Silica particles incorporated into PLGA-based in situ-forming implants exploit the dual advantage of sustained release and particulate delivery.

Poly (lactic-co-glycolic acid) (PLGA) in situ-forming implants are well-established drug delivery systems for controlled drug release over weeks up to months. To prevent initial burst release, which is still a major issue associated with PLGA-based implants, drugs attached to particulate carriers have been encapsulated. Unfortunately, former studies only investigated the resulting release of the soluble drugs and hence missed the potential offered by particulate drug release. In this study, we developed a system capable of releasing functional drug-carrying particles over a prolonged time. First, we evaluated the feasibility of our approach by encapsulating silica particles of different sizes (500 nm and 1 µm) and surface properties (OH or NH2 groups) into in situ-forming PLGA implants. In this way, we achieved sustained release of particles over periods ranging from 30 to 70 days. OH-carrying particles were released much more quickly when compared to NH2-modified particles. We demonstrated that the underlying release mechanisms involve size-dependent diffusion and polymer-particle interactions. Second, particles that carried covalently-attached ovalbumin (OVA) on their surfaces were incorporated into the implant. We demonstrated that OVA was released in association with the particles as functional entities over a period of 30 days. The released particle-drug conjugates maintained their colloidal stability and were efficiently taken up by antigen presenting cells. This system consisting of particles incorporated into PLGA-based in situ-forming implants offers the dual advantage of sustained and particulate release of drugs as a functional unit and has potential for future use in many applications, particularly in single-dose vaccines.

Preparation and Characterization of an Oral Vaccine Formulation Using Electrosprayed Chitosan Microparticles.

Chitosan particles loaded with the antigen ovalbumin (OVA) and the adjuvant Quil-A were produced by electrospray, using mixtures of water/ethanol/acetic acid as a solvent. Three different chitosans designed as HMC+70, HMC+85, and HMC+90 (called as 705010, 855010, and 905010) were tested and its efficacy to be used in oral vaccine delivery applications was investigated. The morphology, size, and zeta potential of the produced particles were investigated, together with the encapsulation efficiency and release of OVA from the three chitosan formulations. Moreover, the mucoadhesion and cytotoxicity of the chitosan microparticles was examined. All the three formulations with OVA and Quil-A were in the micrometer size range and had a positive zeta potential between 46 and 75 mV. Furthermore, all the three formulations displayed encapsulation efficiencies above 80% and the release of OVA over a period of 80 h was observed to be between 38 and 47%. None of the developed formulations exhibited high mucoadhesive properties, either cytotoxicity. The formulation prepared with HMC+70, OVA, and Quil-A had the highest stability within 2 h in buffer solution, as measured by dynamic light scattering. The electrosprayed formulation consisting of HMC+70 with OVA and Quil-A showed to be the most promising as an oral vaccine system.

Lymph Node-Targeting Nanovaccine through Antigen-CpG Self-Assembly Potentiates Cytotoxic T Cell Activation.

Therapeutic vaccines that arouse the cytotoxic T cell immune response to reject infected cells have been investigated extensively for treating disease. Due to the large amounts of resident antigen-presenting cells (APCs) and T cells in lymph nodes, great efforts have been made to explore the strategy of targeting lymph nodes directly with nanovaccines to activate T cells. However, these nanovaccines still have several problems, such as a low loading efficiency and compromised activity of antigens and adjuvants derived from traditional complicated preparation. There are also safety concerns about materials synthesized without FDA approval. Herein, we construct an assembled nanoparticle composed of an antigen (ovalbumin, OVA) and adjuvant (CpG) to ensure its safety and high loading efficiency. The activity of both components was well preserved due to the mild self-assembly process. The small size, narrow distribution, negative charge, and good stability of the nanoparticle endow these nanovaccines with superior capacity for lymph node targeting. Correspondingly, the accumulation at lymph nodes can be improved by 10-fold. Subsequently, due to the sufficient APC internalization and maturation in lymph nodes, ~60% of T cells are stimulated to proliferate and over 70% of target cells are specifically killed. Based on the effective and quick cellular immune response, the assembled nanoparticles exhibit great potential as therapeutic vaccines.

Vaccine delivery by penetratin: mechanism of antigen presentation by dendritic cells.

