Method for rapid conjugation of recombinant photoprotein aequorin with streptavidin and application as a universal detection reagent for binding assays

Zerefos, Panayotis G.; Ioannou, Penelope C.; Christopoulos, Theodore K.

doi:10.1016/j.aca.2005.10.064

Cited by 6 publications

(3 citation statements)

References 17 publications

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“…(His) 6 -aequorin 54 has been exploited not only for the conjugation of oligonucleotides to aequorin 55 but also for the conjugation of streptavidin to aequorin. 56 The aequorin-streptavidin conjugate may be exploited as a universal reporter molecule for bioluminometric DNA hybridization assays because streptavidin non-covalently binds to biotin with high affinity and biotin can be easily attached to practically any biomolecule. Protected sulfhydryl groups were introduced into (His) 6 -aequorin whereas streptavidin was derivatized with maleimide groups.…”

Section: Conjugation Strategiesmentioning

confidence: 99%

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Gene Assays Based on Bio(Chemi)luminescence

Laios

Ioannou²,

Christopoulos

2010

Chemiluminescence and Bioluminescence

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One of the first reports describing the use of recombinant aequorin as a reporter molecule in a bioluminometric nucleic acid hybridization assay was in 1996. 1 Microtiter wells were coated with anti-digoxigenin antibody. The target DNA was hybridized simultaneously with an immobilized digoxigenin-labeled capture probe and a biotinylated detection probe. The hybrids were determined using an aequorin-streptavidin conjugate followed by the measurement of luminescence in the presence of excess Ca 21 (Figure 9.1). The linearity of the assay was in the range 0.1-200 pM (5 amol well À1 to 10 fmol well À1) and the S/B ratio at 0.1 pM (5 amol well À1) was 5.3. A configuration in which the aequorinstreptavidin conjugate was replaced by a preformed complex of biotinylated aequorin to streptavidin resulted in equivalent signals and detectability. An advantage of using the preformed complex is that it was conveniently prepared by simply mixing the two components (streptavidin and biotinylated aequorin), thus providing a practical alternative to the use of aequorin-streptavidin conjugates prepared by covalent crosslinking techniques. The aequorin-biotin and the aequorin-streptavidin conjugates in this study were obtained commercially (the same group later reported the construction of a plasmid suitable for bacterial expression of in vivo-biotinylated aequorin, facilitating further the development of highly sensitive hybridization assays, 2 as well as a method for

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Section: Conjugation Strategiesmentioning

confidence: 99%

“…rapid conjugation of streptavidin to aequorin, 3 as discussed in Section 9.5). The proposed assay was applied to the detection and semi-quantitative assessment of the mRNA for prostate-specific antigen (PSA) as a potential marker for the staging of prostate cancer.…”

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Gene Assays Based on Bio(Chemi)luminescence

Laios

Ioannou²,

Christopoulos

2010

Chemiluminescence and Bioluminescence

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“…Τα συζεύγματα εκπλύθηκαν από τα ελεύθερα ολιγονουκλεοτίδια με φυγοκέντρηση και υποβλήθηκαν σε κατεργασία με διάλυμα Tris για την απενεργοποίηση των αλδεΰδομάδων (εναλλακτικά μπορεί να χρησιμοποιηθεί γλυκίνη) καθώς και με BSA για την κάλυψη των ελεύθερων θέσεων στην επιφάνειά αναφέρονται, επίσης, ο μεγάλος χρόνος επώασης των αντιδράσεων, αλλά και η πιθανότητα σχηματισμού συσσωματωμάτων σφαιριδίων με γέφυρα γλουταραλδεΰδης, παρά την τεράστια περίσσεια αντιδραστηρίου που προστίθεται στο πρώτο στάδιο για την κάλυψη της επιφάνειας. πορεία της αντίδρασης αποτελεί παραλλαγή πρωτοκόλλου σύζευξης πρωτεϊνών[157] και εφαρμόζεται με δύο τρόπους: ((i) ενεργοποίηση ολιγονουκλεοτιδίου με SATA και της επιφάνειας των σφαιριδίων-ΝΗ 2 με Sulfo-SMCC (σχήμα 6.5) και (ii) ενεργοποίηση επιφάνειας των σφαιριδίων-ΝΗ 2 με SATA και ενεργοποίηση του ολιγονουκλεοτιδίου με Sulfo-SMCC. Γενικά, το ολιγονουκλεοτίδιο ενεργοποιείται με το ένα αντιδραστήριο σύζευξης, από την περίσσεια του οποίου καθαρίζεται με χρωματογραφία διήθησης πηκτής, ενώ η επιφάνεια των σφαιριδίων με το δεύτερο, από το οποίο καθαρίζεται με φυγοκέντρηση και απομάκρυνση του υπερκείμενου.…”

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Ανάπτυξη Μεθόδων Πολλαπλής Ανίχνευσης Νουκλεϊκών Οξέων Και Εφαρμογές Στη Μοριακή Διαγνωστική

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Genotyping of single nucleotide polymorphisms by primer extension reaction and a dual-analyte bio/chemiluminometric assay

2007

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Primer extension reaction (PEXT) is the most widely used approach to genotyping of single nucleotide polymorphisms (SNP). It is based on the high accuracy of nucleotide incorporation by the DNA polymerase. We propose a dual-analyte bio/chemiluminometric method for the simultaneous detection of the PEXT reaction products of the normal and mutant allele in a high sample-throughput format. PCR-amplified DNA fragments that span the SNP of interest are subjected to two PEXT reactions using normal and mutant primers in the presence of digoxigenin-dUTP and biotin-dUTP. Both primers contain a d(A)30 segment at the 5'-end but differ in the final nucleotide at the 3'-end. Under optimized conditions only the primer that is perfectly complementary with the interrogated DNA will be extended by DNA polymerase and lead to a digoxigenin- or biotin-labeled product. The products of the PEXT reactions are mixed, denatured, and captured in microtiter wells through hybridization with immobilized oligo(dT) strands. Detection is performed by adding a mixture of antidigoxigenin-alkaline phosphatase (ALP) conjugate and a streptavidin-aequorin conjugate. The flash-type bioluminescent reaction of aequorin is triggered by the addition of Ca2+. ALP is then measured by adding the appropriate chemiluminogenic substrate. The method was evaluated by genotyping two SNPs of the human mannose-binding lectin gene (MBL2) and one SNP of the cytochrome P450 gene CYP2D6. Patient genotypes showed 100% concordance with direct DNA sequencing data.

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Method for rapid conjugation of recombinant photoprotein aequorin with streptavidin and application as a universal detection reagent for binding assays

Cited by 6 publications

References 17 publications

Gene Assays Based on Bio(Chemi)luminescence

Gene Assays Based on Bio(Chemi)luminescence

Ανάπτυξη Μεθόδων Πολλαπλής Ανίχνευσης Νουκλεϊκών Οξέων Και Εφαρμογές Στη Μοριακή Διαγνωστική

Genotyping of single nucleotide polymorphisms by primer extension reaction and a dual-analyte bio/chemiluminometric assay

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