One-pot sample preparation approach for profiling spatial distribution of gibberellins in a single shoot of germinating cereal seeds.

Sample preparation remains a bottleneck in the rapid and reliable quantification of gibberellins (GAs) for obtaining an insight into the physiological processes mediated by GAs. The challenges arise from not only the extremely low content of GAs in complex plant matrices, but the poor detectability of GAs by mass spectrometry (MS) in negative ion mode. In an effort to solve these urgent difficulties, we present a spatial-resolved analysis method to investigate the distribution of GAs in tiny plant tissues based on a simplified one-pot sample preparation approach coupled with ultrahigh-performance liquid chromatography-tandem MS. By integrating extraction and derivatization into one step, target GAs were effectively extracted from plant materials and simultaneously reacted with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, the sample preparation time was largely shortened, the probability of sample loss was minimized and the detection sensitivity of MS was also greatly improved compared with underivatized GAs. Under optimal conditions, the method was validated from the quantification linearity, limits of detection and limits of quantification in the presence of plant matrices, recoveries, and precision. With the proposed method, 15 endogenous GAs were detected and, among these, 11 GAs could be quantified in 0.50 mg fresh weight (FW) wheat shoot samples, and five GAs were quantified in only 0.15 mg FW developing seed samples of Arabidopsis thaliana. The distribution patterns of GAs along both the non-13-hydroxylation pathway and the early 13-hydroxylation pathway in a single shoot of germinating wheat, rice and maize seeds were finally profiled with a spatial resolution down to approximately 1 mm2 .

Mass spectrometry footprinting reveals the structural rearrangements of cyanobacterial orange carotenoid protein upon light activation.

The orange carotenoid protein (OCP), a member of the family of blue light photoactive proteins, is required for efficient photoprotection in many cyanobacteria. Photoexcitation of the carotenoid in the OCP results in structural changes within the chromophore and the protein to give an active red form of OCP that is required for phycobilisome binding and consequent fluorescence quenching. We characterized the light-dependent structural changes by mass spectrometry-based carboxyl footprinting and found that an α helix in the N-terminal extension of OCP plays a key role in this photoactivation process. Although this helix is located on and associates with the outside of the ß-sheet core in the C-terminal domain of OCP in the dark, photoinduced changes in the domain structure disrupt this interaction. We propose that this mechanism couples light-dependent carotenoid conformational changes to global protein conformational dynamics in favor of functional phycobilisome binding, and is an essential part of the OCP photocycle.

A novel targeted iron oxide nanocarrier for inhibiting M2-type macrophages in the tumor microenvironment.

Background: Tumor-associated macrophages (TAMs) are vital to the tumor microenvironment. They are classified as antitumor M1-type or protumor M2-type macrophages. M2-type macrophages accumulate in the tumor stroma and are related to poor prognosis. Iron oxide nanoparticles are used as drug delivery vehicles because of the structure of carboxyl groups on their surface and their ability to be easily phagocytosed by macrophages. Aim: The signal transducer and activator of transcription 6 (STAT6) signaling pathway controls M2 macrophage polarization, but the STAT6 signaling pathway inhibitor AS1517499 lacks efficient targeting in vivo. Thus, our study aimed to block the polarization of TAMs to M2-type macrophages. Methods and Material: We used ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) as drug carriers coated with the STAT6 signaling pathway inhibitors AS1517499 and CD163 monoclonal antibodies to synthesize the targeted nanocomplex AS1517499-USPION-CD163 utilizing the carbodiimide method. Then, we determined its physicochemical properties, including hydrodynamic size distribution, ultrastructure, iron concentration, protein content and activity of the CD163 monoclonal antibody, AS1517499 content, and selectivity for M2-type macrophages, and its biological applications. Results: The hydrodynamic size distribution was stable (average size = 95.37 nm). Regarding biological applications, the targeted nanocomplex selectively inhibited M2-type macrophages. Conclusions: The targeted nanocomplex AS1517499-USPION-CD163 showed high selectivity for M2-type macrophages. Therefore, iron oxide nanoparticles targeting TAMs may be an effective approach to TAM therapy.

Production and characterization of a functional putidaredoxin reductase-putidaredoxin covalent complex.

