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The development of effective cancer vaccines is an important direction in the area of cancer immunotherapy. Although certain types of preventive cancer vaccines have already been used in the clinic, therapeutic cancer vaccines for treatment of already established tumors are still in high demand. In this study, we develop a new type of cancer vaccine by mixing cell-penetrating peptide (CPP) conjugated antigen as the enhanced antigen, together with CpG as the immune adjuvant. A special CPP, cytosol-localizing internalization peptide 6 (CLIP6), which has the ability to enter cells exclusively via a nonendosomal mechanism, i.e., direct translocation across the cell membrane, is conjugated with model antigen ovalbumin (OVA). Compared to naked OVA, the obtained CLIP6-OVA conjugates show greatly increased uptake by dendritic cells (DCs) and, more importantly, remarkably enhanced antigen cross-presentation, eliciting stronger cytotoxic T lymphocyte (CTL) mediated immune responses with the help of CpG. This CLIP6-OVA/CpG formulation offers effective protection for mice against challenged B16-OVA tumors, and is able to further function as a therapeutic vaccine, which, in combination with immune checkpoint blockade therapy, can significantly suppress the already-established tumors. Such a CLIP6-based cancer vaccine developing strategy shows promising potential toward clinical practice owing to its features of easy preparation, low cost, and remarkable biocompatibility.

Cross-presentation, the process that facilitates display of exogenous antigens on MHC-I molecules, is a crucial step in the cascade of CD8 T cell activation. Potentiation of cross-presentation therefore represents an essential design criterion for development of subunit vaccines that target the induction of CD8 T cell immunity. Covalent conjugation of CpG-DNA to antigenic proteins has shown the potential to promote cross-presentation and has attracted great interest as a promising approach for vaccine development. However, heterogeneous product mixtures that result from typical conjugation schemes precluded identification of active conjugate species and impeded optimization of cross-presentation activity. In this report, we explore the effect of molecular composition of antigen-CpG conjugates on their cross-presentation activity using model Ovalbumin (OVA)-CpG conjugates. We developed a method to generate antigen-CpG conjugates with defined molecular compositions and leveraged this method to produce a series of OVA-CpG conjugates with one, two, and three CpG molecules linked to OVA. We observed that conjugates containing one CpG per OVA enhanced cross-presentation by 4-fold compared to native OVA, while conjugates with higher contents of CpG provided no cross-presentation enhancement. These differences are likely due to enhanced aggregation propensity observed for conjugates that carry more than one CpG per OVA. Our findings suggest that tuning molecular composition of antigen-CpG conjugates to maintain physical stability may be essential for achieving potent cross-presentation activity. Our method to generate defined conjugates could facilitate such molecular tuning and may be useful for continued development of antigen-CpG vaccines.

Short peptides are the minimal modality of antigen recognized by cellular immunity and are therefore considered a safe and highly specific source of antigen for vaccination. Nevertheless, successful peptide immunotherapy is limited by the short half-life of peptide antigens in vivo as well as their weak immunogenicity. We recently reported a vaccine strategy based on dendritic cell-recruiting Mesoporous Silica Rod (MSR) scaffolds to enhance T-cell responses against subunit antigen. In this study, we investigated the effect of covalently conjugating peptide antigens to MSRs to increase their retention in the scaffolds. Using both stable thioether and reducible disulfide linkages, peptide conjugation greatly increased peptide loading compared to passive adsorption. In vitro, Bone Marrow derived Dendritic Cells (BMDCs) could present Ovalbumin (OVA)-derived peptides conjugated to MSRs and induce antigen-specific T-cell proliferation. Stable conjugation decreased presentation in vitro while reducible conjugation maintained levels of presentation as high as soluble peptide. Compared to soluble peptide, in vitro, expansion of OT-II T-cells was not affected by adsorption or stable conjugation to MSRs but was enhanced with reversible conjugation to MSRs. Both conjugation schemes increased peptide residence time in MSR scaffolds in vivo compared to standard bolus injections or a simple adsorption method. When MSR scaffolds loaded with GM-CSF and CpG-ODN were injected subcutaneously, recruited dendritic cells could present antigen in situ with the stable conjugation increasing presentation capacity. Overall, this simple conjugation approach could serve as a versatile platform to efficiently incorporate peptide antigens in MSR vaccines and potentiate cellular responses.

