Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 5 de 5
Filtrar
1.
Glycoconj J ; 41(1): 47-55, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38224414

RESUMO

The Streptococcus pneumoniae bacteria has over 100 known serotypes that display a continuous change in prevalence by patients' age and geographical location and therefore necessitate continued efforts toward development of new vaccines with broader protection. Glycoconjugate vaccines have been instrumental in reducing global morbidity and mortality caused by Streptococcus pneumoniae infections. In these vaccines, the bacterial polysaccharide is conjugated to a carrier protein to enhance immunogenicity. To ensure well defined immunogenicity and stability of conjugated vaccines, reliable quantification of non-conjugated (free) polysaccharide is a critical, albeit challenging step during vaccine clinical dosing, release and stability monitoring. Multivalent preparations of Cross-reactive material 197 (CRM197)- conjugated pneumococcal polysaccharide materials often contain only nanogram levels of each individual free polysaccharide at final container concentrations. We have developed a novel method for the separation of free polysaccharides from conjugated material that requires no sample derivatization, employing instead an approach of quantitative immunoprecipitation of CRM197 with 3 different monoclonal antibodies and magnetic beads. A mix of antibodies against both linear and conformational epitopes enables successful removal of conjugates regardless of the protein folded state. The remaining free polysaccharide is subsequently measured in a serotype-specific ELISA.


Assuntos
Polissacarídeos Bacterianos , Streptococcus pneumoniae , Humanos , Sorogrupo , Vacinas Conjugadas , Vacinas Pneumocócicas , Anticorpos Antibacterianos
2.
Vaccines (Basel) ; 12(5)2024 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-38793720

RESUMO

Multivalent pneumococcal vaccines have been developed successfully to combat invasive pneumococcal diseases (IPD) and reduce the associated healthcare burden. These vaccines employ pneumococcal capsular polysaccharides (PnPs), either conjugated or unconjugated, as antigens to provide serotype-specific protection. Pneumococcal capsular polysaccharides used for vaccine often contain residual levels of cell wall polysaccharides (C-Ps), which can generate a non-serotype specific immune response and complicate the desired serotype-specific immunity. Therefore, the C-P level in a pneumococcal vaccine needs to be controlled in the vaccine process and the anti C-P responses need to be dialed out in clinical assays. Currently, two types of cell-wall polysaccharide structures have been identified: a mono-phosphocholine substituted cell-wall polysaccharide C-Ps1 and a di-phosphocholine substituted C-Ps2 structure. In our effort to develop a next-generation novel pneumococcal conjugate vaccine (PCV), we have generated a monoclonal antibody (mAb) specific to cell-wall polysaccharide C-Ps2 structure. An antibody-enhanced HPLC assay (AE-HPLC) has been established for serotype-specific quantification of pneumococcal polysaccharides in our lab. With the new anti C-Ps2 mAb, we herein extend the AE-HPLC assay to the quantification and identification of C-Ps2 species in pneumococcal polysaccharides used for vaccines.

3.
NPJ Vaccines ; 8(1): 2, 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36690697

RESUMO

Bacterial infection remains as one of the major healthcare issues, despite significant scientific and medical progress in this field. Infection by Streptococcus Pneumoniae (S. Pneumoniae) can cause pneumonia and other serious infectious diseases, such as bacteremia, sinusitis and meningitis. The pneumococcal capsular polysaccharides (CPS) that constitute the outermost layer of the bacterial cell are the main immunogens and protect the pathogen from host defense mechanisms. Over 90 pneumococcal CPS serotypes have been identified, among which more than 30 can cause invasive pneumococcal diseases that could lead to morbidity and mortality. Multivalent pneumococcal vaccines have been developed to prevent diseases caused by S. Pneumoniae. These vaccines employ either purified pneumococcal CPSs or protein conjugates of these CPSs to generate antigen-specific immune responses for patient protection. Serotype-specific quantitation of these polysaccharides (Ps) antigen species are required for vaccine clinical dosage, product release and quality control. Herein, we have developed an antibody-enhanced high-performance liquid chromatography (HPLC) assay for serotype-specific quantitation of the polysaccharide contents in multivalent pneumococcal conjugate vaccines (PCVs). A fluorescence-labeled multiplex assay format has also been developed. This work laid the foundation for a serotype-specific antigen assay format that could play an important role for future vaccine research and development.

4.
J Immunoassay Immunochem ; 32(4): 296-317, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21728822

RESUMO

Pharmacokinetic data derived from assays that accurately and precisely quantitate a therapeutic drug in circulation are critical to appropriately designing suitable dosing schedules. This manuscript describes the validation and implementation of methods to quantitate a therapeutic anti-human PCSK9 monoclonal antibody in rat and monkey sera as well as immunogenicity methods to screen the possible presence of rat and monkey antibodies directed against the antibody. As soluble, endogenous PCSK9 can interfere with a PCSK9-mediated capture step in ELISA, an indirect target-capture assay was used that potentially could capture free and target-engaged therapeutic mAb. Immunogenicity assays were based on a standard bridge ELISA using the therapeutic antibody for capture and detection. Both pharmacokinetic and immunogenicity assays were implemented in preclinical studies of the therapeutic antibody. The methods presented here may enable further pharmacokinetic studies.


Assuntos
Anticorpos Monoclonais/análise , Serina Endopeptidases/análise , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Ensaio de Imunoadsorção Enzimática/métodos , Haplorrinos , Humanos , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Serina Endopeptidases/imunologia , Serina Endopeptidases/farmacocinética , Solubilidade
5.
Int J Biol Sci ; 8(3): 310-27, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22355267

RESUMO

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticolesterolemiantes/uso terapêutico , LDL-Colesterol/sangue , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Imunização Passiva , Síndrome Metabólica/terapia , Pró-Proteína Convertases/antagonistas & inibidores , Pró-Proteína Convertases/imunologia , Serina Endopeptidases/imunologia , Sinvastatina/uso terapêutico , Proteínas de Ligação a Elemento Regulador de Esterol/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Afinidade de Anticorpos , Anticolesterolemiantes/administração & dosagem , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/genética , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Perfilação da Expressão Gênica , Células Hep G2/efeitos dos fármacos , Células Hep G2/metabolismo , Hepatócitos/metabolismo , Humanos , Metabolismo dos Lipídeos/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Macaca mulatta , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/genética , Camundongos , Camundongos Transgênicos , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/biossíntese , Pró-Proteína Convertases/genética , RNA Mensageiro/metabolismo , Receptores de LDL/biossíntese , Receptores de LDL/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Sinvastatina/administração & dosagem
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA