Allosteric molecular sensing of anti-HIV antibodies by an immobilized engineered β-galactosidase

Ferraz, Rosa María; Arı́s, Anna; González, Gerardo; López-Santı́n, Josep; Villaverde, Antonio; Álvaro, Gregorio

doi:10.1016/j.enzmictec.2007.04.001

Cited by 3 publications

(5 citation statements)

References 15 publications

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“…The immobilization procedure included two steps: support activation 26 and immobilization process. 25 Glyoxal-agarose gels (agarose-O-CH 2 -CHO) were prepared by etherification of 4BCL agarose beads (Hispanagar, Torrejón de Ardoz, Spain) with glycidol (2,3-epoxypropanol) (Sigma-Aldrich, St. Louis, MO) during 19 h at 20 °C and oxidized using NaIO 4 0.1 M (Sigma, St. Louis, MO). The number of aldehyde groups available to link the ligand was calculated after 35 min by analyzing the consumed periodate through an enabled reaction with KI 10% and saturated NaHCO 3 .…”

Section: Methodsmentioning

confidence: 99%

“…The immobilization procedure included two steps: support activation and immobilization process . Glyoxal-agarose gels (agarose-O-CH 2 -CHO) were prepared by etherification of 4BCL agarose beads (Hispanagar, Torrejón de Ardoz, Spain) with glycidol (2,3-epoxypropanol) (Sigma-Aldrich, St. Louis, MO) during 19 h at 20 °C and oxidized using NaIO 4 0.1 M (Sigma, St. Louis, MO).…”

Section: Methodsmentioning

confidence: 99%

“…In particular, Escherichia coli β-galactosidase has been successfully tailored for the diagnosis of either foot-and-mouth disease 21 and human immunodeficiency virus (HIV) infection, 22,23 and conditions for assays in liquid and immobilized form have been completely established. 24,25 As a proof of concept, the LTCC-β-galactosidase system will be tested by measuring the conversion of ortho-nitrophenyl β-Dgalactopyranoside (ONPG), an usual substrate employed for β-galactosidase activity determinations. Immobilized E. coli β-galactosidase in glyoxal-agarose supports was introduced in a microcolumn monolithically integrated in a LTCC microfluidic system which also incorporates a flow cell for spectrophotometric detection.…”

Section: Low-temperature Cofired Ceramics (Ltcc) Technology Is a Vers...mentioning

confidence: 99%

“…In addition, β-galactosidase is an enzyme which can be modified to exhibit allosteric responses. In particular, Escherichia coli β-galactosidase has been successfully tailored for the diagnosis of either foot-and-mouth disease and human immunodeficiency virus (HIV) infection, , and conditions for assays in liquid and immobilized form have been completely established. , …”

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confidence: 99%

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Ceramic Microsystem Incorporating a Microreactor with Immobilized Biocatalyst for Enzymatic Spectrophotometric Assays

et al. 2009

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Low-temperature cofired ceramics (LTCC) technology is a versatile fabrication technique used to construct microflow systems. It permits the integration of several unitary operations (pretreatment, separation, (bio)chemical reaction, and detection stage) of an analytical process in a modular or monolithic way. Moreover, because of its compatibility with biological material, LTCC is adequate for analytical applications based on enzymatic reactions. Here we present the design, construction, and evaluation of a LTCC microfluidic system that integrates a microreactor (internal volume, 24.28 microL) with an immobilized beta-galactosidase from Escherichia coli (0.479 activity units) and an optical flow cell to measure the product of the enzymatic reaction. The enzyme was immobilized on a glyoxal-agarose support, maintaining its activity along the time of the study. As a proof of concept, the LTCC-beta-galactosidase system was tested by measuring the conversion of ortho-nitrophenyl beta-D-galactopyranoside, the substrate usually employed for activity determinations. Once packed in a monolithically integrated microcolumn, the miniaturized flow system was characterized, the operational conditions optimized (flow rate and injection volume), and its performance successfully evaluated by determining the beta-galactosidase substrate concentration at the millimolar level.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Low-temperature Cofired Ceramics (Ltcc) Technology Is a Vers...mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Ceramic Microsystem Incorporating a Microreactor with Immobilized Biocatalyst for Enzymatic Spectrophotometric Assays

