Comparative Diagnostics of Allergy Using Quantitative Immuno-PCR and ELISA

Simonova, Maria; Pivovarov, V. D.; Ryazantsev, D. Yu.; Dolgova, Anna; Berzhets, V M; Завриев, С. К.; Svirshchevskaya, E. V.

doi:10.4155/bio-2017-0194

Cited by 7 publications

(8 citation statements)

References 16 publications

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“…When analyzing the allergic patient serum by ELISA, a similar nonspecific signal was also observed in the 20-fold dilution, but at higher concentrations of ODN-BE5 conjugate, the signal was on the scale of micrograms per milliliter (Fig 8c). We did not observe this phenomenon in the case of unconjugated antibody (Fig 8c) and BE5-streptavidin-DNA complex [18], as well as in the case of incubation with unconjugated oligonucleotide. Therefore, we believe that the observed nonspecific binding is a result of changes in the structure of the antibody molecule during its chemical modification.…”

Section: Resultsmentioning

confidence: 97%

“…To eliminate nonspecific binding, we tried to optimize the conditions of the immunoassay and introduced an additional blocking step between the stages of the sorption of allergen and the addition of the analyzed serum (compared to the method described for the DNA-streptavidin complex [18]). Blocking was performed with TBS buffer with various components: 1% BSA, 10% goat serum, 0.73 mM sucrose, 0.5% tween-20, 0.02% tween-20 with 1% BSA, 2% skim milk, 0.5% triton X100, 0.1% gelatin, and 1% human immunoglobulin.…”

Section: Resultsmentioning

confidence: 99%

“…This was confirmed by the fact that we obtained conjugates with the MA2 antibody with preserved antibody specificity if the antibody amino groups were modified, and so did other authors with several other antibodies. It is should be noted that the biotinylated BE5 antibody did not change in specificity after streptavidin was added to the supramolecular complex of 5'- and 3'-biotinylated ODN and streptavidin [18]. The biotinylation of antibody BE5 was carried out with the N-hydroxysuccinimide ester of biotin attached to the same reactive amino groups as STP-N3.…”

Section: Discussionmentioning

confidence: 99%

“…iPCR was performed using DNA-streptavidin complex in accordance with previous studies for the detection of IgE [18] and the detection of antibodies to Le C [19].…”

Section: Methodsmentioning

confidence: 99%

“…iPCR was performed with direct DNA-antibody conjugate as reported previously [18–20] except for the conjugate addition stage. The IgE analysis was carried out by coating plate wells with the recombinant allergen Alt a 1 in carbonate-bicarbonate buffer at pH 9.6 with a concentration of 2 μg/ml (50 μl per well).…”

Section: Methodsmentioning

confidence: 99%

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Development of the covalent antibody-DNA conjugates technology for detection of IgE and IgM antibodies by immuno-PCR

Maerle¹,

Simonova

Pivovarov

et al. 2019

PLoS ONE

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Immuno-PCR (iPCR) is one of the methods used for the detection of a wide range of analytes and features the high sensitivity of the polymerase chain reaction (PCR) method. iPCR uses antibodies coupled to DNA, followed by the amplification of the attached DNA using RT-PCR. Two major types of antibody-DNA conjugates are currently used, which are obtained as a result of non-covalent (biotin-streptavidin) or covalent interactions. Using a strain-promoted azide-alkyne cycloaddition (SPAAC), we synthesized covalent DNA-antibody conjugates, optimized the reaction conditions, and developed an efficient protocol for the purification of conjugates, with which all unreacted antibodies and oligonucleotides are separated. Covalent DNA-antibody conjugates were tested with iPCR assays that were previously developed for the detection of IgE and IgM antibodies with the use of the supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin. The results show that the modification of antibodies with amino groups did not allow us to obtain monolabeled antibodies or antibodies with a strictly defined number of DNA-labels. The degree of labeling determined by the dyes introduced through the azido group reflects the actual labeling degree statistically. If the average labeling degree for azido groups is 1.1, the conjugates contain 25% mono-labeled antibodies, 50% double-labeled antibodies, and 25% unlabeled ones. The specificity of the monoclonal antibody to human IgE (BE5) changed after conjugation with the oligonucleotide. The sensitivity of iPCR in the detection of IgM antibodies produced against the LeC disaccharide using a covalent conjugate was similar to that of a supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin, but the new procedure is two steps shorter.

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…iPCR was performed using DNA-streptavidin complex in accordance with previous studies for the detection of IgE [18] and the detection of antibodies to Le C [19].…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Development of the covalent antibody-DNA conjugates technology for detection of IgE and IgM antibodies by immuno-PCR

Maerle¹,

Simonova

Pivovarov

et al. 2019

PLoS ONE

View full text Add to dashboard Cite

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Immunoproteomics Methods and Techniques

Fulton

Baltat

Twine

2019

Methods in Molecular Biology

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Predisposition of hypersensitivity in patients with exfoliative cheilitis

Cai

Jiao

et al. 2022

Journal of Dental Sciences

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Background/purpose Exfoliative cheilitis (EC) is a chronic and reversible inflammatory disease of the lips without definite etiology. Clinically, different types of allergens can be found in exfoliative cheilitis patients, however, few studies have focused on the relationship between exfoliative cheilitis and hypersensitivity. This research aimed to investigate the prevalence of hypersensitivity in EC patients. Materials and methods A prospective study was conducted in 30 patients with exfoliative cheilitis and 30 healthy controls, matched in age and sex. Laboratory tests included serum total IgE, allergen-specific IgE, and food-specific IgG. Results Increased serum total IgE level, positive food-specific IgG were seen more frequently in exfoliative cheilitis patients than in healthy control (P < 0.05). Special IgE level to FX5 and the degree of food-specific IgG to wheat were seen higher in exfoliative cheilitis patients than in healthy control (P < 0.05). Conclusion This study suggests that patients with exfoliative cheilitis may have predisposition of hypersensitivity. The detection of allergens should be strengthened in the future clinical work.

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Comparative Diagnostics of Allergy Using Quantitative Immuno-PCR and ELISA

Abstract: Higher sensitivity of qiPCR in identification of low titer IgE is a result of a higher linearity of qiPCR data.

Cited by 7 publications

References 16 publications

Development of the covalent antibody-DNA conjugates technology for detection of IgE and IgM antibodies by immuno-PCR

Development of the covalent antibody-DNA conjugates technology for detection of IgE and IgM antibodies by immuno-PCR

Immunoproteomics Methods and Techniques

Predisposition of hypersensitivity in patients with exfoliative cheilitis

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