Cell-penetrating peptides (CPP) or membrane-translocating peptides such as penetratin from Antennapedia homeodomain or TAT from human immunodeficiency virus are useful vectors for the delivery of protein antigens or their cytotoxic (Tc) or helper (Th) T cell epitopes to antigen-presenting cells. Mice immunized with CPP containing immunogens elicit antigen-specific Tc and/or Th responses and could be protected from tumor challenges. In the present paper, we investigate the mechanism of class I and class II antigen presentation of ovalbumin covalently linked to penetratin (AntpOVA) by bone marrow-derived dendritic cells with the use of biochemical inhibitors of various pathways of antigen processing and presentation. Results from our study suggested that uptake of AntpOVA is via a combination of energy-independent (membrane fusion) and energy-dependent pathways (endocytosis). Once internalized by either mechanism, multiple tap-dependent or independent antigen presentation pathways are accessed while not completely dependent on proteasomal processing but involving proteolytic trimming in the ER and Golgi compartments. Our study provides an understanding on the mechanism of antigen presentation mediated by CPP and leads to greater insights into future development of vaccine formulations.

Comparison between ovalbumin and ovalbumin peptide 323-339 responses in allergic mice: humoral and cellular aspects.

Ovalbumin (OVA) is widely used in allergy research. OVA peptide 323-339 has been reported to be responsible for 25-35% of isolated BALB/c mouse T-cell response to intact OVA. An investigation of whether OVA and OVA 323-339 molecules can induce equivalent in vivo and in vitro immune responses was conducted. Eight-week-old BALB/c mice were randomly divided into three groups: OVA, OVA 323-339 and saline. On days 0, 7, 14, mice were intraperitoneally injected with 25 microg OVA or OVA 323-339 absorbed on 300 microg Alum, or saline; on days 21-23, all groups were challenged intranasally with either 20 microl of 1% OVA, 1% OVA 323-339 or saline. On day 28, after killing, splenocytes were isolated and cultured under the stimulus of each allergen or medium. Evaluated by hematoxylin/eosin and major basic protein immunohistochemical stainings, OVA and OVA 323-339 induced similar lung inflammation. Interestingly, significant serum total IgE and OVA-specific IgE were observed in OVA mice when compared to saline control. OVA 323-339 mice showed higher serum OVA-specific IgE, OVA 323-339-specific IgE, IL-4 and lower IFN-gamma similar to OVA mice. The proliferative response to OVA was found in cultured splenocytes of both OVA and OVA 323-339 mice, while the similar proliferative response to OVA 323-339 was only observed in the splenocytes of OVA 323-339-sensitized and challenged mice. Although OVA 323-339 induced a Th2-like response in the mouse model as did OVA, OVA 323-339 has clearly limited immunogenic potency to activate OVA-sensitized and challenged mice splenocytes, unlike OVA.

Quantification of nitrotyrosine in nitrated proteins.

For kinetic studies of protein nitration reactions, we have developed a method for the quantification of nitrotyrosine residues in protein molecules by liquid chromatography coupled to a diode array detector of ultraviolet-visible absorption. Nitrated bovine serum albumin (BSA) and nitrated ovalbumin (OVA) were synthesized and used as standards for the determination of the protein nitration degree (ND), which is defined as the average number of nitrotyrosine residues divided by the total number of tyrosine residues in a protein molecule. The obtained calibration curves of the ratio of chromatographic peak areas of absorbance at 357 and at 280 nm vs. nitration degree are nearly the same for BSA and OVA (relative deviations <5%). They are near-linear at low ND (< 0.1) and can be described by a second-order polynomial fit up to ND=0.5 (R2>0.99). A change of chromatographic column led to changes in absolute peak areas but not in the peak area ratios and related calibration functions, which confirms the robustness of the analytical method. First results of laboratory experiments confirm that the method is applicable for the investigation of the reaction kinetics of protein nitration. The main advantage over alternative methods is that nitration degrees can be efficiently determined without hydrolysis or digestion of the investigated protein molecules.

Determination of fluoranthene by antigen-coated indirect competitive real-time immuno-PCR assay.