In the cytochrome P450cam-dependent monooxygenase system from Pseudomonas putida, putidaredoxin (Pdx) shuttles electrons between putidaredoxin reductase (Pdr) and P450cam and, thus, must form transient complexes with both partners. 1-Ethyl 3-[3-(dimethylamino)propyl]carbodiimide (EDC) was found to promote formation of stoichiometric Pdr-Pdx complexes only when carboxyl groups on Pdx were activated. The yield of the EDC-mediated cross-link depended on the Pdx variant used and the redox state of both partners, decreasing in the following order: Pdr(ox)-Pdx(ox) > Pdr(ox)-Pdx(red) > or = Pdr(red)-Pdx(red). The Pdr-Pdx C73S/C85S conjugate was purified and characterized. Compared to the equimolar mixture of intact Pdr and Pdx, the fusion protein was more efficient in electron transfer to cytochrome c and, in the presence of saturating levels of P450cam, more effectively supported camphor hydroxylation. On the basis of our results, we conclude that (i) the cross-linked complex is physiologically relevant and represents a suitable model for mechanistic studies, (ii) molecular recognition between Pdr and Pdx is redox-controlled and assisted by the Glu72(Pdx)-Lys409(Pdr) charge-charge interactions, and (iii) the high specificity of the Pdr-Pdx couple may be due to finely tuned interactions at the protein-protein interface resulting in only one strongly preferred docking orientation leading to efficient FAD-to-[2Fe-2S] electron transfer.

Type II collagen-hyaluronan hydrogel--a step towards a scaffold for intervertebral disc tissue engineering.

Intervertebral disc regeneration strategies based on stem cell differentiation in combination with the design of functional scaffolds is an attractive approach towards repairing/regenerating the nucleus pulposus. The specific aim of this study was to optimise a composite hydrogel composed of type II collagen and hyaluronic acid (HA) as a carrier for mesenchymal stem cells. Hydrogel stabilisation was achieved by means of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) cross-linking. Optimal hydrogel properties were determined by investigating different concentrations of EDC (8 mM, 24 mM and 48 mM). Stable hydrogels were obtained independent of the concentration of carbodiimide used. The hydrogels cross-linked by the lowest concentration of EDC (8 mM) demonstrated high swelling properties. Additionally, improved proliferation of seeded rat mesenchymal stem cells (rMSCs) and hydrogel stability levels in culture were observed with this 8 mM cross-linked hydrogel. Results from this study indicate that EDC/NHS (8 mM) cross-linked type II collagen/HA hydrogel was capable of supporting viability of rMSCs, and furthermore their differentiation into a chondrogenic lineage. Further investigations should be conducted to determine its potential as scaffold for nucleus pulposus regeneration/repair.

Crucial role of Asp408 in the proton translocation pathway of multidrug transporter AcrB: evidence from site-directed mutagenesis and carbodiimide labeling.

The three-component AcrA/AcrB/TolC efflux system of Escherichia coli catalyzes the proton motive force-driven extrusion of a variety of cytotoxic compounds. The inner membrane pump component AcrB belongs to the resistance nodulation and cell division (RND) superfamily and is responsible for drug specificity and energy transduction of the entire tripartite efflux system. Systematic mutational analysis of titratable and polar membrane-located amino acids revealed four residues, D407, D408, K940, and, R971, to be of prime importance for AcrB function. Using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, D408 was shown to specifically react with dicyclohexylcarbodiimide (DCCD) in a pH-dependent manner. The apparent pK(a) of D408 of 7.4 would enable binding and release of protons under physiological conditions. In contrast to other secondary transporters, D408 was not protected from carbodiimide modification in the presence of drugs, which supports the notion of spatially separated transport pathways for drugs and protons. This study provides evidence for a substantial role of membrane-located carboxylates as a central element of the proton translocation pathway in AcrB and other members of the RND superfamily.

Biofunctionalized magnetic hydrogel nanospheres of magnetite and kappa-carrageenan.

Magnetic hydrogel kappa-carrageenan nanospheres were successfully prepared via water-in-oil (w/o) microemulsions combined with thermally induced gelation of the polysaccharide. The size of the nanospheres (an average diameter of about 50 and 75 nm) was modulated by varying the concentration of surfactant. The nanospheres contained superparamagnetic magnetite nanoparticles (average diameter 8 nm), previously prepared by co-precipitation within the biopolymer. Carboxyl groups, at a concentration of about 4 mmol g(-1), were successfully grafted at the surface of these magnetic nanospheres via carboxymethylation of the kappa-carrageenan. The carboxylated nanospheres were shown to be thermo-sensitive in the 37-45 degrees C temperature range, indicating their potential as thermally controlled delivery systems for drugs and/or magnetic particles at physiological temperatures. Finally, preliminary results have been obtained for IgG antibody conjugation of the carboxylated nanospheres and the potential of these systems for bio-applications is discussed.

