Selective cytotoxicity of an antibody-ricin A chain conjugate for human tumor cells

The toxic subunit of a plant ricin has been conjugate by a disulfide bond to a polyclonal rabbit antibody specific for the L-chain of human IgG. Both the antibody and ricin A-chain retained their original biological activity after conjugation. This conjugate proved to be a potent cytotoxin for surface Ig positive Burkitt lymphoma EB-3 cells, growing in vitro and produced 50% inhibition of protein synthesis at level of 1.4 x 10(-9) M. When tested for cytotoxic action on target cells, the composite conjugate molecule was at least 100 times more effective than antibodies alone, ricin A-chain alone or a conjugate ricin A-chain--normal rabbit IgG.     

Kinetics of development and characteristics of antibodies induced in cancer patients against yeast expressed rDNA derived granulocyte macrophage colony stimulating factor (GM-CSF)

We have determined the presence and kinetics of granulocyte macrophage colony stimulating factor (GM-CSF) antibodies induced after repeated administration of a yeast expressed GM-CSF product in prostate cancer patients with minimal recurrent disease using a panel of assays for detection and characterization of antibodies. Results showed that all 15 prostate cancer patients treated with GM-CSF developed GM-CSF reactive antibodies during the course of therapy. Most patients (87%) developed GM-CSF reactive antibodies within 3 months while in other patients (13%), these antibodies were induced after additional cycles of GM-CSF treatment. For most patients, the timing of occurrence of these antibodies was the same regardless of whether the ELISA or surface plasmon resonance (SPR) assays were used for detection. However, in two patients, the recognition of GM-CSF reactive antibodies by SPR assays preceded their detection by ELISA. A significant number of patients (n=9, 60%) developed GM-CSF antibodies which neutralized the biological activity of GM-CSF in vitro in a cell-line based bioassay. These antibodies also recognized GM-CSF protein from different expression systems including the non-glycosylated protein from E. coli indicating that the antibody response is directed towards the amino acid backbone of the protein. A significant effect of GM-CSF antibodies on PSA modulation was not observed in this small cohort of patients despite an alteration in PSA levels in some treated patients. The study design used here did not allow conclusions regarding the relationship between neutralizing antibodies and the PSA levels which were used as a marker for clinical outcome. Implementation of a clinical strategy which permits monitoring for antibody development and for levels of a relevant pre-determined clinical marker at appropriate time-points is necessary for assessing the impact of antibody development on the therapeutic efficacy of the protein.     

Quantitation of aflatoxin B1 and aflatoxin B1 antibody by an enzyme-linked immunosorbent microassay.

A specific microtest plate enzyme immunoassay has been developed for the rapid quantitation of aflatoxin B1 at levels as low as 25 pg per assay. Multiple-site injection of rabbits with an aflatoxin B1 carboxymethyloxime-bovine serum albumin conjugate was used for the production of hyperimmune sera. Dilutions of the purified antibody were air dried onto microplates previously treated with bovine serum albumin and glutaraldehyde and then incubated with an aflatoxin B1 carboxymethyloxime-horseradish peroxidase conjugate. The amount of enzyme bound to antibody was determined by monitoring the change in absorbance at 414 nm after the addition of a substrate solution consisting of hydrogen peroxide and 2,2'-azino-di-3-ethyl-benzthiazoline-6-sulfonate. Antibody titers determined in this manner closely correlated with those determined by radioimmunoassay. Competition assays as performed by incubation of different aflatoxin analogs with the peroxidase conjugate showed that aflatoxins B1 and B2 and aflatoxicol caused the most inhibition of conjugate binding to antibody. Aflatoxins G1 and G2 inhibited the conjugate binding to a lesser degree, whereas aflatoxins M1 and B2a had no effect of the assay.     