A reliable and sensitive antigen-coated indirect competitive real-time immuno-PCR (RT-IPCR) assay was developed for the determination of fluoranthene (FL). 9-Fluoranthenebutanoic acid, gamma-oxo-(FLA) was synthesized as the hapten for FL. Mixed anhydride reaction (MAR) was used to couple the FLA to ovalbumin (OVA) to form artificial coating antigen. Active ester method (AEM) was used to couple the FLA to bovine serum albumin (BSA) to form artificial immune antigen. Male New Zealand white rabbits were immunized with immune antigen to obtain polyclonal antibodies (pAbs), with which, a novel RT-IPCR assay for determination of FL was described. Under best conditions, FL can be determined in a concentration range between 10 fg mL(-1) and 100 pg mL(-1) with a detection limit of 5 fg mL(-1). The cross-reactivities of the anti-FL antibody to seven structurally related compounds were below 11%. Some environmental water samples were analyzed with satisfactory results, which shows a good accuracy and suitability to analyze FL in environmental water. Recovery was lower or higher with agitation but would still be acceptable for use in an on-site field test to provide rapid, semi-quantitative, and reliable test results for making environmental decisions.

Determination of anthracene by real-time immuno-polymerase chain reaction assay.

A reliable and sensitive competitive real-time immuno-PCR (RT-IPCR) assay for the determination of anthracene (AN) was developed. 9-Anthracenebutanoic acid, gamma-oxo-(ANA) was synthesized as the hapten of AN. Mixed anhydride reaction (MAR) was used to couple the ANA to ovalbumin (OVA) to form artificial coating antigen. Active ester method (AEM) was used to couple the ANA to bovine serum albumin (BSA) to form artificial immune antigen. Male New Zealand white rabbits were immunized with immune antigen to obtain polyclonal antibodies (pAbs), with which, a novel RT-IPCR assay for determination of AN was described. Under optimized assay conditions, AN can be determined in the concentration range from 1 fgmL(-1) to 100 pgmL(-1) with a detection limit of 0.5 fgmL(-1). The cross-reactivities of the anti-AN antibody to seven structurally related compounds were below 15%. Environmental water samples were successfully analyzed, showing a good accuracy and suitability to analyze AN in field samples. Recovery was between 93.3% and 120.0% and would be acceptable for use in an on-site field test to provide rapid, semiquantitative, and reliable test results for making environmental decisions.

The adjuvanticity of a mannosylated antigen reveals TLR4 functionality essential for subset specialization and functional maturation of mouse dendritic cells.

The evidence that dendritic cell (DC) subsets produce differential cytokines in response to specific TLR stimulation is robust. However, the role of TLR stimulation in Ag presentation and phenotypic maturation among DC subsets is not clear. Through the adjuvanticity of a novel mannosylated Ag, mannosylated dendrimer OVA (MDO), as a pathogen-associated molecular pattern Ag, we characterized the functionality of GM-CSF/IL-4-cultured bone marrow DC and Flt3 ligand (Flt3-L) DC subsets by Ag presentation and maturation assays. It was demonstrated that both bone marrow DCs and Flt3-L DCs bound, processed, and presented MDO effectively. However, while Flt3-L CD24(high) (conventional CD8(+) equivalent) and CD11b(high) (CD8(-) equivalent) DCs were adept at MDO processing by MHC class I and II pathways, respectively, CD45RA(+) plasmacytoid DCs presented MDO poorly to T cells. Successful MDO presentation was largely dependent on competent TLR4 for Ag localization and morphological/phenotypic maturation of DC subsets, despite the indirect interaction of MDO with TLR4. Furthermore, Toll/IL-1 receptor-domain-containing adaptor-inducing IFN-beta, but not MyD88, as a TLR4 signaling modulator was indispensable for MDO-induced DC maturation and Ag presentation. Taken together, our findings suggest that DC subsets differentially respond to a pathogen-associated molecular pattern-associated Ag depending on the intrinsic programming and TLRs expressed. Optimal functionality of DC subsets in Ag presentation necessitates concomitant TLR signaling critical for efficient Ag localization and processing.

Immunization with mannosylated peptide induces poor T cell effector functions despite enhanced antigen presentation.