The efficient induction of antigen-specific immune responses requires not only promotion of the uptake of antigens and adjuvant molecules into antigen-presenting cells but also control of their intracellular behavior. We previously demonstrated that the β-glucan schizophyllan (SPG) can form complexes with CpG oligonucleotides with attached dA 40 (CpG-dA/SPG), which can accumulate in macrophages in the draining inguinal lymph nodes and induce strong immune responses. In this study, we prepared various conjugates composed of antigenic peptide (OVA 257-264 ) and dA 40 and made complexes with SPG. The conjugates with a disulfide bond between OVA 257-264 and dA 40 were easily cleaved by glutathione. The resultant peptides with a hydrophobic amino acid at the C-terminal end was recognized by puromycin-insensitive leucine aminopeptidase (PILS-AP), which trims antigenic peptide precursors and prepares peptides of eight or nine amino acids in length, which is the optimal length for binding to major histocompatibility complex (MHC)-I. The conjugate exposed to such enzymes induced a high antigen presentation level. The antigen presentation level was almost the same before and after the complexation with SPG. Immunization with a mixture of dA-OVA 257-264 /SPG and CpG-dA/SPG induced high antigen-specific cytotoxic T-lymphocyte activity at a much lower peptide dose than in previous studies. These results can be strongly ascribed to not only the cell-specific delivery by SPG but also the control of the intracellular behavior by the introduction of cleavage sites. Therefore, peptide-dA/SPG complexes could be used as potent vaccine antigens for the treatment of cancers and infectious diseases.

The new generation vaccines are safe but poorly immunogenic, and thus they require the use of adjuvants. Adjuvants that can control the balance and induction level of cellular and humoral immunities are urgently required for the treatment of and/or protection from infectious diseases and cancers. However, there are no adjuvants which can achieve these requirements. In this study, amphiphilic poly(γ-glutamic acid) (γ-PGA) with various kinds of hydrophobic amino acid ethyl esters (AAE) was synthesized (γ-PGA-AAE) and used to prepare antigen-encapsulated nanoparticles (NPs). γ-PGA-graft-Leu (γ-PGA-Leu, where Leu = leucine ethyl ester), γ-PGA-graft-Phe (γ-PGA-Phe, where Phe = phenylalanine ethyl ester), and γ-PGA-graft-Trp (γ-PGA-Trp, where Trp = tryptophan ethyl ester) formed monodispersed NPs that encapsulated ovalbumin (OVA). The type and the induction level of the antigen-specific cellular and humoral immunities could be controlled by the kinds of hydrophobic segments and vaccine formulation (encapsulation or mixture) used. When OVA was encapsulated into NPs, the cellular immunity was dominantly induced, while humoral immunity was dominant when OVA was mixed with NPs. These results are a first report to demonstrate that the balance and induction level of cellular and humoral immunities could be controlled by modifying compositions of NPs and vaccine formulation. Our results suggest that γ-PGA-AAE NPs can provide safe and efficient nanoparticle-based vaccine adjuvants, and the results also provide guidelines in the rational design of amphiphilic polymers as vaccine adjuvants which can control the balance of immune responses.

