Increasing the sensitivity of immunochromatographic assay for penicillin determination in milk due to oriented immobilization of penicillin-binding protein on the surface of colloidal gold

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Abstract — The sensitivity of competitive immunochromatographic assay (ICA) for the determination of penicillin in milk using colloidal gold (CG) conjugates with recombinant penicillin-binding protein (PBP) prepared by two methods, passive non-oriented immobilization and oriented immobilization with antibodies to the histidine tag, was compared. The detection limit of the optimized ICA with non-oriented PBP immobilization was 5 ng/mL. It was possible to achieve a detection limit of 0.3 ng/mL with visual interpretation due to antibodies to the histidine tag with the use of oriented PBP immobilization. The analysis time was 10 min. The efficiency of two PBP immobilization methods was compared. It was shown that the sensitivity of the assay increased due to an improved PBP orientation and a simultaneous decrease in the number of binding sites on the CG. Thus, targeted immobilization of specific proteins on sensor surfaces using antibodies to the histidine tag can increase the sensitivity of the assay and simultaneously decrease the amount of biospecific reagents used. This approach can be applied to the simple conjugation of recombinant proteins with a histidine tag to nanoparticles.

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Sobre autores

I. Maksin

MIREA – Russian Technological University (Lomonosov Institute of Fine Chemical Technologies); Rapid Bio LLC

Autor responsável pela correspondência
Email: maxinivanv@gmail.com
Rússia, 86, Vernadskogo Prospekt, 119571 Moscow; Bolshoy Boulevard., 42, b.1, 121205 Moscow

A. Kuandykova

Rapid Bio LLC

Email: maxinivanv@gmail.com
Rússia, Bolshoy Boulevard., 42, b.1, 121205 Moscow

T. Luzyanin

MIREA – Russian Technological University (Lomonosov Institute of Fine Chemical Technologies); Rapid Bio LLC

Email: maxinivanv@gmail.com
Rússia, 86, Vernadskogo Prospekt, 119571 Moscow; Bolshoy Boulevard., 42, b.1, 121205 Moscow

V. Ivanov

Rapid Bio LLC

Email: maxinivanv@gmail.com
Rússia, Bolshoy Boulevard., 42, b.1, 121205 Moscow

Y. Kirillova

MIREA – Russian Technological University (Lomonosov Institute of Fine Chemical Technologies)

Email: maxinivanv@gmail.com
Rússia, 86, Vernadskogo Prospekt, 119571 Moscow

G. Khunteev

Rapid Bio LLC

Email: maxinivanv@gmail.com
Rússia, Bolshoy Boulevard., 42, b.1, 121205 Moscow

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2. Fig. 1. (a) TEM image of the obtained colloidal gold nanoparticles, (b) light absorption spectrum of colloidal gold nanoparticles, (c) histogram of the size distribution of colloidal gold nanoparticles.

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3. Fig. 2. Curves of the dependence of the aggregation coefficient K of conjugates (a) K3-a-His and (b) K3-PSB on pH and protein concentration.

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4. Fig. 3. MALDI-TOF mass spectra of conjugates with bovine serum albumin.

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5. Fig. 4. Calibration curve of the dependence of the test line staining intensity on the concentration of penicillin in a milk sample using K3-PSB and K3-aHis-PSB conjugates with different amounts of penicillin-binding protein.

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6. Fig. 5. Results of immunochromatographic determination of penicillin using conjugates KZ-PSB4 μg and KZ-PSB1 μg. The concentration of penicillin, which corresponds to the detection limit for visual interpretation of the result, is shown in bold.

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7. Fig. 6. Results of immunochromatographic determination of penicillin using conjugates KZ-PSB1 μg, KZ-PSB0.5 μg and KZ-PSB0.2 μg. The concentration of penicillin, which corresponds to the detection limit for visual interpretation of the result, is shown in bold.

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8. Fig. 7. Orientation of the penicillin-binding protein on the surface of gold nanoparticles with non-oriented or oriented immobilization. Due to the small size of penicillin, its effective binding to the penicillin-binding protein is expected even in the case of a hindered orientation of the latter (the active center faces the surface of the nanoparticle). In this case, the sterically hindered penicillin-binding protein will be inactive towards the BSA-antibiotic conjugate, since the conjugated ampicillin is shielded by the bulky BSA molecule.

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9. Fig. 8. Schematic illustration of the experiment to determine the amount of unbound penicillin-binding protein after conjugation with colloidal gold or K3-a-His.

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10. Fig. 9. The amount of penicillin-binding protein in the supernatant after incubation with K3 and K3-a-His for different times.

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11. Fig. 10. Schematic illustration of the experiment to determine the amount of penicillin after incubation with K3-PSB1 μg and K3-a-His-PSB1 μg.

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12. Fig. 11. Data from an experiment to determine the concentration of penicillin required to inhibit the conjugate KZ-PSB1 μg and KZ-a-His-PSB1 μg.

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