In this study, we investigated the development of T cell responses in mice after administration of a mannosylated ovalbumin peptide (M-OVA(323-339)). Immunization with M-OVA(323-339) in complete adjuvant resulted in enhanced antigen presentation in draining lymph nodes. Monitoring the fate of CFSE-labeled ovalbumin peptide-specific TCR transgenic CD4(+) T cells revealed that immunization with M-OVA(323-339) induced normal clonal expansion, recirculation and CD62L expression of antigen-specific T cells in vivo. However, these T cells developed only poor effector functions, reflected by minimal IFN-gamma production, low IgG2a levels in serum and poor peptide-specific delayed-type hypersensitivity (DTH) responses. This diminished inflammatory response was associated with decreased infiltration of T cell blasts and macrophages. Importantly, also mice with functional effector T cells did not mount a robust DTH response after a challenge with M-OVA(323-339) in the ear, although their T cells responded normally to M-OVA(323-339) in vitro. In conclusion, mannosylated peptide induces proliferation of T cells with impaired T(h)1 cell effector functions and additionally abrogates the activity of pre-existing effector T cells.

Effect of oral administration of CpG ODN-OVA on WBB6F1-W/Wv mice.

BACKGROUND: We have already reported that antigen-specific IgG1 antibody production in WBB6F1-W/Wv (W/Wv) mice after oral administration of ovalbumin (OVA) was extremely high. Active systemic anaphylaxis (ASA) was induced in these mice after intraperitoneal (i.p.) administration of OVA, and Th2-dominant helper T-cell activation occurred. In this study, we examined the effect of CpG oligodeoxynucleotide (ODN) conjugation of OVA on oral immunization of W/Wv mice. METHODS: W/Wv mice were sensitized by administration of 0.1 mg OVA or CpG ODN-OVA by gavage every day for 4 weeks, and the serum titers of OVA-specific IgG1, IgE, and IgG2a antibody were determined. ASA was induced by i.p. injection of OVA, and the changes in body temperature were monitored. In vitro production of Th1- and Th2- type cytokines by splenocytes re-stimulated with antigen was also measured. RESULTS: The antigen-specific IgG1 antibody titer in the CpG ODN-OVA-sensitized W/Wv mice was lower than in the OVA-sensitized group, but the IgG2a titer was higher. ASA was not induced by i.p. OVA challenge. There were significant increases in the production of Th1-type cytokine (IFN-gamma) by splenocytes in the CpG ODN-OVA-sensitized mice, but the Th2-type cytokine (IL-4) level in the splenocyte culture medium was lower. CONCLUSIONS: These results indicated that oral administration of CpG ODN-OVA conjugate significantly induced antigen-specific Th1 responses and reduced Th2 responses (allergic reactions) on re-stimulation. These findings suggest that CpG ODN-antigen conjugate may be useful as an oral vaccine.

Chemical processing as a tool to generate ovalbumin variants with changed stability.

Processing of ovalbumin may result in proteins that differ more than 23 degrees C in denaturation temperature while the structural fold is not significantly affected. This is achieved by 1) conversion of positive residues into negative ones (succinylation); 2) elimination of negative charges (methylation); 3) reducing the proteins hydrophobic exposure (glycosylation); 4) increasing the hydrophobic exposure (lipophilization); or by 5) processing under alkaline conditions and elevated temperature (S-ovalbumin). The effect on the structural fold was investigated using a variety of biochemical and spectroscopic tools. The consequences of the modification on the thermodynamics of the protein was studied using differential scanning calorimetry and by monitoring the tryptophan fluorescence or ellipticity at 222 nm of protein samples dissolved in different concentrations of guanidine-HCl. The impact of the modification on the denaturation temperature scales for all types of modifications with a free energy change of about 1 kJ per mol ovalbumin per Kelvin (or 0.0026 kJ per mol residue per K). The nature of the covalently coupled moiety determines the impact of the modification on the protein thermodynamics. It is suggested that especially for lipophilized protein the water-binding properties are substantially lowered. Processing of globular proteins in a controlled manner offers great opportunities to control a desired functionality, for example, as texturizer in food or medical applications.

Mimicry of native peptide antigens by the corresponding retro-inverso analogs is dependent on their intrinsic structure and interaction propensities.