Sensitivity enhancement of surface plasmon resonance biosensing of small molecules

Surface plasmon resonance (SPR) biosensor formats using gold nanoparticle or protein signal amplification for the sensitive assay of small molecules were developed using progesterone as a model compound. Progesterone was immobilized to a dextran surface in the Biacore biosensor through in situ covalent immobilization using an oligoethylene glycol linker attached to the 4 position of the steroid. This surface produced stable antibody binding for in excess of 1100 assay cycles. Using this surface, assays were developed for progesterone using 10- and 20-nm gold-streptavidin labels attached to biotinylated monoclonal antibody in both label prebinding and sequential binding formats. Prelabeling formats gave no signal enhancement but produced assays with limits of detection of 143 pg/ml, compared with approximately 1 ng/ml in previous studies. Sequential binding formats gave signal enhancements of 2.2-fold over the monoclonal antibody and a limit of detection of 23.1 pg/ml. It was found that secondary antibody labeling gave 8.1-fold signal enhancements and a limit of detection of 20.1 pg/ml, whereas use of secondary antibody-25 nm gold complexes provided more signal enhancement (13-fold) and a further improvement in limit of detection of 8.6 pg/ml.     

Aequorin-based homogeneous cortisol immunoassay for analysis of saliva samples

Homogeneous assays are attractive because they are performed in only one phase, namely, the liquid phase, and thus, they do not require separation of phases as their heterogeneous counterparts do. As opposed to heterogeneous assays, the signal generation in a homogeneous assay is a direct result of analyte binding, which allows the multiple washing and incubation steps required in an indirect heterogeneous assay format to be eliminated. Moreover, homogeneous assays are usually fast and amenable to miniaturization and automation. In this article, we describe the development of a homogeneous assay for the hormone cortisol using the bioluminescent photoprotein aequorin as a reporter molecule. A cortisol derivative was chemically conjugated to the lysine residues of a genetically modified aequorin in order to prepare an aequorin-cortisol conjugate capable of binding anticortisol antibodies. The binding of anticortisol antibodies to the aequorin-cortisol conjugate resulted in a linear response reflected in the emission of bioluminescence by aequorin. A competitive binding assay was developed by simultaneously incubating the aequorin-cortisol conjugate, the anticortisol antibodies, and the sample containing free cortisol. Dose-response curves were generated relating the intensity of the bioluminescence signal with the concentration of free cortisol in the sample. The optimized homogeneous immunoassay produced a detection limit of 1 x 10 (-10) M of free cortisol, with a linear dynamic range spanning from 1 x 10 (-5) to 1 x 10 (-9) M. Both serum and salivary levels of cortisol fall well within this assay's linear range (3.0 x 10 (-7) M to 7.5 x 10 (-7) M and 1.0 x 10 (-8) M to 2.5 x 10 (-8) M, respectively), thereby making this assay attractive for the analysis of this hormone in biological samples. To that end, it was demonstrated that the assay can be reliably used to measure the concentration of free cortisol in saliva without significant pretreatment of the sample.     

Homogeneous enzyme immunoassay for lipoic acid based on the pyruvate dehydrogenase complex: a model for an assay using a conjugate with one ligand per subunit.

A homogeneous enzyme immunoassay for lipoic acid was developed by using an enzyme-ligand conjugate containing only one ligand per enzyme subunit. Theoretical studies have shown that the traditional use of multisubstituted enzyme-ligand conjugates has limited the detection limits and sensitivity obtainable with these assays. The use of conjugates with a smaller number of ligands should allow for improved assays. The pyruvate dehydrogenase complex was chosen for this study because each polypeptide chain of dihydrolipoyl transacetylase contains one lipoic acid as a covalently attached prosthetic group. Thus, the naturally occurring enzyme can be considered as an enzyme-lipoic acid conjugate. Anti-lipoic acid antibodies were developed in New Zealand White rabbits to be used as the analyte-specific binders. Association and binder dilution curves were prepared in order to optimize the reagent concentrations and the analytical conditions. Unexpected inhibition by free lipoic acid resulted in a biphasic dose-response curve with a detection limit of 5 x 10(-6) M lipoic acid. This technique has several advantages over previous electrochemical and chromatographic techniques for lipoic acid determination.     

