Stabilization of Dichloromethane-Induced Protein Denaturation During Microencapsulation

Raghuvanshi, Rajeev Singh; Goyal, Sandhya; Singh, Om; Panda, Amulya K.

doi:10.3109/10837459809028504

Cited by 61 publications

(33 citation statements)

References 26 publications

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“…2204398), polyvinyl alcohol-glutaraldehyde (2-4), and PVP (5,20) have been shown to have significant blocking abilities and do not interfere with specific binding. In addition, PVA has been shown to stabilize the immunoreactive activities of proteins (8,32) and improve antibody adsorption. Rodda and Yamazaki (33) demonstrated a significant improvement in the specificity of the secondary antibody reaction using PVA with rabbit IgG compared with the specificity obtained with traditional blocking agents (e.g., BSA and milk).…”

Section: Discussionmentioning

confidence: 99%

Enhanced Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Virus-Like Particles of Human Papillomavirus

Studentsov¹,

Schiffman

Strickler³

et al. 2002

J Clin Microbiol

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Measurement of antibodies to human papillomavirus (HPV) is complicated by many factors. Although enzyme-linked immunosorbent assays (ELISAs) that use virus-like particles (VLPs) have proved useful, the assays have, in general, had moderate sensitivities and low signal-to-noise ratios. To enhance the performance of the assay, a systematic investigation was undertaken to examine key variables used in ELISAs for the detection of antibodies to VLPs of HPV. Incorporation of two vinyl polymers, polyvinyl alcohol (molecular weight, 50,000) (PVA-50) and polyvinylpyrrolidone (molecular weight, 360,000) (PVP-360), was found to increase the sensitivity as well as the specificity of the assay for the detection of antibodies to VLPs of HPV. In particular, the addition of PVA-50 to the blocking solution reduced the amount of nonspecific binding of antibodies to VLPs and the microplate surface, whereas the addition of PVP-360 increased the sensitivity of antibody detection. The new ELISA demonstrated increased sensitivity and specificity for the detection of cervical HPV type 16 infection compared to those of a prototype assay with coded clinical serum samples from women with known cervicovaginal HPV infection status. It is anticipated that the enhanced ELISA conditions will have wide application to a large number of clinical diagnostic assays.Human papillomavirus (HPV) infection of the cervicovaginal region is the most common sexually transmitted disease in young adults and adolescents (6, 10). The association of HPV with preinvasive and invasive cervical cancer (26) makes genital HPV infection of particular medical importance. Initial attempts to characterize the humoral response to HPV virions were made as early as 1965 (1). However, research in this area has been impeded by an inability to grow large amounts of HPV virions in the laboratory and the poor sensitivities and specificities of antibody assays based on denatured recombinant HPV proteins reported to date.A major innovation in HPV serology was the use of recombinant DNA technology to produce HPV capsid proteins that assembled into virus-like particles (VLPs) (18,29,37,42). These methods produce conformationally intact HPV capsids in adequate amounts for use in enzyme-linked immunosorbent assays (ELISAs) (12, 28). Of major significance was the observation that antibodies to conformational epitopes on these synthetically produced HPV VLPs develop in response to typespecific infections and that such antibodies were neutralizing in cell culture systems and in an animal model (13-15, 34, 35, 40, 41).Clinical applications of assays for HPV antibody detection, however, have been limited due to the lack of highly sensitive and reproducible assay systems. The majority of serum specimens have low titers of antibodies to VLPs of HPV, perhaps due to the immune isolation of the cervix and/or the fact that infection with HPV has no viremic phase. In addition, low titers of antibodies to conformational epitopes of HPV make background reactivity a major barrier to the development...

