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Toward a prostate specific antigen-based prostate cancer diagnostic assay: preparation of keyhole limpet hemocyanin-conjugated normal and transformed prostate specific antigen fragments.

ABSTRACT: Prostate specific antigen (PSA) molecules secreted by cancerous and normal prostate cells differ in their N-linked glycan composition, while the peptide backbone appears to be conserved. Antibodies selectively recognizing such differentially glycosylated PSA structures could form a basis for a new diagnostic assay for prostate cancer. Twenty-amino acid PSA fragments carrying di-, tri-, and tetrabranched complex-type glycans were prepared by total synthesis and conjugated to maleimide-modified keyhole limpet hemocyanin (KLH) carrier protein through backbone Cys residues. These glycopeptide/KLH conjugates were then used for antibody generation.

SUBMITTER: Dudkin VY

PROVIDER: S-EPMC2646745 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Toward a prostate specific antigen-based prostate cancer diagnostic assay: preparation of keyhole limpet hemocyanin-conjugated normal and transformed prostate specific antigen fragments.

Dudkin Vadim Y VY Miller Justin S JS Dudkina Anna S AS Antczak Christophe C Scheinberg David A DA Danishefsky Samuel J SJ

Journal of the American Chemical Society 20080918 41

Prostate specific antigen (PSA) molecules secreted by cancerous and normal prostate cells differ in their N-linked glycan composition, while the peptide backbone appears to be conserved. Antibodies selectively recognizing such differentially glycosylated PSA structures could form a basis for a new diagnostic assay for prostate cancer. Twenty-amino acid PSA fragments carrying di-, tri-, and tetrabranched complex-type glycans were prepared by total synthesis and conjugated to maleimide-modified ke ...[more]

PMID: 18798614

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Cryo-electron tomography of keyhole limpet hemocyanin

Project description: Not available

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Keyhole Limpet Hemocyanin-Conjugated Peptides from Hepatitis C Virus Glycoproteins Elicit Neutralizing Antibodies in BALB/c Mice.

Project description:Currently, no vaccine to prevent hepatitis C virus (HCV) infection is available. A major challenge in developing an HCV vaccine is the high diversity of HCV sequences. The purpose of immunization with viral glycoproteins is to induce a potent and long-lasting cellular and humoral immune response. However, this strategy only achieves limited protection, and antigen selection plays a crucial role in vaccine design. In this study, we investigated the humoral immune responses induced by intraperitoneal injection of keyhole limpet hemocyanin conjugated with 4 highly conserved peptides, including amino acids [aa]317-325 from E1 and aa418-429, aa502-518, and aa685-693 from E2, or 3 peptides from hypervariable region 1 (HVR1) of E2, including the N terminus of HVR1 (N-HVR1, aa384-396), C terminus of HVR1 (C-HVR1, aa397-410), and HVR1 in BALB/c mice. The neutralizing activity against HCV genotypes 1-6 was assessed using the cell culture HCV (HCVcc) system. The results showed that the 4 conserved peptides efficiently induced antibodies with potent neutralizing activity against 3 or 4 genotypes. Antibodies induced by aa685-693 conferred potent protection (>50%) against genotypes 2, 4, and 5. Peptide N-HVR1 elicited antibodies with the most potent neutralization activities against 3 HCV genotypes: TNcc(1a), S52(3a), and ED43(4a). These findings suggested that peptides within HCV glycoproteins could serve as potent immunogens for vaccine design and development.

| S-EPMC7834783 | biostudies-literature

Monitoring of dynamic changes in Keyhole Limpet Hemocyanin (KLH)-specific B cells in KLH-vaccinated cancer patients.