A tctex1-Ca2+ channel complex for selective surface expression of Ca2+ channels in neurons.

Voltage-gated Ca(2+) channels (VGCCs) are important in regulating a variety of cellular functions in neurons. It remains poorly understood how VGCCs with different functions are sorted within neurons. Here we show that the t-complex testis-expressed 1 (tctex1) protein, a light-chain subunit of the dynein motor complex, interacts directly and selectively with N- and P/Q-type Ca(2+) channels, but not L-type Ca(2+) channels. The interaction is insensitive to Ca(2+). Overexpression in hippocampal neurons of a channel fragment containing the binding domain for tctex1 significantly decreases the surface expression of endogenous N- and P/Q-type Ca(2+) channels but not L-type Ca(2+) channels, as determined by immunostaining. Furthermore, disruption of the tctex1-Ca(2+) channel interaction significantly reduces the Ca(2+) current density in hippocampal neurons. These results underscore the importance of the specific tctex1-channel interaction in determining sorting and trafficking of neuronal Ca(2+) channels with different functionalities.

Ethylene carbodiimide-fixed donor splenocytes combined with cordycepin induce long-term protection to mice cardiac allografts.

Infusion of ethylene carbodiimide-fixed donor splenocytes (ECDI-SPs) is an effective method to induce donor-specific protection to allografts. However, the ischemia reperfusion (I/R) injury during transplant leads to abundant of pro-inflammatory cytokines, which negates the effect of ECDI-SPs. Therefore, suppressing pro-inflammatory cytokine secretion while promoting anti-inflammatory cytokine release would enhance the graft protective efficacy of ECDI-SPs. In this study, we aimed to determine the effect of ECDI-SPs combined with a short course of cordycepin (an anti-inflammatory agent) on the long-term outcomes of mice cardiac allografts. Our results demonstrated that ECDI-SPs combined with cordycepin significantly promoted mice cardiac allograft survival compared with ECDI-SPs monotherapy. This effect was accompanied by decreased production of pro-inflammatory cytokines (IL-1ß, IL-6, IL-17 and TNFα), increased secretion of anti-inflammatory cytokines (IL-10 and TGFß), inhibition of Th17 and expansion of Tregs, and prevention of I/R injury. We concluded that cordycepin appeared to enhance the effect of modulating cytokine profile and regulate the Teff:Treg balance so as to strengthen the graft protective effect of ECDI-SPs. Our study of ECDI-SPs combined with cordycepin may provide a promising approach for prolong allograft survival.

PEG-stabilized carbodiimide crosslinked collagen-chitosan hydrogels for corneal tissue engineering.

Implantable biomaterials that mimic the extracellular matrix (ECM) in key physical and physiological functions require components and microarchitectures that are carefully designed to maintain the correct balance between biofunctional and physical properties. Our goal was to develop hybrid polymer networks (HPN) that combine the bioactive features of natural materials and physical characteristics of synthetic ones to achieve synergy between the desirable mechanical properties of some components with the biological compatibility and physiological relevance of others. In this study, we developed collagen-chitosan composite hydrogels as corneal implants stabilized by either a simple carbodiimide cross-linker or a hybrid cross-linking system comprised of a long-range bi-functional cross-linker (e.g. poly(ethylene glycol) dibutyraldehyde (PEG-DBA)), and short-range amide-type cross-linkers (e.g. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS)). Optimum hybrid hydrogel demonstrated significantly enhanced mechanical strength and elasticity by 100 and 20%, respectively, compared to its non-hybrid counterpart. It demonstrated excellent optical properties, optimum mechanical properties and suturability, and good permeability to glucose and albumin. It had excellent biocompatibility and when implanted into pig corneas for 12 months, allowed seamless host-graft integration with successful regeneration of host corneal epithelium, stroma, and nerves.

Differentiation of dicyclohexylcarbodiimide reactive sites of the ATPase complex bovine heart mitochondria.