et al. 2009

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“…Altogether, these data showed that the two novel peptides were highly promising allosteric receptors whose use in multiple sensing arrays would widen the spectrum of anti-HIV-1 antibodies detectable by enzymatic reactions. In this regard, the recent finding that allosteric beta-galactosidase sensors show high performance upon solid-phase immobilization [Ferraz et al, 2007] offers the possibility to develop miniaturized devices in which several sensing elements reactive to complementary analytes (in this case, different populations of antibodies) could offer matricidal data profiling the immune response. The null responses of JX795-p17 and JX795-p24 are probably due to low titres of antibodies directed against p17 and p24 in most of the tested sera, since such proteins were also hardly recognized in western blot (data not shown).…”

Section: Antibody-mediated Sensor Activationmentioning

confidence: 97%

Screening HIV‐1 antigenic peptides as receptors for antibodies and CD4 in allosteric nanosensors

Ferraz

Rodríguez‐Carmona

Ferrer-Miralles

et al. 2009

J of Molecular Recognition

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We have analyzed the suitability of six antigenic peptides from several HIV-1 structural proteins (namely gp41, gp120, p17, and p24), as anti-HIV-1 antibody receptors in an allosteric enzymatic biosensor. These peptides were inserted in a solvent-exposed surface of Escherichia coli (E. coli) beta-galactosidase by means of conventional recombinant DNA technology. The resulting enzymes were tested to allosterically respond to sera from HIV-1-infected individuals. Only stretches from gp41 and gp120 envelope proteins were able to transduce the molecular contact signal in the presence of immunoreactive sera. Intriguingly, the enzyme displaying the CD4 binding site segment KQFINMWQEVGKAMYAPP was activated by soluble CD4, suggesting that it produces conformational modifications on the allosteric enzyme as those occurring during antibody-promoted induced fit. This fact is discussed in the context of the design of smart protein drugs and markers targeted to CD4+ cells.

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Discriminating Foot-and-Mouth Disease Virus-Infected and Vaccinated Animals by Use of β-Galactosidase Allosteric Biosensors

Sánchez-Aparicio

Rosas

Ferraz

et al. 2009

Clin Vaccine Immunol

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Recombinant ␤-galactosidases accommodating one or two different peptides from the foot-and-mouth disease virus (FMDV) nonstructural protein 3B per enzyme monomer showed a drastic enzymatic activity reduction, which mainly affected proteins with double insertions. Recombinant ␤-galactosidases were enzymatically reactivated by 3B-specific murine monoclonal and rabbit polyclonal antibodies. Interestingly, these recombinant ␤-galactosidases, particularly those including one copy of each of the two 3B sequences, were efficiently reactivated by sera from infected pigs. We found reaction conditions that allowed differentiation between sera of FMDV-infected pigs, cattle, and sheep and those of naïve and conventionally vaccinated animals. These FMDV infection-specific biosensors can provide an effective and versatile alternative for the serological distinction of FMDV-infected animals.

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Allosteric molecular sensing of anti-HIV antibodies by an immobilized engineered β-galactosidase

Cited by 3 publications

References 15 publications

Ceramic Microsystem Incorporating a Microreactor with Immobilized Biocatalyst for Enzymatic Spectrophotometric Assays

Ceramic Microsystem Incorporating a Microreactor with Immobilized Biocatalyst for Enzymatic Spectrophotometric Assays

Screening HIV‐1 antigenic peptides as receptors for antibodies and CD4 in allosteric nanosensors

Discriminating Foot-and-Mouth Disease Virus-Infected and Vaccinated Animals by Use of β-Galactosidase Allosteric Biosensors

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