Project description:Keyhole limpet hemocyanin (KLH) is used as an immunogenic neo-antigen for various clinical applications and during vaccine development. For advanced monitoring of KLH-based interventions, we developed a flow cytometry-based assay for the ex vivo detection, phenotyping and isolation of KLH-specific B cells. As proof-of-principle, we analyzed 10 melanoma patients exposed to KLH during anti-cancer dendritic cell vaccination. Our assay demonstrated sensitive and specific detection of KLH-specific B cells in peripheral blood and KLH-specific B cell frequencies strongly correlated with anti-KLH serum antibody titers. Profiling of B cell subsets over the vaccination course revealed that KLH-specific B cells matured from naïve to class-switched memory B cells, confirming the prototypic B cell response to a neo-antigen. We conclude that flow-cytometric detection and in-depth phenotyping of KLH-specific B cells is specific, sensitive, and scalable. Our findings provide novel opportunities to monitor KLH-specific immune responses and serve as a blueprint for the development of new flow-cytometric protocols.

| S-EPMC5361210 | biostudies-literature

Evaluation of human antibody responses to keyhole limpet hemocyanin on a carbohydrate microarray.

Project description:PurposeKeyhole limpet hemocyanin (KLH) is used as a vaccine adjuvant, as a carrier protein for small haptens, and as a treatment for bladder cancer. Immunization with KLH produces antibodies to tumor-associated carbohydrate antigens (TACAs) in animals, and these antibodies have been postulated as the basis of efficacy for bladder cancer treatment. The purpose of this study was to evaluate antibody responses to KLH in humans.Experimental designA carbohydrate microarray was used to profile antibody responses in 14 individuals immunized with KLH plus alum adjuvant.ResultsEight out of fourteen individuals produced antibodies to at least one TACA. Increases to Lewis X, Lewis Y, GA1di, GM3, and sialyl Lewis A were observed in certain individuals, but, in general, antibody profiles were highly variable. Pre-immunization antibody levels to a subset of array antigens had a statistically significant correlation with the magnitude of the antibody response to KLH.Conclusions and clinical relevanceAntibodies to TACAs can be produced in humans, but antibody profiles differ considerably from person to person, which may contribute to variable clinical responses with KLH. Pre-treatment antibody levels to certain antigens may be useful for predicting which patients will respond favorably to KLH.

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Cryo iDPC-STEM single particle analysis of keyhole limpet hemocyanin

Project description: Not available

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Localization of keyhole limpet hemocyanin-like immunoreactivity in the nervous system of Biomphalaria alexandrina.

Project description:Recent years have led to increased effort to describe and understand the peripheral nervous system and its influence on central mechanisms and behavior in gastropod molluscs. This study revealed that an antibody raised against keyhole limpet hemocyanin (KLH) cross-reacts with an antigen(s) found extensively in both the central and the peripheral nervous systems of Biomphalaria alexandrina. The results revealed KLH-like immunoreactive (LIR) neurons in the cerebral, pedal, buccal, left pleural, right parietal, and visceral ganglion within the CNS with fibers projecting throughout all the peripheral nerves. Numerous KLH-LIR peripheral sensory neurons located in the foot, lips, tentacles, mantle, esophagus, and penis exhibited a bipolar morphology with long tortuous dendrites. KLH-LIR cells were also present in the eye and statocyst, thus suggesting the labeling of multiple sensory modalities/cell types. KLH-LIR cells did not co-localize with tyrosine hydroxylase (TH)-LIR cells, which have previously been described in this and other gastropods. The results thus provide descriptions of thousands of peripheral sensory neurons, not previously described in detail. Future research should seek to pair sensory modalities with peripheral cell type and attempt to further elucidate the nature of KLH-like reactivity. These findings also emphasize the need for caution when analyzing results obtained through use of antibodies raised against haptens conjugated to carrier proteins, suggesting the need for stringent controls to help limit potential confounds caused by cross-reactivity. In addition, this study is the first to describe neuronal cross-reactivity with KLH in Biomphalaria, which could provide a substrate for host-parasite interactions with a parasitic trematode, Schistosoma.

| S-EPMC10401489 | biostudies-literature

Genetic and Non-Genetic Inheritance of Natural Antibodies Binding Keyhole Limpet Hemocyanin in a Purebred Layer Chicken Line.

