High Molecular Weight Conjugates Research Articles

Encapsulation of probiotic Lacticaseibacillus casei with whey protein isolate/soybean hull polysaccharide enhances cell viability in harsh conditions

Whey protein isolate (WPI)-soybean hull polysaccharide (SHP) Maillard reaction conjugates were used to encapsulate Lacticaseibacillus casei and determine the effect of encapsulation on bacterial viability during freeze drying, simulated pasteurization simulated gastrointestinal digestion and storage. The WPI-SHP conjugates were characterized by degree of conjugation, size-exclusion chromatography, infrared and fluorescence spectroscopy and other analyses. The degree of conjugation, the formation of Maillard reaction products and the content of high molecular weight conjugates increased with reaction time. The emulsification capacity and glass transition temperature (Tg) of the WPI-SHP conjugates peaked after 4 days of reaction, at a Tg of 140.51 ± 2.76 °C, an emulsification activity index of 9.12 m2/g and emulsion stability index of 87.01%. WPI-SHP4d had the best protection effect of probiotics under freeze drying, simulated pasteurization and gastrointestinal digestion. These findings provide valuable insights into the selection of encapsulating agents to improve probiotic viability in functional foods.

Lactobacillus plantarum 21805 encapsulated by whey protein isolate and dextran conjugate for enhanced viability

Probiotics incorporated into food products are often insufficiently potent because of unfavorable interference during processing. This study addressed these issues by encapsulating Lactobacillus plantarum into whey protein isolate (WPI) and dextran (DX) conjugates using an encapsulator. The objective of this work is to investigate the effect of encapsulation using WPI and DX conjugates on the viability of Lactobacillus plantarum. The formation of the conjugates was confirmed with absorbance of the intermediate- and late-stage products, degree of grafting, browning index, and gel electrophoresis. Extending the heating time from 1 to 5 h increased the content of high molecular weight conjugates. These conjugates provided better protection of probiotics under processing stress conditions, gastrointestinal conditions, and storage. Furthermore, encapsulated L. plantarum exhibited a reduction of only 0.33 log CFU/mL after 90 d of storage at 4 °C. Thus, microencapsulation of probiotics can enhance the viability of functional food products and facilitate corresponding research.

Origin and Function of Structural Diversity in the Plant Specialized Metabolome.

The plant specialized metabolome consists of a multitude of structurally and functionally diverse metabolites, variable from species to species. The specialized metabolites play roles in the response to environmental changes and abiotic or biotic stresses, as well as in plant growth and development. At its basis, the specialized metabolism is built of four major pathways, each starting from a few distinct primary metabolism precursors, and leading to distinct basic carbon skeleton core structures: polyketides and fatty acid derivatives, terpenoids, alkaloids, and phenolics. Structural diversity in specialized metabolism, however, expands exponentially with each subsequent modification. We review here the major sources of structural variety and question if a specific role can be attributed to each distinct structure. We focus on the influences that various core structures and modifications have on flavonoid antioxidant activity and on the diversity generated by oxidative coupling reactions. We suggest that many oxidative coupling products, triggered by initial radical scavenging, may not have a function in se, but could potentially be enzymatically recycled to effective antioxidants. We further discuss the wide structural variety created by multiple decorations (glycosylations, acylations, prenylations), the formation of high-molecular weight conjugates and polyesters, and the plasticity of the specialized metabolism. We draw attention to the need for untargeted methods to identify the complex, multiply decorated and conjugated compounds, in order to study the functioning of the plant specialized metabolome.

Characterization of Plasmodium falciparum NEDD8 and identification of cullins as its substrates

A variety of post-translational modifications of Plasmodium falciparum proteins, including phosphorylation and ubiquitination, are shown to have key regulatory roles during parasite development. NEDD8 is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulates diverse cellular processes. Although neddylation is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. We characterized P. falciparum NEDD8 (PfNEDD8) and identified cullins as its physiological substrates. PfNEDD8 is a 76 amino acid residue protein without the C-terminal tail, indicating that it can be readily conjugated. The wild type and mutant (Gly75Ala/Gly76Ala) PfNEDD8 were expressed in P. falciparum. Western blot of wild type PfNEDD8-expressing parasites indicated multiple high molecular weight conjugates, which were absent in the parasites expressing the mutant, indicating conjugation of NEDD8 through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 (rub1Δ) or NEDD8 conjugating E2 enzyme (ubc12Δ). The PfNEDD8 immunoprecipitate also contained S. cerevisiae cullin cdc53, further substantiating cullins as physiological substrates of PfNEDD8. Our findings lay ground for investigation of specific roles and drug target potential of neddylation in malaria parasites.