Peptide-based therapy is a promising strategy for antigen-specific immunosuppression to treat or even heal autoimmune diseases with significantly reduced adverse effects compared to conventional therapies. However, there has been no major success due to the drawbacks of native peptides, i.e., limited bioavailability. Considering the importance and limitations of peptide-based therapies for treatment of autoimmune diseases, we designed and constructed oligoglycerol (OG)- and polyglycerol (PG)-based peptide conjugates. They were evaluated for their biological activity (in vitro and in vivo), bioavailability, and tolerogenic potential. Among the OG- and PG-peptide constructs, PG-peptide constructs exhibited an extended bioavailability compared to OG-peptide constructs and unconjugated peptide. Interestingly, size, structure, and linker chemistry played a critical role for the tolerogenic capacity of the constructs. The PG-peptide construct bound via an ester linkage was the most tolerogenic conjugate, while the PG-peptide construct bound via an amide induced stronger proliferation, but also higher TNF production and lower frequencies of Foxp3(+) regulatory T-cells. Therefore, we conclude that PG-peptide conjugates bound via an ester linkage are not only promising candidates for tolerogenic vaccination, but also open a new avenue toward the application of peptides for the treatment of autoimmune diseases.

Lectins are ubiquitous carbohydrate-binding proteins of nonimmune origin that are characterized by their specific recognition of defined monosaccharide or oligosaccharide structures. However, the use of carbohydrates to study lectin has been restricted by the weak binding affinity and noncovalent character of the interaction between carbohydrates and lectin. In this report, we designed and synthesized a multifunctional photoaffinity reagent composed of a trialkyne chain, a masked latent amine group, and a photoreactive 3-trifluoromethyl-3-phenyl-diazirine group in high overall yield. Two well-defined chemistries, Huisgen-Sharpless click chemistry and amide bond coupling, were the key steps for installing the multivalent character and tag in our designed photoaffinity probe. The photolabeling results demonstrated that the designed probe selectively labeled the target lectin, RCA120 ( Ricinus communis Agglutinin), in an E. coli lysate and an asialoglycoprotein receptor (ASGP-R) on intact HepG2 cell membranes. Moreover, the probe also enabled the detection of weak protein-protein interactions between RCA120 and ovalbumin (OVA).

Previously we reported emulsion polymerization of propylene sulfide with Pluronic F127 as an emulsifier, yielding nanoparticles (NPs) in the 25 nm size range. Immunologically functional NPs were prepared by adding an antigen-Pluronic conjugate to the polymerization mixture ( Reddy , S. T. , et al. ( 2007 ) Nat. Biotechnol. 25, 1159 ). We sought a more flexible scheme for conjugation of antigens and other biomolecules to the NP surfaces that would allow for milder reaction conditions than achievable during the polymerization step. Here, we present the synthesis of such functionalizable NPs in the form of NPs that carry thiol-reactive groups, to which thiol-containing antigens (peptide or protein) or other biomolecules can be conjugated under mild conditions to yield immunofunctional NPs. The Pluronic-stabilized poly(propylene sulfide) (PPS) NPs with thiol-reactive pyridyl disulfide groups are prepared in two steps by (1) emulsion polymerization of propylene sulfide in the presence of a carboxylate-Pluronic and (2) reaction of the carboxylic acid groups on the NP surface with cysteamine pyridyl disulfide and a water-soluble carbodiimide reagent. We choose pyridyl disulfide groups to have a reduction-sensitive disulfide bond linking the antigen to the NP surface, allowing efficient release of antigen inside the cell in response to the reductive conditions within the endosome. The functionalizable NPs are characterized by proton NMR, dynamic light scattering (DLS), UV/vis spectroscopy, and transmission electron microscopy (TEM). Conjugation of small molecules and protein to the NP surface is presented.