An enzyme-linked immunosorbent assay to quantitate the elastin crosslink desmosine in tissue and urine samples

An enzyme-linked immunosorbent assay method has been developed for the determination of desmosine. The method is based on an inhibition immunoassay (under nonequilibrium conditions) and uses rabbit antisera directed against a desmosine-bovine serum albumin conjugate and microtiter plates coated with desmosine-gelatin conjugate. The assay quantitates desmosine in the range 2.5-50 pmol in tissue and urine samples. Important applications of this rapid and sensitive assay are in studying elastin metabolism and in screening for monoclonal antibodies against desmosine. Methods are described for obtaining a constant level of substitution of desmosine per molecule of bovine serum albumin and for preparing a desmosine-gelatin coating antigen. Five different antibody preparations directed against desmosine exhibit 15-20% cross-reactivity toward pyridinoline (3-hydroxypyridinium), a nonreducible collagen crosslinking compound also present in urine and many tissue samples.     

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.     

Sensitive and rapid detection of 2,4-dicholorophenoxyacetic acid in water samples by using evanescent wave all-fiber immunosensor

Sensitive and rapid detection of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was achieved with a newly developed evanescent wave all-fiber immunosensor (EWAI). A reusable functional sensing surface of the immunosensor is prepared by covalent binding of 2,4-D-bovine serum albumin (2,4-D-BSA) conjugate to a self-assembled alkanethiol monolayer formed onto the fiber optic probe through heterobifunctional reagent. The quantification of free 2,4-D in samples was based on indirect competitive immunoreaction principle. Under optimum conditions, calibration curve obtained for 2,4-D had detection limits of 0.07 microg L(-1), the 50% inhibition concentration (IC(50)) was 3.93+/-0.03 microg L(-1) and the quantitative detection range was 0.22-69.5 microg L(-1). The antibodies binding on the sensor surface could be removed simply by the flow of a pepsin solution (pH 1.9), facilitating reuse of the same probe. The regeneration of the sensor surface allowed the performance of more than 100 assay cycles without significant loss of reactivity. The antibody showed negligible cross-reactivity against a few compounds structurally similar to 2,4-D. The immunosensor developed was successfully applied to the monitoring of 2,4-D in spiked water samples without significant effect of the matrix. The proposed portable immunosensor is promising for real-time on-site analysis of small molecules of environmental interest.     

Human immune response to monoclonal antibody-enzyme conjugates in ADEPT pilot clinical trial.

The human immune response to monoclonal antibody-enzyme conjugates has been studied in patients included in the pilot clinical trial of ADEPT. Each patient received murine monoclonal anti-CEA antibody fragments (A5B7-F(ab')2, conjugated to bacterial enzyme, carboxypeptidase G2 (CPG2) followed by a galactosylated monoclonal anti-CPG2 antibody (SB43), 36-48 h after the conjugate. Some patients were also given a dose of 131I-labeled conjugate (4-8 mg, 7-15 mCi) for blood clearance and gamma camera image studies. All patients studied developed human antimouse antibodies (HAMA) and anti-CPG2 antibodies within 10 d after a single course of treatment with the conjugate. In most cases, IgM response was detected at 7 d after the conjugate followed by the IgG response 14 d later. In one patient, HAMA and anti-CPG2 antibodies of the IgG type could still be detected at 10 mo after treatment. Anti-CPG2 antibodies in serum of one patient were found to inhibit CPG2 activity in vitro. Generation of neutralizing antibodies limits the use of repeat cycles of ADEPT in patients. Use of immunosuppressive agents may allow a useful time window for several ADEPT cycle treatments by delaying the appearance of HAMA and anti-CPG2 antibodies. Patients given cyclosporin A before and during ADEPT are currently being studied for HAMA and anti-CPG2 response.     

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.     

