Improved group determination of tetracycline antibiotics in competitive enzyme-linked immunosorbent assay

Abstract

Seven tetracycline (TC) antibiotics were used as haptens for synthesis of conjugated antigens and as standards in indirect competitive enzyme-linked immunosorbent assay (ELISA). Seven conjugates were prepared by formaldehyde condensation (f) and another seven antigens were synthesised by linking TCs with periodate-oxidised transferrin (TF(pi)). Two gelatineÁlymecycline (LC) conjugates were the products of glutaraldehyde and activated esters reactions. To estimate the influence of solid-phase antigen on assay specificity, all the conjugates were absorbed on polystyrene plates and using anti-bovine serum albuminÁTC(f) sera, the ELISAs were developed and compared. The variant with immobilised TFÁ chlortetracycline (CTC(pi)) showed the best group specificity: recognition of seven TCs differed only in 5.3 times. This assay displayed sensitivity 0.1 ng/ml and crossreactivity indexes: TC (100%), CTC (105%), oxytetracycline (19.8%), methacycline (23.6%), minocycline (70%), doxycycline (25%) and LC (50%). The dependence of ELISA specificity on immobilised antigen spatial structure was demonstrated. Solid-phase conjugate selection is a useful tool for modifying assay specificity.

Highly Sensitive Immunochromatographic Identification of Tetracycline Antibiotics in Milk

“…The procedure in [ 20 ] was amended and applied. In this procedure, 30 mg of BSA was dissolved in 2.0 mL of H 2 O and 26 mg TC-HCl solution was added to 200 μ L DMSO and 120 μ L of 37% aqueous formaldehyde solution (BSA molar ratio : TC : formaldehyde = 1 : 120 : 3,600) and stirred at room temperature overnight.…”

“…Implementation of these methods does not require expensive equipment and is therefore of interest for mass contamination screening. The most common methods of microplate enzyme immunoassay (ELISA) for TCs [ 18 – 20 ] are implemented in a laboratory environment, but with the use of relatively inexpensive and easy to use instrumentation. Unlike ELISA, immunochromatography (IC) test systems are nonlaboratory analytical tools.…”

Indirect Competitive Determination of Tetracycline Residue in Honey Using an Ultrasensitive Gold-Nanoparticle-Linked Aptamer Assay

“…The tetracycline-BSA conjugate was obtained by a glutaraldehyde crosslinking method following the procedures reported previously [36]. A brief description of steps is as follows: First, 20 mg tetracycline was dissolved in 3 mL pyridine and 40 mg BSA was dissolved in 5 mL PBS buffer (pH 7.4) solution.…”

New haptens synthesis, antibody production and comparative molecular field analysis for tetracyclines

“…When the results obtained in this work are compared with those achieved by other authors, it can be observed that the ELISA we developed for TC offered signicant advantages in terms of sensitivity. 13,17,20 The IC 50…”

“…12 To date, there have been only a few reports concerning the preparation of antibodies for TCs. [13][14][15][16][17][18][19][20] In this work, we describe the synthesis of two novel haptens and the generation of a specic polyclonal antibody for TC that was found to exhibit signicantly improved sensitivity. A highly specic and sensitive enzyme-linked immunosorbent assay (ELISA) based on the novel antibody was developed and optimized and was then applied to milk samples.…”