active sites of antibodies is used. The antigenic properties of two conjugates differing in the method of their preparation are compared. The conjugates were obtained by the covalent binding of peroxidase, oxidized in its carbohydrate component, with insulin (conjugate 1) or hexamethylene-diamine-modified insulin (conjugate 2). The conjugates represented a mixture of oligomers differing in their molecular weight. Conjugate 1 possessed higher affinity to antibodies and higher enzymatic activity than conjugate 2. The method for evaluating the quality of antisera to insulin used in the assay has been proposed. The time of the insulin assay is 5-16 hours, the limit of insulin detection is 5 microU/ml, the variation factor is 3-12%.     

Electrospray ionisation-cleavable tandem nucleic acid mass tag-peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS

The synthesis and characterization of isotopomer tandem nucleic acid mass tag-peptide nucleic acid (TNT-PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.     

Use of maleimide-thiol coupling chemistry for efficient syntheses of oligonucleotide-enzyme conjugate hybridization probes

Two general methods which exploit the reactivity of sulfhydryl groups toward maleimides are described for the synthesis of oligonucleotide-enzyme conjugates for use as nonradioisotopic hybridization probes. In the first approach, 6-maleimidohexanoic acid succinimido ester was used to couple 5'-thiolated oligonucleotide to calf intestine alkaline phosphatase to provide a 1:1 conjugate in 80-85% yield. The second strategy employed N,N'-1,2-phenylenedimaleimide to cross-link thiolated horseradish peroxidase or beta-galactosidase with a 5'-thiolated oligonucleotide in 58% and 65% yields, respectively. The oligonucleotide-alkaline phosphatase conjugate was able to detect 6 amol of target DNA in 4 h, while the horseradish peroxidase conjugate was found to be 40-fold lower in its sensitivity of detection by using dye precipitation assays.     

Functionalization of covalent DNA-streptavidin conjugates by means of biotinylated modulator components.

Covalent DNA-streptavidin conjugates are versatile biomolecular coupling reagents, since they have binding capacity for both a complementary nucleic acid and four molecules of biotin. The DNA-streptavidin hybrid molecules have been investigated for their capabilities to bind two different types of biotinylated components. Thus, (i) a functional biomolecule, e.g., a single-stranded DNA fragment or an enzyme and (ii) low-molecular weight biotin derivatives ("modulators") were coupled stepwise with the hybrid molecules. Modulators were D-biotin, aminobiotin, and biotin-fluorescein conjugate as well as a lysine-rich 10mer peptide, containing a biotin and a fluorescein substituent. These modulators were chosen to affected the hybridization properties of the DNA-streptavidin conjugates. As investigated by surface-plasmon resonance and microplate solid-phase hybridization measurements, D-biotin, biotin-fluorescein, and aminobiotin decreased the efficiency of hybridization with complementary, surface-bound oligonucleotides to a varying extent. The basic peptide increased the conjugate's hybridization efficiency. Moreover, it was demonstrated in two examples how modulators can be utilized as additional functional domains of streptavidin-based conjugates. First, fluorescein-containing modulators were used as hapten groups, allowing a sensitive detection by means of specific antibodies directed against the modulator. Second, the biotinylated peptide was used as a carrier molecule to attach multiple fluorogenic lanthanide-chelate groups to the streptavidin conjugate, enabling its sensitive detection by time-resolved fluorometry. The applicability of this kind of bioconjugation strategy to generate sensor-probes for gene detection assays was demonstrated.     

Electrospray ionisation-cleavable tandem nucleic acid mass tag–peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS

The synthesis and characterization of isotopomer tandem nucleic acid mass tag–peptide nucleic acid (TNT–PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.     

A novel homogeneous bioluminescence resonance energy transfer element for biomolecular detection with CCD camera or CMOS device

A novel optical signal element based on homogeneous bioluminescence resonance energy transfer (BRET) was developed for biomolecular detection. A fluorescent dye and alkaline phosphatase (AP) conjugate was used as a reporter and light‐generation element for imaging detection platforms that use a CCD camera or CMOS chip‐based devices. In the presence of a luminescence substrate, the energy from the first light emission of a bioluminescence enzymatic reaction was transferred to fluorescent dyes which were conjugated to an enzyme. This resulted in a second light emission with a shorter wavelength. The second light was localized at the position of target molecules without the diffusion problems present in current technology. To optimize energy transfer efficiency, the ratio of enzyme to fluorophore in the conjugates, the fluorescent dyes used in the conjugates and the luminescence substrates used for BRET were investigated. BRET was demonstrated by using both a CCD camera and a CMOS imaging device. Image spatial resolution was greatly improved compared with conventional chemiluminescence detection. This new signal element opens a door for the direct measurement of fluorescent signals on an imaging chip without an external light source and portable instrumentation normally required for the fluorescent detection of biomolecules. Copyright © 2007 John Wiley & Sons, Ltd.     