Retro-inverso (ri) analogs of model T cell and B cell epitopes were predictively designed as mimics and then assayed for activity to understand the basis of functional ri-antigenic peptide mimicry. ri versions of two MHC class I binding peptide epitopes, one from a vesicular stomatitis virus glycoprotein (VSV(p)) and another from OVA (OVAp), exhibit structural as well as functional mimicry of their native counterparts. The two ri peptides exhibit conformational plasticity and they bind to MHC class I (H-2K(b)) similar to their native counterparts both in silico and in vivo. In fact, ri-OVAp is also presented to an OVAp-specific T cell line in a mode similar to native OVAp. In contrast, the ri version of an immunodominant B cell peptide epitope from a hepatitis B virus protein, PS1, exhibits no structural or functional correlation with its native counterpart. PS1 and its ri analog do not exhibit similar conformational propensities. PS1 is less flexible relative to its ri version. These observed structure-function relationships of the ri-peptide epitopes are consistent with the differences in recognition properties between peptide-MHC vs peptide-Ab binding where, while the recognition of the epitope by MHC is pattern based, the exquisitely specific recognition of Ag by Ab arises from the high complementarity between the Ag and the binding site of the Ab. It is evident that the correlation of conformational and interaction propensities of native L-peptides and their ri counterparts depends both on their inherent structural properties and on their mode of recognition.

Utility of azapeptides as major histocompatibility complex class II protein ligands for T-cell activation.

Major histocompatibility complex class II (MHC II) protein binding and antigen specific activation of CD4+ "helper" T cells are demonstrated with peptides composed of the antigenic hen egg ovalbumin 325-339 peptide (OVA) substituted with azaamino acids. AzaAla and azaGly substitutions were made at 10 sequential peptide positions (326Ala-335Asn) that lie in the binding groove. The peptide positions substituted with azaamino acids encompass almost the entire binding groove, including positions where the identity of the amino acid side chain is known to have the most significant effect on MHC binding and the least effect on T-cell recognition. In addition, the T-cell contact 333Glu was substituted with azaGlu to generate a partial agonist ligand for the 3DO-54.8 T-cell hybridoma. Binding to MHC II protein was assayed by measuring the kinetic stability of complexes formed between detergent-solubilized MHC II I-A(d) protein and fluorescein-labeled OVA peptides using a fluorescence-HPLC assay. T-cell activation was also evaluated for aza-substituted peptides with azaamino acid substitutions at the peptide positions known to interact with the MHC II protein. All aza-substituted peptides showed detectable MHC binding, and some were found to show T-cell activation potency equal to the native peptide. Several of these were also found to be weak or partial agonists. Our results demonstrate that azaamino acids substituted into an antigenic peptide cause a subtle, global effect on peptide conformation that can be used to design altered peptide ligands (APL) as T-cell partial agonists. These may have potential as T-cell epitopes for synthetic vaccines and therapeutic agents for autoimmune diseases.

Designing potent derivatives of ovokinin(2-7), an anti-hypertensive peptide derived from ovalbumin.

We obtained a potent anti-hypertensive peptide, RPFHPF, by replacing the amino acid residues of ovokinin(2-7) (RADHPF), an orally active anti-hypertensive peptide derived from ovalbumin. After intravenous administration in anesthetized Wistar rats, the designed peptide [Pro2, Phe3]-ovokinin(2-7) had a long-lasting hypotensive activity at a dose of 10 mg/kg, while that of ovokinin(2-7) was only transient even at a dose of 100 mg/kg. After oral administration in conscious spontaneously hypertensive rats (SHRs), [Pro2, Phe3]-ovokinin(2-7) significantly lowered the systolic blood pressure in a dose-dependent manner. It is noteworthy that the minimum effective dose of [Pro2, Phe3]-ovokinin(2-7) was 0.3 mg/kg, about one-thirtieth of that of ovokinin(2-7). On the other hand, orally administered [Pro2, Phe3]-ovokinin(2-7) did not show any significant hypotensive effect in normotensive Wistar-Kyoto rats (WKYs) even at a dose of 3 mg/kg. Taken together, [Pro2, Phe3]-ovokinin(2-7) proved to be an ideal, potent anti-hypertensive peptide with little effect on normal blood pressure when given orally.

Modulation of Th2 responses by peptide analogues in a murine model of allergic asthma: amelioration or deterioration of the disease process depends on the Th1 or Th2 skewing characteristics of the therapeutic peptide.