This paper describes a method for a single-step, site-specific conjugation of bioactive peptides to proteins that exploits the monitoring advantages provided by the unique UV signature absorbance of a bis-arylhydrazone. The utility of this method is demonstrated by the conjugation of a decapeptide molecular adjuvant, YSFKDMP(MeL)aR (EP67), to two test proteins, ovalbumin (OVA) and bovine serum albumin (BSA), and to proteins expressed on intact influenza virons and fungal arthroconidia (spores) of Coccidioides. Conjugation is accomplished with a version of EP67 in which its N-terminus is modified with succinimidyl-4-benzoylhydrazino-nicotinamide (S4BHyNic) (peptide 7), thus enabling conjugation to these large entities via formation of amide bonds with surface-exposed amino groups. The presence of the strongly absorbing bis-arylhydrazone S4BHyNic (ε(354 nm) = 29 000 L mol(-1) cm(-1)) allows for determination of EP67-to-protein molar substitution ratios (MSR), which are in good agreement with the MSRs determined by amino acid analysis. Conjugation to OVA does not compromise the ability of EP67 to engage C5a receptor bearing antigen presenting cells (APC) as measured by the EP67-mediated release of interleukin-6 (IL-6) from APCs. Mice immunized with the resulting OVA-EP67 vaccine conjugate produce high serum titers of OVA-specific IgG antibodies relative to OVA alone. Also, the conjugation of EP67 does not affect the surface integrity of influenza virons or the biological viability of Coccidioides spores. This method of conjugating bioactive peptides to proteins and other large biological entities may represent a convenient and effective way of generating various bioconjugates for use in mechanistic studies or novel therapeutic entities such as EP67-containing vaccines.

While many infectious diseases are controlled by vaccine strategies, important limitations continue to motivate the development of better antigen delivery systems. This study focuses on the use of a pH-sensitive polymeric carrier based on poly(propylacrylic acid) (PPAA) to address the need for more potent CD8 cytotoxic T-cell (CTL) responses. An MHC-1/CD8 CTL cell model system with ovalbumin as the protein antigen was used to test whether PPAA could enhance the delivery of ovalbumin into the MHC-1 display pathway. Ovalbumin was conjugated to poly(propylacrylic acid-co-pyridyldisulfide acrylate) (PPAA-PDSA) by disulfide exchange to make reversible conjugates that could be reduced by the glutathione redox system in the cytosol of antigen presenting cells. The PPAA-PDSA ovalbumin conjugates displayed the pH-sensitive membrane disruptive properties of the parent polymer as determined by their hemolysis activities (sharply active at the endosomal pH values of 6-6.5). The polymer-ovalbumin conjugates exhibited strong 22-fold increases in the MHC-1 presentation and ovalbumin-specific CTL activation compared to free ovalbumin. No CTL activation was observed with control conjugates of ovalbumin and poly(methylacrylic acid) (PMAA) that do not display membrane disruptive activies, suggesting that it is the membrane destabilizing properties of the polymer that result in increased MHC-1 display and CTL activation. Further mechanistic studies quantitated the time course of stable intracellular localization of radiolabeled conjugates. 52% of initially internalized PPAA-conjugated ovalbumin remained in the cells after 4 h, compared to less than 10% of ovalbumin or PMAA-ovalbumin. These results showing enhanced cytosolic delivery and MHC-1 presentation for the PPAA-antigen conjugates suggest that they warrant future characterization as a CD8-enhancing vaccine delivery system.

The mutant beta1,4-galactosyltransferase (beta4Gal-T1), beta4Gal-T1-Y289L, in contrast to wild-type beta4Gal-T1, can transfer GalNAc from the sugar donor UDP-GalNAc to the acceptor, GlcNAc, with efficiency as good as that of galactose from UDP-Gal. Furthermore, the mutant can also transfer a modified sugar, C2 keto galactose, from its UDP derivative to O-GlcNAc modification on proteins that provided a functional handle for developing a highly sensitive chemoenzymatic method for detecting O-GlcNAc post-translational modification on proteins. We report herein that the modified sugar, C2 keto galactose, can be transferred to free GlcNAc residues on N-linked glycoproteins, such as ovalbumin or asialo-agalacto IgG1. The transfer is strictly dependent on the presence of both the mutant enzyme and the ketone derivative of the galactose. Moreover, the PNGase F treatment of the glycoproteins, which cleaves the N-linked oligosaccharide chain, shows that the modified sugar has been transferred to the N-glycan chains of the glycoproteins and not to the protein portion. The application of the mutant galactosyltransferase, beta4Gal-T1-Y289L, to produce glycoconjugates carrying sugar moieties with reactive groups, is demonstrated. We envision a broad potential for this technology such as the possibilities to link cargo molecules to glycoproteins, such as monoclonal antibodies, via glycan chains, thereby assisting in the glycotargeting of drugs to the site of action or used as biological probes.