An immunochemical assay model system for the sensitive detection of pyruvate dehydrogenase complex (PDHc) and its decarboxylating subunit pyruvate dehydrogenase (E1).

An immunochemical enzyme immunoassay model system was developed and compared for maximum sensitivity with a radioimmunoassay method and the classic enzyme activity method for the detection of pyruvate dehydrogenase complex (PDHc) and its decarboxylating subunit, pyruvate dehydrogenase (E1), isolated from Escherichia coli. Cross-linked large molecular weight antibody-enzyme conjugate systems are compared with heterobifunctional singular antibody conjugates substituted with high levels of horseradish peroxidase. Both polyclonal and monoclonal antibodies generated to the Escherichia coli PDHc and E1 antigens were used to develop a double-antibody sandwich microtiter plate enzyme-linked immunosorbent assay. It is demonstrated that a double sandwich immunochemical assay system can be quantitative for PDHc, can detect PDHc in crude cell lysates and has levels of sensitivity of 2.0.10(-16) mol for the detection of PDHc. This assay model system provides specific antibody selection criteria and coupling methods needed to select specific antisera that cross-react with human PDHc. This rapid and sensitive immunochemical assay method clearly demonstrates that sensitive mass assay systems can be developed for the detection of PDHc. Different from Western blot, this methodology could be used to generate mass assays which could be applied to the rapid detection of mammalian antigens (employing the corresponding antibodies) implicated in a number of pyruvate dehydrogenase deficiencies associated with human disorders.     

A trinitrophenyl(TNP)-poly-L-lysine-horseradish peroxidase conjugate for the detection of anti-TNP antibodies in vivo

A new conjugate for the detection of anti-trinitrophenyl(TNP) antibodies was developed to study the localization pattern of specific antibody containing cells and extracellular antibody in vivo. By means of a bridging molecule, poly-L-lysine, nine TNP groups and six horseradish peroxidase (HRP) groups were joined in one conjugate. Thus a higher specificity (more hapten) was united with a higher staining intensity (more enzyme) in the same conjugate. This conjugate made possible the simultaneous detection of anti-TNP antibody containing cells and establishment of their class (immunoglobulin M (IgM) and IgG). It was also used for the demonstration of anti-TNP antibodies in tissues where a TNP-alkaline phosphate (AP) conjugate could not be used due to high AP (endogenous) background staining. Thus we demonstrated anti-TNP antibody containing cells in gut associated lymphoid tissue and anti-TNP-(TNP-ovalbumin) immune complexes in the glomeruli of the kidney. We suggest that poly-L-lysine is a suitable bridging molecule for the preparation of hapten-HRP conjugates.     

Lipoic acid residues in a take-over mechanism for the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The pyruvate dehydrogenase complex of Escherichia coli contains two lipoic acid residues per dihydrolipoamide acetyltransferase chain, and these are known to engage in the part-reactions of the enzyme. The enzyme complex was treated with trypsin at pH 7.0, and a partly proteolysed complex was obtained that had lost almost 60% of its lipoic acid residues although it retained 80% of its pyruvate dehydrogenase-complex activity. When this complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, the rate of modification of the remaining S-acetyldihydrolipoic acid residues was approximately equal to the accompanying rate of loss of enzymic activity. This is in contrast with the native pyruvate dehydrogenase complex, where under the same conditions modification proceeds appreciably faster than the loss of enzymic activity. The native pyruvate dehydrogenase complex was also treated with lipoamidase prepared from Streptococcus faecalis. The release of lipoic acid from the complex followed zero-order kinetics for most of the reaction, whereas the accompanying loss of pyruvate dehydrogenase-complex activity lagged substantially behind. These results eliminate a model for the enzyme mechanism in which specifically one of the two lipoic acid residues on each dihydrolipoamide acetyltransferase chain is essential for the reaction. They are consistent with a model in which the dihydrolipoamide acetyltransferase component contains more lipoic acid residues than are required to serve the pyruvate decarboxylase subunits under conditions of saturating substrates, enabling the function of an excised or inactivated lipoic acid residue to be taken over by another one. Unusual structural properties of the enzyme complex might permit this novel feature of the enzyme mechanism.     