1. In isolated bovine heart mitochondria, the 14C-labelled dicyclohexylcarbodiimide (DCCD) induced inhibition of the ATPase activity is accompanied by labelling of three polypeptides of Mx 9000, 16 000 and 33 000. Of these, only the 9000 polypeptide reacts with [14C]DCCD proportionally to the inhibitory effect, being saturated when the enzyme is maximally inhibited. 2. The 9000 and 16 000 polypeptides are extracted by neutral chloroform/methanol (2 : 1 v/v) while the 33 000 polypeptide remains in the non-extractable residue. No disaggregation of the polypeptides takes place during the extraction. 3. In the ATPase complex immunoprecipitated with antibody against F1, the 9000 and 16 000 polypeptides are present, but the 33 000 polypeptide is absent. 4. The results obtained indicate that the 33 000 polypeptide is not a component of the ATPase complex. As far as F0 is concerned, two types of the binding sites for DCCD were demonstrated, corresponding to the 9000 and 16 000 polypeptides. Their existence is explained by a non-random arrangement among individual monomers of the DCCD-binding protein.

Interaction of Escherichia coli F1-ATPase with dicyclohexylcarbodiimide-binding polypeptide.

Antibody raised against the N,N'-dicyclohexylcarbodiimide (DCCD)-binding polypeptide of Escherichia coli bound to the cytoplasmic surface of the cell membrane. A weak reaction was seen with everted vesicles of the thermophile PS3. Rat-liver mitochondrial membranes did not react with the antibody. Reaction of the isolated DCCD-binding polypeptide with the antibody was prevented by oxidation of methionine residues or cleavage of the polypeptide with cyanogen bromide. Modification of the arginine residues of the DCCD-binding polypeptide did not affect interaction with the antibody. Purified F1-ATPase of E. coli bound to the isolated DCCD-binding polypeptide as shown by solid-phase radioimmune assay. Binding involved the alpha and/or beta subunits of F1 and the arginine residues of the polar central region of the DCCD-binding polypeptide. Our results are consistent with a looped arrangement of the DCCD-binding polypeptide in the membrane in which the carboxyl- and amino-terminal regions of the molecule are at the periplasmic surface and the polar central region, interacting with F1, is at the cytoplasmic surface of the cell membrane.

Ligand binding properties of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase labelled with N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide.

The (Ca2+ + Mg2+)-ATPase of rabbit sarcoplasmic reticulum, when labelled at two Ca2+-protected sites with N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide (NCD-4) retains Ca2+ binding capacity at the sites with Kd values of approx. 3 microM and 0.12 mM as assessed by fluorescence titration. The sites correspond to the two high-affinity Ca2+ binding sites present in the native ATPase. The NCD-4 labelled ATPase exhibits slow conformational changes at each site on addition of Ca2+. It retains the ability to form phosphoenzyme, and can most likely translocate Ca2+.

Stoicheiometry of dicyclohexylcarbodiimide-ATPase interaction in mitochondria.

1. The oligomeric dicyclohexylcarbodiimide (DCCD)-binding protein of mitochondrial ATPase was studied using (a) the relationship between (14C] DCCD binding and inhibition of ATPase activities and (b) the analysis of the kinetics of inhibition. 2. The [14C]DCCD binding to bovine heart mitochondria is linearly proportional to the inhibition of ATP hydrolysis up to a 50% decrease of the original activity resulting in 0.6 mol DCCD bound covalently to the specific inhibitory site (Houstek, J., Svoboda, P., Kopecký, J., Kuzela, S. and Drahota, Z. (1981) Biochim. Biophys. Acta 634, 331-339) per mol of the fully inhibited enzyme. 3. Kinetics of the inhibition of both the ATPase activity (heart and liver mitochondria) and ADP-stimulated respiration (liver) reveal that 1 mol DCCD per mol ATPase eliminates both the synthetic and the hydrolytic activities. It is inferred that the activity-binding correlation underestimates that number of DCCD-reactive sites. 4. The second-order rate constant of the DCCD-ATPase interaction (k) is inversely related to the concentration of membranes, indicating that DCCD reaches the inhibitory site by concentrating in the hydrophobic (phospholipid) environment. 5. At a given concentration of liver mitochondria, comparable k values are obtained both for the inhibition of ATP hydrolysis (k = 5.35.10(2)M-1.min-1) and ADP-stimulated respiration (k = 5.67.10(2)M-1.min-1). 6. It is concluded that both the synthetic and the hydrolytic functions if ATPase are inhibited via a common single DCCD-reactive site. This site is represented by one of the several polypeptide chains forming the oligomer of the DCCD-binding protein. The inhibitor-ATPase interaction does not exhibit cooperativity, indicating that the preferential reactivity towards DCCD is an inherent property of the inhibitory site.