The development of multicomponent biotherapeutic carriers is an important challenge in the field of drug delivery, particularly in the area of protein-based vaccines. While the delivery of protein antigens to antigen presenting cells (APCs) is crucial for this type of vaccination, the incorporation of additional adjuvants may be just as important in order to generate more potent immune responses. This article presents the synthesis and biological evaluation of carrier particles that both deliver a protein payload to APCs and display receptor ligands for the enhancement of APC immunostimulation. Particles displaying CpG oligonucleotide ligands for Toll-like receptor 9 were synthesized. The addition of CpG DNA to the particles led to a 45-fold increase in the secretion of interleukin-12, a cytokine that aids in T-cell activation, and a significant increase in the expression of costimulatory molecules by APCs. Moreover, vaccination with particles containing both ovalbumin (OVA) and CpG DNA induced a superior OVA-specific CD8 T-cell response in vivo, as measured by increased OVA-specific CD8 T-cell proliferation, secretion of the proinflammatory cytokine IFN-gamma, and the induction of OVA-specific cytotoxicity.

Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) are known as an immune adjuvant. CpG DNAs coupled with a particular antigen enabling both CpG DNA and antigen delivery to the same antigen-presenting cell have been shown to be more effective. Based on our previous finding that beta-(1-->3)-D-glucan schizophyllan (SPG) can be used as a CpG DNA carrier, here we present the synthesis of an antigen-conjugated SPG and the characterization of the conjugate. Ovalbumin (OVA, 43 kDa) was used as a model antigen, and two OVA were conjugated to one SPG molecule (M(w) = 150,000), denoted by OVA-SPG. Circular dichroism and gel electrophoresis showed that OVA-SPG could form a complex with a (dA)(40)-tailed CpG DNA at the 3' end (1,668-(dA)(40)). When OVA-SPG was added to macrophages (J774.A1), the amount of the ingested OVA-SPG was increased compared with that of OVA itself, suggesting that Dectin-1 (proinflammatory nonopsonic receptor for beta-glucans) is involved to ingest OVA-SPG. Furthermore, the complex of the conjugate and DNA was co-localized in the same vesicles, implying that OVA (antigen) and CpG DNA (adjuvant) were ingested into the cell at the same time. This paper shows that OVA-SPG can be used as a CpG DNA carrier to induce antigen-specific immune responses.

The T-cell receptors of CD4(+) T lymphocytes recognize immunogenic peptide sequences bound within the groove of MHC class II molecules, and the peptides that bind to these molecules are known to share common structural motifs. For example, OVA(323-339), an I-A(d)-binding peptide, involves a motif of the I-A(d) peptide-binding groove. In the present study, OVA peptides of up to 26-mer were sequentially synthesized and screened, and two additional I-A(d) binding OVA peptides, OVA(20-43) and OVA(264-286), were found to stimulate CD4(+) T cells of OVA-immune BALB/c mice. OVA(20-43) involved structural motifs of the I-A(d) peptide-binding groove, while OVA(264-286) did not. The ability of these three I-A(d) binding OVA peptides to induce antigen-specific cytokine production was compared among CD4(+) T cells of mice immunized either with alum-adsorbed OVA (OVA-alum) or OVA chemically coupled to the surface of liposome (OVA-liposome). CD4(+) T cells of mice immunized with OVA-alum produced more cytokines when stimulated with OVA(264-286) than with OVA(323-339), while CD4(+) T cells of mice immunized with OVA-liposome conjugates produced more cytokines when stimulated with OVA(323-339) than with OVA(264-286). OVA(20-43) induced production of comparable levels of cytokines in mice immunized either with OVA-alum or OVA-liposome. Confocal laser scanning microscopic analysis demonstrated that chemically coupled OVA and liposomes were colocalized in APCs until OVA received processing. Three-dimensional structural analysis demonstrated that both OVA(264-286) and OVA(323-339) were present on the surface of OVA, but OVA(20-43) was not. These results suggested that the chemical coupling of OVA to liposome affected antigen processing in APCs and thus resulted in the induction of differential T-cell epitopes as compared with those induced by plain OVA.