Pea GH3 acyl acid amidosynthetase conjugates IAA to proteins in immature seeds of Pisum sativum L. – A new perspective on formation of high-molecular weight conjugates of auxin

Gretchen Hagen 3 (GH3) acyl acid amidosynthetases are encoded by early auxin-responsive genes and catalyze an ATP-dependent biosynthesis of IAA-amino acid conjugates. An amide conjugate of IAA, indole-3-acetyl-aspartate (IAA-aspartate, IAA-Asp), is a predominant form of bound auxin in immature seeds of pea. However, there is some evidence that IAA is also able to form high molecular weight amide conjugates with proteins in pea and other plant species. In this short study we report that recombinant PsGH3 IAA-amino acid synthetase, which exhibits a preference for the formation of IAA-Asp, can also conjugate IAA with the protein fraction from immature seeds of pea (S-10 fraction). We studied [14C]IAA incorporation to the S-10 protein fraction by two assays: TLC method and protein precipitation by trichloroacetic acid (TCA). In both cases, radioactivity of [14C]IAA in the protein fraction increases in comparison to the control (without PsGH3), about 9.3- and 3.17-fold, respectively. l-Asp, as a preferred substrate in the IAA conjugation catalyzed by PsGH3, down-regulates [14C]IAA conjugation to the proteins as shown by the TLC assay (∼2.8-fold decrease) and the TCA precipitation variant (∼2-fold decrease). Moreover, l-Trp that competes with Asp for the catalytic site of PsGH3 and inhibits activity of the enzyme, diminished radioactivity of [14C]IAA-proteins about 1.2- and 2.8-fold, respectively. Taking into account that amino group of an amino acid or a protein acts as an acceptor of the indole-3-acetyl moiety from IAA-AMP intermediate during GH3-dependent conjugation, we masked amine groups (α- and ε-NH2) of the S-10 protein fraction from pea seeds by reductive alkylation. The alkylated proteins revealed about 3- and 2.8-fold lower radioactivity of [14C]IAA than non-alkylated fraction for TLC and TCA precipitation variant, respectively. This is a first study demonstrating that formation of high molecular weight IAA conjugates with proteins is catalyzed by a GH3 acyl acid amidosynthetase.

Structure and physiochemical characteristics of whey protein isolate conjugated with xylose through Maillard reaction at different degrees

The purpose of this study is to prepare Maillard reaction products (MRPs) with both good emulsification and antioxidant activity, and the relationship between the Maillard reaction degree and structural changes of whey protein isolate (WPI) conjugated with xylose through wet-heating was explored. Browning intensity didn’t change while the free amino groups reduced significantly at the initial stage of Maillard reaction (MR). Amino acid analysis indicated that the lysine and arginine reduced significantly. The antioxidant property of the MRPs was significantly improved. The best emulsifying properties could be obtained in the middle degree of MR. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis illustrated that WPI and xylose formed high molecular weight conjugates. The circular dichroism spectra suggested that α-helix and random coil were increased while the β-sheet and β-turns were decreased after the MR. All of the MRPs exhibited a marked red shift in the maximum fluorescence intensity, indicating that the hydrophilicity of MRPs was enhanced.

Selected Physicochemical and Pharmaceutical Properties of Poly-ε-caprolactone and Poly(d,l-lactide-co-ε-caprolactone) Conjugates of Lamivudine Synthesized via Ring-Opening Polymerization.