Sensitive determination of estriol-16-glucuronide using surface plasmon resonance sensing

For the quantitative evaluation of low levels of an estriol metabolite of estriol (estriol-16-glucuronide (E3-16G)) in liquid media, we developed a simple and highly sensitive immunoassay using a surface plasmon resonance (SPR) biosensor which did not require any time-consuming sample pretreatment steps. E3-16G was conjugated to ovalbumin (OVA) through an oligoethylene glycol (OEG) linker to form protein conjugates (E3-16G-OEG-OVA), which were then immobilized on a carboxymethyl dextran-coated sensor chip via amine coupling to develop inhibition immunoassays. A limit of detection (LOD) of 76 pg/mL was achieved using a rabbit anti-sheep primary antibody as a binding agent. The detection limit was further improved by using synthesized gold colloids (15 nm) as high mass labels conjugated to the primary antibody. In this Au nanoparticle-enhanced assay, the concentration of E3-16G in aqueous samples could be determined in 7.5 min at a level as low as 14 pg/mL. In addition, the high stability of the E3-16G-OEG-OVA surface gave no obvious drop in antibody-binding capability after more than 1000 binding/regeneration cycles which significantly lowered the research cost.     

Development of an Amperometric Immunosensor for the Determination of Methamphetamine in Urine

An amperometric immunosensor in the competitive format was developed for the detection of methamphetamine in urine. The electrodes consisted of carbon paste and Ag/AgCl screen printed on heat sealing film, respectively, and of monoclonal anti-methamphetamine antibody as the biorecognition element. Optimum amounts of methamphetamine- N -bovine serum albumin conjugate, monoclonal antibody and alkaline phosphatase-goat anti-mouse immunoglobulin G were 20, 10 ng and 1:10,000 dilution in 10 μl each, respectively. Methamphetamine was detected by the conversion of p -aminophenyl phosphate to electroactive p -aminophenol in the range of 200 ng/ml (lower detection limit) to 1,500 ng/ml methamphetamine in a nearly linear dose response curve. Within amphetamine concentrations of 0-1,500 ng/ml cross-reaction with methamphetamine was not observed. Working with urine samples spiked with methamphetamine, the accuracy and precision of the assay were 91.5-104.4% and 15.8-24.4%, respectively. This is a proof of concept in the clinical perspective for an amperometric immunosensor whose electrodes are amenable to future mass production.     

A new type sandwich immunoassay for microcystin: production of monoclonal antibodies specific to the immune complex formed by microcystin and an anti-microcystin monoclonal antibody.

To develop an ultrasensitive immunoassay for microcystins (MCs), a group of heptapeptide hepatotoxins produced by cyanobacteria, we produced monoclonal antibodies (MAbs) which specifically recognize the immune complex (IC) formed by an anti-MC MAb (MC MAb) and MCs. The use of the anti-IC MAb (IC MAb) as the secondary antibody made it possible to develop a sandwich type immunoassay, which is theoretically superior to the widely used competitive immunoassay in sensitivity as well as accuracy. A MC MAb mixed with microcystin-LR (MCLR) to form the IC was immunized to mice. Three IC MAbs were obtained, all of which specifically reacted with the IC, but almost never reacted to MC MAb or MCLR in enzyme-linked immunosorbent assays (ELISAs). Binding kinetics study of one of the IC MAbs, 3F7, by a BIAcore biosensor technique revealed that 3F7 IC MAb could associate with free MC MAb as well as the IC, but the binding to free MC MAb was much more easily dissociated than that to the IC, thus resulting in about 300-fold higher affinity of 3F7 for the IC than for MC MAb alone (1.8 x 10(9) M(-1) and 4.6 x 10(6) M(-1) for the IC and MC MAb, respectively). Finally, 3F7 IC MAb was shown to react with the IC formed by the addition of MCLR to MC MAb-coated plates in a dose-dependent manner. Therefore, a new type sandwich immunoassay, anti-immune complex ELISA (IC ELISA) for MCs, was indeed established. The detection limit of the IC ELISA was 2 pg of MCLR ml(-1) (50 fg per assay), making it the most sensitive of all the methods for detecting MCs reported to date.     