Allergen-specific CD4+ Th2 cells play an important role in the immunological processes of allergic asthma. Previously we have shown that, by using the immunodominant epitope OVA323-339, peptide immunotherapy in a murine model of OVA induced allergic asthma, stimulated OVA-specific Th2 cells, and deteriorated airway hyperresponsiveness and eosinophilia. In the present study, we defined four modulatory peptide analogues of OVA323-339 with comparable MHC class II binding affinity. These peptide analogues were used for immunotherapy by s.c. injection in OVA-sensitized mice before OVA challenge. Compared with vehicle-treated mice, treatment with the Th2-skewing wild-type peptide and a Th2-skewing partial agonistic peptide (335N-A) dramatically increased airway eosinophilia upon OVA challenge. In contrast, treatment with a Th1-skewing peptide analogue (336E-A) resulted in a significant decrease in airway eosinophilia and OVA-specific IL-4 and IL-5 production. Our data show for the first time that a Th1-skewing peptide analogue of a dominant allergen epitope can modulate allergen-specific Th2 effector cells in an allergic response in vivo. Furthermore, these data suggest that the use of Th1-skewing peptides instead of wild-type peptide may improve peptide immunotherapy and may contribute to the development of a successful and safe immunotherapy for allergic patients.

Rapid synthesis and introduction of a protected EDTA-like group during the solid-phase assembly of peptides.

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.

Functional protein-polysaccharide conjugate prepared by controlled dry-heating of ovalbumin-dextran mixtures.

A functional ovalbumin-dextran conjugate was prepared by dry-heated storage at 60 degrees C and 65% relative humidity for 3 weeks. The emulsifying properties of the ovalbumin-dextran conjugate were about three times higher than those of an ovalbumin-glucose conjugate. SDS-electrophoresis patterns showed that the ovalbumin-dextran conjugate obtained by dry-heating was not as polydispersed as that obtained by cyanogen bromide-activated dextran. The average molecular weight of the ovalbumin-dextran conjugate was about 200,000. The excellent emulsifying properties of ovalbumin-dextran conjugate were maintained even at pH 3 and were further improved at pH 10. In addition, the emulsifying properties of the ovalbumin-dextran conjugate were greatly enhanced by preheating the conjugate at 100 degrees C. Thus, it is suggested that an ovalbumin-dextran conjugate prepared by controlled dry-heating can be used as a macromolecular emulsifier for food applications.

I-Ad-binding peptides derived from unrelated protein antigens share a common structural motif.

Purified Ia molecules can specifically bind many unrelated peptide Ag, and such binding appears to be a necessary, albeit not sufficient, prerequisite for the immunogenicity of the proteins from which such peptides are derived. We have recently analyzed the affect of single amino acid substitutions on the I-Ad binding of the immunogenic peptide OVA 323-339. The results obtained demonstrated the very permissive nature of Ag-Ia interaction. We also showed that unrelated peptides that are good I-Ad binders share a common structural motif and speculated that recognition of such motifs could represent a mechanism to achieve a very permissive type of interaction that yet retained some degree of specificity. In the present set of experiments we analyzed the I-Ad binding pattern of a series of overlapping peptides derived from sperm whale myoglobin (residues 102-125) and influenza hemagglutinin (residues 121-146) to determine whether the peptide regions predicted on the basis of structural similarity to be involved in I-Ad binding were in fact involved. In both cases, the I-Ad-interacting determinants were found to contain the sequence motif postulated to be important for I-Ad binding. These data support the hypothesis that I-Ad molecules recognize a large library of Ag by virtue of common structural motifs present in peptides derived from phylogenetically unrelated proteins.

Solid-phase peptide synthesis under continuous-flow conditions.

A system is described for solid-phase synthesis of peptides under continuous-flow conditions with liquid chromatographic equipment, conventional polystyrene supports, and well-defined chemistry. The model tetrapeptide Leu-Ala-Gly-Val was assembled in 99.3% purity in about 4 hr on microporous copoly(styrene-1% divinylbenzene). During coupling, the preformed symmetric anhydrides were conserved by being recycled. Relative yields of the peptide products were determined quantitatively in 20 min by reverse-phase high-pressure liquid chromatography. This rapid assay system was used to examine the influence on product yields of (i) the time and number of couplings per cycle, (ii) microporous versus macroporous polystyrene, and (iii) tert-butoxycarbonyl (Boc) group versus 9-fluorenylmethoxycarbonyl for amine protection. Use of microporous polystyrene and two 30-min couplings of Boc-amino acids per cycle gave the best results. This continuous-flow system provides a rapid and efficient approach to solid-phase peptide synthesis. A 17-residue peptide from chicken ovalbumin was obtained in similar purity and yield from a discontinuous synthesis and from a continuous-flow synthesis.