The modification of drug fate after administration may be achieved by the covalent coupling of active pharmaceutical ingredients with macromolecules. To prolong or delay the release, slowly degrading polymers such as polyesters may be applied for conjugation. The detachment of a covalently conjugated drug from the polymeric matrix relies mostly on the material degradation profile and barely on the weak interaction between the drug and macromolecules. In the present study, lamivudine was conjugated via ring-opening polymerization with poly-ε-caprolactone and poly(d,l-lactide-co-ε-caprolactone). The influence of the reaction parameters on the course of the polymerization and physicochemical properties of obtained conjugates were investigated. Subsequently, selected reaction products were formulated into submicron particles, and drug release profiles in physiological-like conditions were investigated. The course of the reaction was monitored via gel permeation chromatography. The structure and physicochemical properties of products were evaluated via spectroscopic, calorimetric, and diffractometric methods. The profile of the drug release from particles prepared by the slow evaporation of conjugate solution from o/w emulsion was monitored with high-performance liquid chromatography. Both an elevated reaction temperature and higher catalyst concentration increased the polymerization rate and simultaneously promoted the side reactions, resulting in a broad molecular weight distribution of products in the range from 1.30 to 2.15. The physicochemical properties of conjugates obtained in different conditions varied and had a direct influence on the drug release. The release curve of lamivudine from particles based on low molecular weight conjugates achieved a plateau between 18.9 and 22.2 μg per mg of conjugate within a month. Drug detachment from particles composed of high molecular weight conjugates exhibited a distinct delay period preceded by a drug burst release at a maximal level of 13.3 μg per mg of conjugate. Conjugate chemical composition and the degree of crystallinity were also found to influence the release.

Dextranol: An inert xeroprotectant.

Dextranol, a reduced dextran, prevents damage to stored dry protein samples that unmodified dextran would otherwise cause. Desiccation protectants (xeroprotectants) like the polysaccharide dextran are critical for preserving dried protein samples by forming a rigid glass that protects entrapped protein molecules. Stably dried proteins are important for maintaining critical information in clinical samples like blood serum as well as maintaining activity of biologic drug compounds. However, we found that dextran reacts with both dried serum proteins and lyophilized purified proteins during storage, producing high-molecular weight Amadori-product conjugates. These conjugates appeared in a matter of days or weeks when stored at elevated temperatures (37° or 45°C), but also appeared on a timescale of months when stored at room temperature. We synthesized a less reactive dextranol by reducing dextran’s anomeric carbon from an aldehyde to an alcohol. Serum samples dried in a dextranol-based matrix protected the serum proteins from forming high-molecular weight conjugates. The levels of four cancer-related serum biomarkers (prostate specific antigen, neuropilin-1, osteopontin, and matrix-metalloproteinase 7) decreased, as measured by immunoassay, when serum samples were stored for one to two weeks in dextran-based matrix. Switching to a dextranol-based xeroprotection matrix slightly reduced the damage to osteopontin and completely stopped any detectable damage during storage in the other three biomarkers when stored for a period of two weeks at 45°C. We also found that switching from dextran to dextranol in a lyophilization formulation eliminates this unwanted reaction, even at elevated temperatures. Dextranol offers a small and easy modification to dextran that significantly improves the molecule’s function as a xeroprotectant by eliminating the potential for damaging protein-polysaccharide conjugation.

Effect of microencapsulation with Maillard reaction products of whey proteins and isomaltooligosaccharide on the survival of Lactobacillus rhamnosus

Lactobacillus rhamnosus (L. rhamnosus) was microencapsulated with Maillard reaction products of whey protein and isomaltooligosaccharide (WIMRPs) through cold gelation within emulsification system and its survival was evaluated after exposure to simulated gastrointestinal conditions. The Maillard reaction of whey protein and isomaltooligosaccharide (4:1) occurred in wet heating conditions (90 °C, 4 h) forming high molecular weight conjugate. The WIMRPs microspheres showed the highest encapsulation yield (88.88%) after cold gelation within emulsification system compared with the microcapsules produced with the mixture of whey protein and isomaltooligosaccharide (WIMIX). The shape of WIMRPs microparticles was more spherical and smooth. The microparticles WIMRPs and WIMIX had mean diameter of 183 μm and 201 μm, respectively. WIMRPs provided better protection of probiotics after exposure of the microparticles to simulated gastrointestinal conditions, with the count of 7.48 and 7.17 log10 cfu/g respectively after 90 min incubation in Simulated Gastric Fluid (SGF) and 3% bile salt solution. The microspheres could be completely released in 60 min.