A method for preparing protein-liposome conjugates based on micelles as intermediates was developed. Ovalbumin was thiolated with 2-IT and conjugated to the surface of micelles composed of a maleimide-derivatized active lipid and a micelle-forming lipid. These micelles were then incubated with liposomes, allowing the micelle components to exchange into the liposome bilayers. Using this technique we were able to demonstrate that it was possible to saturate the surface of the micelle with protein and use this property to control the level of conjugation. Titration of these protein-micelle conjugates into liposome solutions resulted in reproducible batches of protein-liposome conjugates. Chemical cross-linking could be observed in some cases; however, this was controllable through selection of reagent concentrations. The effects of parameters such as thiolation levels, micelle lipid composition, active lipid structure, micelle-forming lipid structure, and micelle/liposome/protein ratios were examined. The method represents a general approach to the preparation of well defined and reproducible protein-liposome-based drug formulations.

We previously reported that liposomes having differential lipid components displayed differential adjuvant effects when antigen was coupled with liposomes via glutaraldehyde. In the present study, antigen-liposome conjugates prepared using liposomes having differential lipid components were added to the macrophage culture, and phagocytosis and the antigen digest of liposome-coupled antigen by macrophages were then investigated. Antigen presentation by macrophages to an antigen-specific T-cell clone was further investigated using the same conjugates. Antigen-liposome conjugates which induced higher levels of antibody production in vivo were recognized more often, and the liposome-coupled antigen was digested to a greater degree by macrophages than antigen-liposome conjugates which induced lower levels of antibody production. These results correlated closely with those regarding antigen presentation by macrophages; when antigen was coupled to liposomes showing higher adjuvant effect, macrophages cocultured with antigen-liposome conjugates activated antigen-specific T-cells at a higher degree. The concentration of OVA in the macrophage culture added as antigen-liposome conjugates was approximately 32 microg/mL. However, the extent of T-cell activation was almost equal to that when 800 microg/mL of soluble OVA was added to the culture. The results of the present study demonstrated that the adjuvant activity of liposomes observed primary in vivo correlated closely with the recognition of antigen-liposome conjugates and antigen presentation of liposome-coupled antigen by macrophages, suggesting that the adjuvant effects of liposomes are exerted at the beginning of the immune response, i.e., recognition of antigen by antigen-presenting cells.

A solid-phase conjugation method utilizing carrier protein bound to an ion exchange matrix was developed. Ovalbumin was adsorbed to an anion exchange matrix using a batch procedure, and the immobilized protein was then derivatized with iodoacetic acid N-hydroxysuccinimid ester. The activated protein was conjugated with glutathione, the conjugation ratio determined by acid hydrolysis, and amino acid analysis performed with quantification of carboxymethyl cysteine. Elution of conjugates from the resin by a salt gradient revealed considerable heterogeneity in the degree of derivatization, and immunization experiments with the eluted conjugates showed that the more substituted conjugates gave rise to the highest titers of glutathione antibodies. Direct immunization with the conjugates adsorbed to the ion exchange matrix was possible and gave rise to high titers of glutathione antibodies. Conjugates of ovalbumin and various peptides were prepared in a similar manner and used for production of peptide antisera by direct immunization with the conjugates bound to the ion exchanger. Advantages of the method are its solid-phase nature, allowing fast and efficient reactions and intermediate washings, and the ability to release conjugates from the solid phase under mild conditions.