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Section: Discussionmentioning

confidence: 99%

Enhanced Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Virus-Like Particles of Human Papillomavirus

Studentsov¹,

Schiffman

Strickler³

et al. 2002

J Clin Microbiol

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“…Thus, it is possible that in the applied method, the polar wax compounds involved in the adhesion of the conidia are removed from the epicuticle, thereby releasing the adhered conidia. Dichloromethane is also known as a protein-denaturing solvent (34).…”

Section: Discussionmentioning

confidence: 99%

Novel Technique for Quantifying Adhesion of Metarhizium anisopliae Conidia to the Tick Cuticle

Ment

Gindin

Rot³

et al. 2010

Appl Environ Microbiol

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The present study describes an accurate quantitative method for quantifying the adherence of conidia to the arthropod cuticle and the dynamics of conidial germination on the host. The method was developed using conidia of Metarhizium anisopliae var. anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae) and engorged Rhipicephalus annulatus (Say) (Arachnida: Ixodidae) females and was also verified for M. anisopliae var. acridum Driver et Milner (Hypocreales: Clavicipitaceae) and Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) larvae. This novel method is based on using an organic solvent (dichloromethane [DCM]) to remove the adhered conidia from the tick cuticle, suspending the conidia in a detergent solution, and then counting them using a hemocytometer. To confirm the efficacy of the method, scanning electron microscopy (SEM) was used to observe the conidial adherence to and removal from the tick cuticle. As the concentration of conidia in the suspension increased, there were correlating increases in both the number of conidia adhering to engorged female R. annulatus and tick mortality. However, no correlation was observed between a tick's susceptibility to fungal infection and the amount of adhered conidia. These findings support the commonly accepted understanding of the nature of the adhesion process. The mechanism enabling the removal of the adhered conidia from the host cuticle is discussed.The entomopathogenic fungus Metarhizium anisopliae var. anisopliae (Metschn.) Sorokîn (1883) infects a broad range of arthropod hosts and can be used as a biopesticide against different insect and tick species (8,22,35,36). The adhesion of the conidia of entomopathogenic fungi to the host cuticle is the initial stage of the pathogenic process and includes both passive and active events (5, 10). The hydrophobic epicuticular lipid layer plays an important role during both the attachment process and the germination of the conidia on the surface of the host (15,19). According to Boucias et al. (7), the attachment of conidia to the host cuticle is based on nonspecific hydrophobic and electrostatic forces. The conidia of most entomopathogenic fungi, including M. anisopliae, have an outer cell layer made up of rodlets (6). The hydrophobins, specific proteins present in the rodlet layer, mediate the passive adhesion of conidia to hydrophobic surfaces, such as the cuticles of arthropods (16,45,46). However, as germination commences, the hydrophobins are replaced by an adhesion-like protein, Mad1, which promotes tighter and more-specific adhesion between the conidia and the host (44). Many factors may affect the adhesion and persistence of conidia on the host cuticle (i.e., characteristics of the pathogen, including its virulence [2,18,48], conditions under which the pathogen is cultured [17], type of spores [7,16], topographical and chemical properties of the host cuticle [9,38,42], host surface hydrophobicity [15,23], host behavior [31,33], and environmental conditions [33]). Conidia of M. anisopliae have show...

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“…Anti-TT, antibodies in rat sera were estimated as reported previously (Raghuvanshi et al,1998). Briefly 1 µg of TT in 100 µl of PBS (50 mM, pH 7.4) was coated in each well of 96-well flat bottom Nunc immunoplates.…”

Section: Elisa Protocolmentioning

confidence: 99%

Potentiation of Immune Response from Polymer-Entrapped Antigen: Toward Development of Single Dose Tetanus Toxoid Vaccine

et al. 2003

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Stabilization of Dichloromethane-Induced Protein Denaturation During Microencapsulation

Cited by 61 publications

References 26 publications

Enhanced Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Virus-Like Particles of Human Papillomavirus

Enhanced Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Virus-Like Particles of Human Papillomavirus

Novel Technique for Quantifying Adhesion of Metarhizium anisopliae Conidia to the Tick Cuticle

Potentiation of Immune Response from Polymer-Entrapped Antigen: Toward Development of Single Dose Tetanus Toxoid Vaccine

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