SUMO1 Affects Synaptic Function, Spine Density and Memory

Small ubiquitin-like modifier-1 (SUMO1) plays a number of roles in cellular events and recent evidence has given momentum for its contributions to neuronal development and function. Here, we have generated a SUMO1 transgenic mouse model with exclusive overexpression in neurons in an effort to identify in vivo conjugation targets and the functional consequences of their SUMOylation. A high-expressing line was examined which displayed elevated levels of mono-SUMO1 and increased high molecular weight conjugates in all brain regions. Immunoprecipitation of SUMOylated proteins from total brain extract and proteomic analysis revealed ~95 candidate proteins from a variety of functional classes, including a number of synaptic and cytoskeletal proteins. SUMO1 modification of synaptotagmin-1 was found to be elevated as compared to non-transgenic mice. This observation was associated with an age-dependent reduction in basal synaptic transmission and impaired presynaptic function as shown by altered paired pulse facilitation, as well as a decrease in spine density. The changes in neuronal function and morphology were also associated with a specific impairment in learning and memory while other behavioral features remained unchanged. These findings point to a significant contribution of SUMO1 modification on neuronal function which may have implications for mechanisms involved in mental retardation and neurodegeneration.

Polymer conjugates of doxorubicin bound through an amide and hydrazone bond: Impact of the carrier structure onto synergistic action in the treatment of solid tumours

In this study, we describe the synthesis, physico-chemical characterisation and results of the in vitro and in vivo evaluation of the biological behaviour of N-(2-hydroxypropyl)methacrylamide-based (HPMA) copolymer conjugates bearing doxorubicin (DOX) partly bound via a pH-sensitive hydrazone and partly via enzymatically degradable amide bonds, each contributing to a different anti-tumour mechanism of action of the polymer–doxorubicin conjugate. The following two types of HPMA copolymer drug carriers designed for passive tumour targeting were synthesised and compared: the linear non-degradable copolymer and the biodegradable high-molecular-weight (HMW) diblock copolymer. The HMW diblock copolymer carrier containing a degradable disulphide bond between the polymer blocks showed a rapid degradation in a buffer containing glutathione within the first few hours of incubation. In contrast to the conjugate with the amide bond-bound DOX requiring the presence of lysosomal enzymes to release DOX, the polymer–drug conjugate with the DOX bound via a hydrazone bond released DOX by pH-sensitive hydrolysis, which was significantly faster in a buffer of pH 5.0 (intracellular pH) than pH 7.4, mimicking the conditions in the bloodstream. The significant and comparable in vivo anti-tumour activity of the diblock HMW conjugate and an equimolar mixture of the conjugates differing in the DOX attachment method along with the development of cancer resistance during treatment with these conjugates demonstrated the high potential of these compounds in the development of new nanomedicines suitable for the treatment of solid tumours.

Synthesis of Long‐Circulating, Backbone Degradable HPMA Copolymer–Doxorubicin Conjugates and Evaluation of Molecular‐Weight‐Dependent Antitumor Efficacy

Backbone degradable, linear, multiblock N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (DOX) conjugates are synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization followed by chain extension via thiol-ene click reaction. The examination of molecular-weight-dependent antitumor activity toward human ovarian A2780/AD carcinoma in nude mice reveals enhanced activity of multiblock, second-generation, higher molecular weight conjugates when compared with traditional HPMA copolymer-DOX conjugates. The examination of body weight changes during treatment indicates the absence of non-specific adverse effects.

Polyvalent Side Chain Peptide–Synthetic Polymer Conjugates as HIV-1 Entry Inhibitors

This report describes the synthesis and properties of a series of polyvalent side chain peptide-synthetic polymer conjugates designed to block the CD4 binding site on gp120 and inhibit HIV-1 entry into a host cell. The peptide sequences in the conjugates are based on the CDR H3 region of the neutralizing anti-HIV-1 antibody IgG1 b12. Using a consecutive ester-amide/thiol-ene postpolymerization modification strategy, a library of polymer conjugates was prepared. Evaluation of the HIV-1 inhibitory properties revealed that midsized polymer conjugates displayed the highest antiviral activity, while shorter and longer conjugates proved to be less efficacious inhibitors. The lower molecular weight conjugates may not have sufficient length to span the distance between two neighboring gp120 containing spikes, while the higher molecular weight conjugates may be compromised due to a higher entropic penalty that would accompany their binding to the viral envelope. Although the IC(50) values for these polymer conjugates are higher than that of the parent IgG1 b12 antibody, the strategy presented here may represent an interesting antiviral approach due to the attractive properties of such polymer therapeutics (relatively inexpensive production and purification costs, high thermal and chemical stability in storage conditions, long half-life in biological tissues, low immunogenicity, and protection from proteolytic degradation).

Immunogenicity and thermal stability of a combined vaccine against Haemophilus influenzae type b and Neisseria meningitidis serogroup C diseases

The immunogenicity, structure and stability of a combined conjugate vaccine against Haemophilus influenzae type b and meningococcal serogroup C (Hib/MenC) were investigated. A rat model for immunogenicity showed that antibody responses to Hib and MenC in the combined vaccine were similar to or higher than those of individual conjugates given alone, or concomitantly at separate sites. At elevated temperatures, the combination vaccine was slightly more stable than a monovalent Hib-TT vaccine, with respect to molecular size, which could be attributed to differences in the formulations. Following 5 weeks incubation at 56 °C, there was some dissociation of high molecular weight conjugate without significant loss of saccharide integrity; however, this did not significantly affect the vaccine immunogenicity, demonstrating the stability of this lyophilized vaccine.

High molecular weight blood group A trisaccharide-polyacrylamide glycoconjugates as synthetic blood group A antigens for anti-A antibody removal devices.

Specific immunoadsorption of blood group antibodies by synthetic antigens immobilized on support matrices in the peri-transplantation period provides a promising solution to hyperacute rejection risk following ABO-incompatible transplantation. In this study, we investigated binding interactions between anti-A antibodies and synthetic blood group A trisaccharide conjugated with polyacrylamide of different molecular weights (30 and 1000 kDa). The glycopolymers were equipped with biotin tags and deposited on streptavidin-coated sensor chips. The affinity and kinetics of anti-A antibodies binding to glycoconjugates were studied using surface plasmon resonance (SPR). The high molecular weight conjugate (Atri-PAA(1000)-biotin) enhanced antibody binding capacity by two to three fold compared with the low molecular weight conjugate (Atri-PAA(30)-biotin), whereas varying the carbohydrate content in Atri-PAA(1000)-biotin (20 mol % or 50 mol %) did not affect antibody binding capacity of the glycoconjugate. The obtained results suggest that immunoadsorption devices, especially hollow fiber-based antibody filters which are limited in available surface area for antigen immobilization, may greatly benefit from the new synthetic high molecular weight polyacrylamide glycoconjugates.

Iron Chelation by Deferoxamine Induces Autophagy

Deferoxamine (DFO) is a high affinity Fe (III) chelator produced by Streptomyces pilosus that is used clinically to remove systemic iron in secondary iron overload disorders. DFO cannot be absorbed through the intestine and must be injected. As shown previously, De Domenico et al. EMBO J (2006), expression of Ferroportin (Fpn), the only mammalian iron exporter, can deplete cells of ferritin by lowering cytosolic iron and by exporting iron from cells. Fpn-mediated iron loss induces ferritin degradation by the proteosome. In this study we show that permeable iron chelators, desferirax or deferriprone also induce the proteosomal degradation of ferritin. In contrast, DFO-mediated iron chelation at clinically useful concentrations, leads to ferritin degradation in lysosomes. Immunochemical analysis revealed that DFO-treated cells show increased levels of LC3B, a protein required for autophagy, suggesting that DFO induces autophagy. Treatment of cells with desferasirox or deferriprone did not lead to accumulation of LC3B. Studies using high molecular weight conjugates of DFO or inhibitors of endocytosis showed that the presence of DFO in lysosomes was responsible for the induction of autophagy. Incubation of DFO-treated cells with 3-methyladenine, an autophagy inhibitor, does not, however, prevent ferritin loss suggesting there may be an alternate route for ferritin degradation. This hypothesis was confirmed by examining the effect of the proteosome inhibitor, MG132, on DFOinduced autophagy in cells treated with DFO and 3-methyladenine. Addition of MG132 to 3-methyladenine treated cells prevents ferritin degradation. These results indicate that ferritin degradation occurs by two routes: a DFO-induced entry of ferritin into lysosomes and a cytosolic route in which iron is extracted from ferritin prior to degradation by the proteosome.