Evidence for the presence of one carboxyl group in the catalytic center of D-beta-hydroxybutyrate dehydrogenase. Inactivation and binding studies with carbodiimide reagents.

D-beta-Hydroxybutyrate dehydrogenase D-3-hydroxybutyrate: NAD+ oxidoreductase, EC 1.1.1.30), a phosphatidylcholine-requiring enzyme, was irreversibly inactivated by a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) or a hydrophobic carbodiimide, N,N'-dicyclohexylcarbodiimide (DCCD). The inactivation is pseudo-first-order with a kinetic stoichiometry of about 1. Phospholipid-free apoenzyme was more sensitive towards these reagents than reconstituted phospholipid-enzyme or membrane-bound enzyme forms. Reduced coenzyme (NADH) protected the enzyme against the inactivation, while oxidized coenzyme (NAD+) in presence of 2-methylmalonate (a pseudo-substrate) gave a better protection. It was found that the phospholipid-free apoenzyme bound about 1 mol [14C]DCCD. This incorporation was prevented by EDAC, indicating that both reagents react at the same site. [14C]Glycine ethyl ester, a nucleophilic compound which reacts specifically with the carboxylcarbodiimide derivative was incorporated to the enzyme (1 mol [14C]glycine ethyl ester per polypeptide chain), whatever its form, in the presence of DCCD or EDAC. These results indicate the presence of one carboxyl group probably located at or near the coenzyme-binding site and near the interacting domain of the enzyme with phospholipid.

Evaluation of the specific dicyclohexylcarbodiimide binding sites in brown adipose tissue mitochondria.

1. The content of the membrane sector of the ATPase complex (Fo) in brown adipose tissue mitochondria was determined by means of specific [14C]-DCCD binding. 2. The specific DCCD binding to the F0 protein was distinguished from the nonspecific binding to the other membrane proteins and phospholipids by: (a) Scatchard plot analysis of the equilibrium binding data, (b) SDS-polyacrylamide gel electrophoresis of the 14C-labelled membrane proteins, (c) partial purification of the chloroform-methanol extractable DCCD-binding protein. It was found that the specific DCCD binding was present in three polypeptides of a relative molecular weight of 9000, 16 000 and 32 000. In brown adipose tissue mitochondria the specific binding was 10-times lower than in heart or liver mitochondria. The binding to the other membrane proteins and to phospholipids was quite similar in all mitochondrial preparations studied. 3. The decreased quantity of the specific binding sites in brown adipose tissue mitochondria demonstrated that the reduction of F0 parallels the reduction of the F1-ATPase and revealed that in these mitochondrial membranes the ratio between the respiratory chain enzymes and the ATPase complex is 10- to 20- times higher than in heart or liver mitochondria.

Modulation of lysozyme charge influences interaction with phospholipid vesicles.