Isolation and use in virological research of a peroxidase conjugate with protein A

On the basis of proteins A produced at the Mechnikov Central Research Institute for Vaccines and Sera (Moscow) and obtained from Pharmacia AB (Sweden), several batches of peroxidase conjugate have been prepared by a modified method of periodate oxidation. The preparations thus obtained have been evaluated by means of the corresponding enzyme immunoassay systems for the detection of antibodies to herpes simplex and measles viruses. The results of this investigation indicate that serum antibody titers, determined in the assays with antispecific conjugates and with protein A-based based conjugates, coincide. The comparative study of conjugates prepared on the basis of Soviet and imported proteins A has demonstrated their similar activities and specificities.     

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.     

Theoretical models for predicting the effect of bridging group recognition and conjugate substitution on hapten enzyme immunoassay dose-response curves

Models for predicting the effect of immunological recognition of the bridge group on the dose-response curves obtained with heterogeneous hapten enzyme immunoassays are presented. Appropriate theoretical treatment shows that the greater affinity of antibodies toward the enzyme-labeled species than for the unlabeled hapten analyte results in assays with limited detection capabilities. This problem is compounded when enzyme conjugates possessing multiple haptens are used. In equilibrium type competitive arrangements, the concentrations of binder and labeled hapten may be optimized to some extent to improve assay performance. However, the results presented show that only when assays are performed in a sequential binding mode using carefully controlled timing of reagent incubations can the detection capabilities of the assays be fully maximized for analyte measurements. Unfortunately, it is also shown that such sequential binding approaches render the assays essentially nonselective. The effect of decreasing the affinity of the binder to the enzyme-labeled hapten relative to the unlabeled analyte by using heterologous conjugates in equilibrium arrangements is shown to improve detection capabilities but also at the expense of reduced selectivity. Suggestions for reagent concentrations and conjugate substitution (degree of conjugation), which provide optimized dose-response curves at a given ED50 value, are also presented as are proposals for using different binders which do not exhibit bridging group recognition.     

Structurally colored thiol chitosan thin films as a platform for aqueous heavy metal ion detection

Thin films of the polysaccharide chitosan and several chitosan derivatives, including conjugates of l-cysteine, thioglycolic acid, and 2-iminothiolane, were produced from dilute acidic solutions. Attempts to produce a fourth conjugate using lipoic acid resulted in the synthesis of partially N-acetylated chitosan ethanoate. These biopolymer films were exposed to solutions containing 50 ppm concentrations of various metal ion and counterion analytes. Analyte-induced changes in film thicknesses and refractive indices were measured using a spectroscopic ellipsometer, and shifts in film color were quantified using a reflectance spectrometer. The modified chitosans were generally more sensitive to change in response to pure water but also showed varied response to several ions of interest, including Cr(III) and Cr(VI), Hg(II), Ni(II), and others. The potential for tuning film response was demonstrated by varying the concentration of sulfur groups in the thioglycolic acid conjugate, leading to increased specificity for Hg(II).     

Evaluation of the use of chromogenic and fluorogenic substrates in solid-phase enzyme linked immunosorbent assays (ELISA)

Fluorogenic and chromogenic substrates were used in direct and trapping enzyme-linked immunosorbent assays (ELISA) for the detection of mouse IgG and foot-and-mouth disease virus (FMDV). The detection limits for both antigens were compared using different combinations of enzymes and substrates. Various times and concentrations of chemicals were used to obtain maximum sensitivity for both systems. Similar sensitivities were found using fluorogenic and chromogenic substrates. Tetramethyl benzidine substrate for horse-radish peroxidase enzyme conjugates was found to attain the highest sensitivity levels for chromogenic assays (0.12 ng IgG/ml and 1.0 ng/ml FMDV respectively), after 10 min incubation. Of the two fluorogenic enzyme/substrates studied, B-galactosidase was the most sensitive but required extended incubation times (2-3 h) as compared with chromogenic systems. Special microplates for fluoro-immunoassay (FIA) were compared with conventional microplates and no advantage was found to justify their use. An alkaline phosphatase anti-guinea-pig conjugate was used to confirm the equivalence of fluorogenic and chromogenic substrates in terms of sensitivity. A comparison of the amount of signal generated using various concentrations of enzyme in the absence of antigen was made for two different alkaline phosphatase conjugates to obtain theoretical sensitivity limits. One possible advantage of fluorogenic substrates is that high binding ratio can improve the confidence in discrimination of positive results.