The E3 ligase Topors induces the accumulation of polysumoylated forms of DNA topoisomerase I in vitro and in vivo

Human Topors has originally been identified as binding partner of p53 and DNA topoisomerase I (TOP1). It can function as both an ubiquitin and SUMO-1 E3 ligase for p53. Here we demonstrate that Topors enhances the formation of high-molecular weight SUMO-1 conjugates of TOP1 in a reconstituted in vitro system and also in human osteosarcoma cells, similar to treatment with CPT. In contrast to the situation observed with p53, overall sumoylation levels were rather unaffected. Experiments with TOP1 point mutants strongly suggest that the high-molecular weight conjugates represent SUMO-1 chains formed on a limited number of SUMO-1 acceptor sites.

Substrate Modification with Lysine 63-linked Ubiquitin Chains through the UBC13-UEV1A Ubiquitin-conjugating Enzyme

Protein modification with lysine 63-linked ubiquitin chains has been implicated in the non-proteolytic regulation of signaling pathways. To understand the molecular mechanisms underlying this process, we have developed an in vitro system to examine the activity of the ubiquitin-conjugating enzyme UBC13-UEV1A with TRAF6 in which TRAF6 serves as both a ubiquitin ligase and substrate for modification. Although TRAF6 potently stimulates the activity of UBC13-UEV1A to synthesize ubiquitin chains, it is not appreciably ubiquitinated. We have determined that the presentation of Lys(63) of ubiquitin by UEV1A suppresses TRAF6 modification. Based on our observations, we propose that the modification of proteins with Lys(63)-linked ubiquitin chains occurs through a UEV1A-independent substrate modification and UEV1A-dependent Lys(63)-linked ubiquitin chain synthesis mechanism.

Comparison of Neisseria meningitidis serogroup W135 polysaccharide–tetanus toxoid conjugate vaccines made by periodate activation of O-acetylated, non- O-acetylated and chemically de- O-acetylated polysaccharide

Polysaccharide (PS) and tetanus toxoid (TT) protein conjugate vaccines were prepared using O-acetylated (OAc +), O-acetyl negative (OAc −) and chemically de- O-acetylated (de-OAc) meningococcal W135 PS. The PSs were activated by periodate oxidation and coupled to hydrazine derivatized TT. High performance anion exchange chromatography of acid hydrolysates of periodate activated W135 PSs, showed that galactose residues in OAc + PS were more sensitive to the periodate oxidation step than they were in the OAc − PS or de-OAc PS. Mouse antisera against OAc −-TT conjugate vaccines recognized both OAc − and OAc + PS by ELISAs and had high bactericidal titers against both OAc + and OAc − W135 strains. Purified high molecular weight (HMW) conjugates showed higher PS to protein ratios in OAc −-TT (HMW) and de-OAc-TT (HMW) indicating better conjugation efficiency than OAc +-TT (HMW) conjugate. Antisera against the HMW fractions gave higher bactericidal titers than antisera against unfractionated conjugates. Inhibition ELISAs indicated that OAc − and OAc + HMW conjugates induced antibodies that bound both OAc + and OAc − PS. Thus, for W135, PS O-acetylation does not contribute a dominant immunogenic epitope. The OAc − PS may be a good starting material for preparing W135 PS–TT conjugate vaccines using periodate oxidation.

Flow immunoassay of trinitrophenol based on a surface plasmon resonance sensor using a one-pot immunoreaction with a high molecular weight conjugate

A surface plasmon resonance (SPR) immunosensor based on a competitive immunoreaction for the determination of trinitrophenol (TNP) is described. A goat anti-mouse IgG (1st antibody), which recognizes an Fc moiety of an antibody, was immobilized on a gold film of an SPR sensor chip by physical adsorption. A TNP solution containing a fixed concentration of a mouse anti-TNP monoclonal antibody (2nd antibody) and a TNP-keyhole limpet hemocyanin (KLH) conjugate was incubated in one-pot and introduced into the sensor chip. The TNP-KLH conjugate competes with TNP for binding with the 2nd antibody. The resulting complex of the 2nd antibody with the TNP-KLH conjugate was bound to the 1st antibody, which is immobilized on the sensor chip. The SPR sensor signal based on resonance angle shift is dependent on the concentration of TNP in the incubation solution in the range from 25 ppt to 25 ppb, and the coefficient of variation of the SPR signals for the 25 ppb TNP solution was determined to be 13% ( n = 4). The experimental results for the adsorption constant of the 1st antibody on the sensor chip and the binding constant of the 1st antibody complex with the 2nd antibody are discussed, together with theoretical considerations.