Project description:Natural antibodies (NAb) are defined as antibodies present in individuals without known antigenic challenge. Levels of NAb binding keyhole limpet hemocyanin (KLH) in chickens were earlier shown to be heritable, and to be associated with survival. Selective breeding may thus provide a strategy to improve natural disease resistance. We phenotyped 3,689 white purebred laying chickens for KLH binding NAb of different isotypes around 16 weeks of age. Heritabilities of 0.12 for the titers of total antibodies (IgT), 0.14 for IgM, 0.10 for IgA, and 0.07 for IgG were estimated. We also estimated high, positive genetic, and moderate to high, positive phenotypic correlations of IgT, IgM, IgA, and IgG, suggesting that selective breeding for NAb can be done on all antibody isotypes simultaneously. In addition, a relatively substantial non-genetic maternal environmental effect of 0.06 was detected for IgM, which may reflect a transgenerational effect. This suggests that not only the genes of the mother, but also the maternal environment affects the immune system of the offspring. Breaking strength and early eggshell whiteness of the mother's eggs were predictive for IgM levels in the offspring, and partly explained the observed maternal environmental effects. The present results confirm that NAb are heritable, however maternal effects should be taken into account.

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Cardiovascular exercise intervention improves the primary antibody response to keyhole limpet hemocyanin (KLH) in previously sedentary older adults.

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| S-EPMC2576741 | biostudies-literature

Novel Polyclonal Antibody Raised against Tetrodotoxin Using Its Haptenic Antigen Prepared from 4,9-anhydrotetrodotoxin Reacted with 1,2-Ethaneditiol and Further Reacted with Keyhole Limpet Hemocyanin.

Project description:A novel polyclonal antibody against tetrodotoxin (TTX) was raised using its haptenic antigen, where 4,9-anhydroTTX was reacted with 1,2-ethanedithiol and this derivative was further reacted with keyhole limpet hemocyanin (KLH). This newly designed antigen (KLH-TTX) was inoculated into rabbits, resulting in the production of the specific polyclonal antibody, which reacted well with TTX and its analogs, 4-epiTTX, 11-oxoTTX and 5,6,11-trideoxyTTX, except for 4,9-anhydroTTX. The enzyme-linked immunosorbent assay (ELISA) system using this specific antibody was also developed in the present study. This newly developed polyclonal antibody with analytical procedures using direct one-step ELISA is useful to detect TTX and its analogs in toxic organisms and also disclose the mechanisms involved in their metabolic pathways and accumulation of TTX.

| S-EPMC6832204 | biostudies-literature

Phase III multicenter clinical trial of the sialyl-TN (STn)-keyhole limpet hemocyanin (KLH) vaccine for metastatic breast cancer.

Project description:PurposeThis double-blind, randomized, phase III clinical trial evaluated time to progression (TTP) and overall survival in women with metastatic breast cancer (MBC) who received sialyl-TN (STn) keyhole limpet hemocyanin (KLH) vaccine. Secondary endpoints included vaccine safety and immune response.Experimental designThe study population consisted of 1,028 women with MBC across 126 centers who had previously received chemotherapy and had had either a complete or a partial response or no disease progression. All women received one-time i.v. cyclophosphamide (300 mg/m(2)) 3 days before s.c. injection of 100 ?g STn-KLH plus adjuvant (treatment group) or 100 ?g KLH plus adjuvant (control group) at weeks 0, 2, 5, and 9. Subsequently, STn-KLH without adjuvant or KLH without adjuvant was then administered monthly for 4 months, and then quarterly until disease progression, without cyclophosphamide.ResultsSTn-KLH vaccine was well tolerated; patients had mild to moderate injection-site reactions and reversible flu-like symptoms. Week-12 antibody testing revealed high specific IgG titers and a high rate of IgM-to-IgG seroconversion; the median IgG titers in STn-KLH recipients were 320 (anti-ovine submaxillary mucin) and 20,480 (anti-STn), with no detectable antimucin antibodies in the control group. The TTP was 3.4 months in the treatment group and 3.0 months in the control group. The median survival times were 23.1 months and 22.3 months, respectively.ConclusionsAlthough STn-KLH was well tolerated in this largest to date metastatic breast cancer vaccine trial, no overall benefit in TTP or survival was observed. Lessons were learned for future vaccine study designs.

| S-EPMC3228158 | biostudies-literature

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