Development of aflatoxin B(1)-lysine adduct monoclonal antibody for human exposure studies

Mouse monoclonal antibodies were developed against a synthetic aflatoxin B(1) (AFB)-lysine-cationized bovine serum albumin conjugate. The isotype of one of these antibodies, IIA4B3, has been classified as immunoglobulin G1(lambda). The affinity and specificity of IIA4B3 were further characterized by a competitive radioimmunoassay. The affinities of IIA4B3 for AFB and its associated adducts and metabolites are ranked as follows: AFB-lysine > 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-AFB > AFB = 8,9-dihydro-8-(N(7)-guanyl)-9-hydroxy-AFB > aflatoxin M(1) > aflatoxin Q(1). IIA4B3 had about a 10-fold higher affinity for binding to AFB-lysine adduct than to AFB when (3)H-AFB-lysine was used as the tracer. The concentration for 50% inhibition for AFB-lysine was 0.610 pmol; that for AFB was 6.85 pmol. IIA4B3 had affinities at least sevenfold and twofold higher than those of 2B11, a previously developed antibody against parent AFB, for the major aflatoxin-DNA adducts 8,9-dihydro-8-(N(7)-guanyl)-9-hydroxy-AFB and 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-AFB, respectively. An analytical method based on a competitive radioimmunoassay with IIA4B3 and (3)H-AFB-lysine was validated with a limit of detection of 10 fmol of AFB-lysine adduct. The method has been applied to the measurement of AFB-albumin adduct levels in human serum samples collected from the residents of areas at high risk for liver cancer.     

Engineering receptors and antibodies for biosensors

Biosensor sensitivity and selectivity depend essentially on the properties of the biorecognition elements to be used for analyte binding. Two principally different applications are considered, (1) effects monitoring with biological components as targets for bioeffective substances, among them endocrine disruptors; and (2) immunochemical analysis employing antibodies as binding proteins for a wide variety of analytes such as pesticides. Genetic engineering provides an elegant way not only for providing unlimited amounts of biorecognition molecules but also for the alteration of existing properties and the supplementation with additional functions. Instrumental applications were carried out with the optical sensor BIAcore. The first example deals with the characterization of receptors. For this purpose, the human estrogen receptor alpha was used. Binding studies were carried out with natural as well as xenoestrogens. An equilibrium dissociation constant K(d) of 2.3x10(-10) (M) was derived for 17beta-estradiol. A competition assay was performed with a bovine serum albumin (BSA)-17beta-estradiol conjugate, immobilized at the optical sensor surface, and the free estrogen. The signals obtained represent estradiol equivalents. This format was transferred to a microplate-based enzyme-linked receptor assay. It reached a detection limit of 0.02 microg l(-1) 17beta-estradiol and proved suitable for the detection of natural and synthetic estrogens as well as xenoestrogens in field studies. The second example is targeted at kinetic and affinity measurements of recombinant antibody fragments derived from antibody libraries with s-triazine selectivities. Different strategies for the synthesis of antibody fragment libraries, followed by the selection of specific antibody variants, were examined. An antibody library was derived from a set of B cells. Chain shuffling of the heavy and light chains provided the best binders. An enzyme linked immunosorbent assay (ELISA) was achieved for atrazine with an IC(50) of 0.9 microg l(-1) and a detection limit of 0.2 microg l(-1). The close relations between the optimization of recombinant antibodies by evolutionary strategies and genetic algorithms are considered.     

Direct ring conjugation of catecholamines and their immunological interactions

Catecholamine derivatives were synthesized with potential applications as coating antigens in biosensors or in the raising of specific antibodies. Thioether-bridged derivatives of the catecholamines dopamine, norepinephrine, and epinephrine that attach carboxylic acid functionalities directly to the aromatic ring via an easily incremented linker chain were synthesized by an electrochemical method. These derivatives were purified by convenient ion-exchange chromatography, exact positions of conjugation determined by NMR, and a dopamine derivative immobilized in situ in a BIAcore surface plasmon resonance (SPR) biosensor and its antibody binding studied in comparison with immobilization via the catecholamine primary amine. Binding of an antibody raised to an amine-conjugated protein conjugate showed clear distinction between conjugations at different positions on the catecholamine, illustrating the importance of rational conjugate design in immunosensing of the catecholamines.     