With the aim of producing novel antibodies to domoic acid (DA), an original, rapid, and simple procedure for preparing minute amount of hapten-protein conjugates was developed. The amide-bond-generating mixed anhydride method of Erlanger was performed using 0.32-0.64 micromol of DA in a reversed micellar medium allowing strong carrier haptenization as determined by spectrophotometric measurement. Bovine serum albumin (BSA) and ovalbumin (OVA) conjugates were, respectively, used for immunization of BALB/c mice and antibody screening by enzyme-linked immunosorbent assay (ELISA). Specific polyclonal antibodies were produced upon multiple injections of (DA)(17)-BSA conjugate administered by three different routes: (i) intraperitoneal (i.p.), (ii) intraperitoneal + subcutaneous (i.p. + s.c.), (iii) footpad (f.p.). The i.p. route induced antisera of higher titer (1:350000) than did the other protocols (approximately 1:72900) and was selected throughout further experiments. Using a competitive ELISA format with a peroxidase immunoconjugate and a chromogenic substrate, no significant cross-reactivity was observed with glutamic acid, aspartic acid and kainic acid (KA), a structural analogue of DA. The sensitivity of this assay could be enhanced by 1 order of magnitude by using a beta-galactosidase immunoconjugate with a fluorogenic substrate while preserving DA specificity. The calculated dissociation constant (K(D)) for the interaction of the antibodies with free DA was 5 x 10(-)(7) M (chromogenic assay) and 5 x 10(-)(8) M (fluorogenic assay). Using the optimized assay the limit of detection (LOD) and the limit of quantitation (LOQ) in the ELISA buffer were 1.4 and 3 ng/mL, respectively. Moreover this assay was found applicable for measuring DA levels in spiked mussel extracts pre-cleaned through a solid-phase extraction column, as a very good correlation (r(2) = 0.96) was observed between the actual amounts of DA added and amounts detected by ELISA. Thus, accurate determinations of DA in clean extracts could be achieved between 2 and 180 ng/mL in spiked samples which corresponds to 0.02-1.8 microg/g of original mussel tissue. Owing to the regulation limits of 20 microg DA/g of shellfish tissue, these extraction and assay procedures should provide a useful complement to the standard HPLC analytical technique currently employed in monitoring DA in shellfish tissue.

A minute amount (0.446 micromol) of cholesterol (Chol) was converted into an hemisuccinate derivative (Chol HS) using an excess of succinic anhydride. The optimal conditions for synthesis of Chol HS were explored by checkerboard experiments in which various succinic anhydride/Chol molar ratios ranging from 5:1 to 30:1 were assayed over a wide temperature range (50-85 degrees C) and for various incubation times (3-8 h). Total conversion was obtained at the higher reagent ratios, temperatures, and incubation times. Subsequently, this carboxylic derivative was first covalently linked to bovine serum albumin (BSA) then to various proteins (casein, ovalbumin, and hemocyanins) or to a synthetic homopolymer (poly-DL-Lysine) via a modified version of the mixed anhydride method of Erlanger, performed in a reversed micellar medium. The assessment of the number of haptenic groups per mole of BSA (epitope density) was achieved chromatographically by two methods according to a Chol standard curve established at 207 nm with linearity in the range 0-50 microg. These procedures involving an alkaline hydrolysis of a sample of either the conjugate (direct method) or the unreacted Chol HS (indirect method) yielded an acceptable level of agreement and concordant results in all cases. The influence of the activated hapten/BSA molar ratio on the coupling efficiency was investigated by the direct method within the range 10:1 to 250:1. Using the optimal conditions determined for Chol HS synthesis (a molar reagent ratio of 30:1 with incubation at 65 degrees C for 6 h) and for BSA haptenation (a 100-fold molar excess of activated hapten, with a carrier stock concentration of 5 mg/mL), epitope density of the conjugates lied between 23 and 27. By reacting the same amount of activated hapten ( approximately 216 microg) with identical amounts of various carriers (300 microg), conjugation efficiency was found similar on a microgram of Chol bound per milligram of carrier basis. This simple and reproducible conjugation and analysis procedures should provide a general method applicable to poorly available and weakly immunogenic haptens bearing hydroxyl groups such as polyether-type marine toxins.