Lysozyme is a globular protein which is known to bind to negatively charged phospholipid vesicles. In order to study the relationship between charge state of the protein and its interaction with negatively charged phospholipid membranes chemical modifications of the proteins were carried out. Succinylation and carbodiimide modification was used to shift the isoelectric point of lysozyme to lower and higher pH values, respectively. The binding of the modified lysozyme to phospholipid vesicles prepared from phosphatidic acid (PA) was determined using microelectrophoresis and ultracentrifugation. At acidic pH of the solution all lysozyme species reduced the surface charges of PA vesicles. Succinylated lysozyme (succ lysozyme) reduced the electrophoretic mobility (EPM) to nearly zero, whereas native lysozyme and carboxylated lysozyme (carbo lysozyme) changed the surface charge to positive values. At neutral pH, the reduction of surface charges was less for carbo lysozyme and unmodified lysozyme. Succ lysozyme did not change the EPM. Unmodified and carbo lysozyme decreased the magnitude of EPM, but the whole complex was still negatively charged. The bound fraction of all modified lysozyme to PA vesicles at high lysozyme/PA ratios was nearly constant at acidic pH. At low lysozyme/PA ratios the extent of bound lysozyme is changed in the order carbo>unmodified>succ lysozyme. Increasing the pH, the extent of bound lysozyme to PA large unilamellar vesicles (LUV) is reduced, at pH 9.0 only 35% of carbo lysozyme, 23% of unmodified lysozyme is bound, whereas succ lysozyme does not bind at pH 7.4 and 9.0. At low pH, addition of all lysozyme species resulted in a massive aggregation of PA liposomes, at neutral pH aggregation occurs at much higher lysozyme/PA ratios. Lysozyme binding to PA vesicles is accompanied by the penetration of lysozyme into the phospholipid membrane as measured by monolayer techniques. The penetration of lysozyme into the monolayer was modulated by pH and ionic strengths. The interaction of lysozyme with negatively charged vesicles leads to a decrease of the phospholipid vesicle surface hydration as measured by the shift of the maximum of the fluorescence signal of a headgroup labeled phospholipid. The binding of bis-ANS as an additional indicator for the change of surface hydrophobicity is increased at low pH after addition of lysozyme to the vesicles. More hydrophobic patches of the lysozyme-PA complex are exposed at low pH. At low pH the binding process of lysozyme to PA vesicles is followed by an extensive intermixing of phospholipids between the aggregated vesicles, accompanied by a massive leakage of the vesicle aqueous content. The extent of lysozyme interaction with PA LUV at neutral and acidic pH is in the order carbo lysozyme>lysozyme>succ lysozyme.

Preparation and characterization of cross-linked carboxymethyl chitin porous membrane scaffold for biomedical applications.

Porous dermal scaffold membrane (PDSM) was successfully prepared by using a so-called sol-gel freeze-drying method. In this method, the carboxymethyl chitin (CMC) hydrosol was first cross-linked by 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and then lyophilized to form the PDSM. For the first time, this research focused on the cross-linked CMC as the only component for three-dimensional PDSM. The effects of cross-linking conditions on the performance of the PDSM were investigated. And PDSM with optimal performance was obtained through 4-h cross-linking at 4 wt% of CMC concentration in the hydrosol, where the mass ratio of EDC to NHS to CMC was 5:3:10. The porosity of the optimized PDSM was more than 90% and the water swelling rate was above 4000%. The pore size was well distributed and was between 100 µm and 200 µm. And the tensile strength was above 0.09 MPa. The as-made PDSM could be degraded above 80% in 12 days in the presence of a 0.2mg/mL lysozyme solution. Very importantly, the PDSM had no cytotoxicity and good biocompatibility from MTT assays. Our results showed the application possibility of the as-prepared PDSM as dermal scaffold for skin tissue engineering.

Hairpin-shaped DNA duplexes with disulfide bonds in sugar-phosphate backbone as potential DNA reagents for crosslinking with proteins.

Convenient approaches were described to incorporate -OP(=O)O(-)-SS-O(-)(O=)PO- bridges in hairpin-shaped DNA duplexes instead of regular phosphodiester linkages: (i) H2O2- or 2,2'-dipyridyldisulfide-mediated coupling of 3'- and 5'-thiophosphorylated oligonucleotides on complementary template and (ii) more selective template-guided autoligation of a preactivated oligonucleotide derivative with an oligomer carrying a terminal thiophosphoryl group. Dithiothreitol was found to cleave completely modified internucleotide linkage releasing starting oligonucleotides. The presence of complementary template as an intrinsic element of the molecule protects the hairpin DNA analog from spontaneous exchange of disulfide-linked oligomer fragments and makes it a good candidate for auto-crosslinking with cysteine-containing proteins.

Development of solid-phase immunoassay using chemiluminescent IgG conjugates.

Solid-phase luminescent immunoassay (LIA) was studied using mainly aminobutyl-ethylisoluminol-IgG conjugates. Different solid-phase supports such as immunobeads, polystyrene balls and tubes gave comparable results although tubes were preferred in most of the assays because of the better linearity and reproducibility obtained and their ease of handling. The properties of the conjugates were tested using both direct and sandwich LIA. Direct LIAs performed using sheep-anti-rabbit IgG coated balls provided information on the antigenicity of the conjugates while the sandwich LIA was the actual working system. A sensitivity of 1 ng was obtained in this system. The stability, easy use, safe handling and low cost of the conjugates coupled with a short assay time make this luminescent system a potential alternative to RIA.