Evaluation of progesterone-ovalbumin conjugates with different length linkers in enzyme-linked immunosorbant assay and surface plasmon resonance-based immunoassay

A series of progesterone-4-ovalbumin (OVA) conjugates with different length linkers (4-, 11-, and 18-atoms long) were synthesized by successive aminocaproic acid homologation of 3-(pregn-4-ene-3,20-dione-4-yl)thiopropanoic acid (1) before conjugation to ovalbumin. The performance studies of these progesterone-4-ovalbumin conjugates showed that the effects of the length of linker on the antibody binding are dependent upon different immunoassay formats. In a rapid flow biosensor surface, on a BIAcore Surface Plasmon Resonance (SPR) instrument, antibody-binding capacities and response rate were dramatically increased for progesterone-4-ovalbumin conjugates when the length of the linker was incremented from 4 atoms to 11 or 18 atoms. Thus, highly sensitive SPR-based immunoassays for progesterone over a range of 0.1-50 ng ml(-1) were developed using biosensor surfaces immobilized with progesterone-ovalbumin conjugates having extended linkers. The SPR-based assays were fully competitive with conventional enzyme-linked immunosorbant assay (ELISA) but much more rapid and simple. However, there were little changes in antibody-binding performance using a conventional ELISA for the same conjugates. The progesterone-4-ovalbumin conjugate (1-OVA) had better antibody binding than its progesterone-7alpha-ovalbumin analog (2-OVA) in the SPR-based assay, but with a conventional ELISA there was no significant difference between these two isomeric conjugates.     

Antibodies specific for 1-methylguanosine as a probe of tRNA conformation

Antibodies specific for 1-methylguanosine (m1G) were produced by immunization of rabbits with a bovine serum albumin conjugate of m1G. Antibodies specificity was determined by measuring the inhibition of binding of 3H-m1G trialcohol by various nucleosides or related derivatives. The relative affinities of the unpurified antibodies for various nucleosides showed that m1G trialcohol had an 8-fold higher affinity than m1G; further, guanosine and 2'-O-methylguanosine had at least a 500-fold lower affinity than m1G. The antibodies were purified on m1G-AH-Sepharose column and subsequently immobilized to Sepharose. Immobilized m1G antibodies quantitatively and exclusively retained m1G-containing oligonucleotides derived from ribonuclease A digests of 32P-labeled phage T4 tRNAPro. On the other hand, intact 32P-labeled T4 tRNAPro or its precursor RNA(s) did not bind to the same column. These findings indicate that at least a portion of m1G adjacent to the 3' end of the anticodon in intact T4 tRNAPro is not accessible for antibody binding.     

Estrogen conjugation and antibody binding interactions in surface plasmon resonance biosensing

Thioether-linked 3-mercaptopropionic acid derivatives of 17beta-estradiol and estrone were formed at the A-ring 4-position of the steroids by substitution of their 4-bromo analogues. The carboxylic acid terminal was used to link to an oligoethylene glycol (OEG) chain of 15-atoms in length. The OEG derivative of 17beta-estradiol was then in situ immobilized on a carboxymethylated dextran-coated gold sensor surface used to detect refractive index changes upon protein binding to the surface by surface plasmon propagation in a BIAcore surface plasmon resonance (SPR) instrument. Two other estradiol-OEG derivatives with Mannich reaction linkage at the 2-position and hemisuccinate linkage at the 3-position were also immobilized on the sensor surfaces for comparison. Binding performance between these immobilized different positional conjugates and monoclonal anti-estradiol antibody, raised from a 6-position conjugate, clearly demonstrated that both 2- and 4-conjugates, not conjugated through existing functional groups, gave strong antibody bindings, whereas the 3-conjugate through an existing functional group (3-OH) gave very little binding (2% compared to the 2-conjugate). Both 2- and 4-position conjugates were then applied in a highly sensitive estradiol SPR immunoassay with secondary antibody mediated signal enhancement that gave up to a 9.5-fold signal enhancement of primary antibody binding, and a detection limit of 25 pg/mL was achieved for a rapid and convenient flow-through immunoassay of estradiol.