The effectiveness of the carrier protein in eliciting antigen-specific antibodies was investigated. The effect of the carrier protein was independent of the conjugation chemistry involved. Keyhole limpet hemocyanin (KLH), purified protein derivative (PPD), and ovalbumin (OVA) were used as carrier proteins in the immunization of mice. Three antigens were studied: LY170881 (a small drug molecule), 4-[1'-cyanobenz(f)isoindolyl]butyric acid (CBI-butyric acid), and a seven residue peptide GPGRGPG (KLE1). The serum antibody response to the antigen or antigen:BSA conjugate was superior in the case where the PPD:antigen conjugates were used as the immunogen when compared to KLH and OVA. The specificity of the antibodies to the respective antigens vs cross-reactivity with the carrier protein was investigated. PPD-coupled antigen immunized mice generated a higher percentage of antigen-specific hybridomas compared to the other carrier proteins. These findings confirmed PPD as the best carrier molecule for the production of both polyclonal and monoclonal antibodies.

In this paper are reported the synthesis and characterization of three LSD derivatives. On the basis of several analytical characterization studies, the most stable derivative has been selected and a procedure to covalently link the derivative to polystyrene microparticles through a carrier protein has been developed. In addition, two new LSD immunogens have been synthesized and characterized, and from these immunogens antibodies that recognize not only LSD but also several major LSD metabolites have been generated. Using the selected derivative and antibody, a homogeneous microparticle-based immunoassay has been developed for the detection of LSD in human urine with the required sensitivity and specificity for an effective screening assay. The performance of this LSD OnLine assay has been evaluated using the criteria of precision, cross-reactivity, correlation to the Abuscreen LSD RIA and GC/MS/MS, assay specificity, and limit of detection.

A series of ruthenium polypyridyl complexes has been covalently bound to poly(L-lysine), bovine serum albumin, human serum albumin, ovalbumin, and immunoglobulin G using different binding methods. The conjugation ratios and the luminescence properties of the bioconjugates are reported. All conjugates show nonsingle-exponential decay curves. Quenching of the emission by oxygen has been studied.

An original procedure which preserves the structure of the sugar ring is described to link a plant hormone as N6-isopent-2-enyladenosine [( 9R]iP) onto a protein carrier to prepare a more specific immunogen. This cytokinin is bound to bovine serum albumin (BSA) and ovalbumin by a five-step procedure. These [9R]iP-protein conjugates have a maximal absorption at 269 nm and show molar ratios of hormone bound to proteins in the range of 12:1 and 18:1 for BSA and ovalbumin, respectively. Polyclonal antibodies were raised in rabbits against [9R]iP-BSA and were purified by affinity chromatography. Titers and specificity of the antisera and purified antibodies were determined by ELISA and RIA. These antibodies are highly specific for [9R]iP and do not cross-react with zeatin and ribosylzeatin. An immunoaffinity matrix was prepared with a capacity of 1 microgram of [9R]iP/mL of gel.

A simple two-step procedure is reported for the synthesis of a tert-butyl ester protected form of an EDTA-like bifunctional chelating agent. This reagent can be easily introduced on any available amino group during the assembly of peptides on solid-phase supports. Using the model tetradecapeptide OVA(323-336), we have introduced an EDTA group at the N-terminus of this T-cell epitope and confirmed that the EDTA group is present on the molecule, can chelate metals, and does not affect the biological activity of the peptide.