Development of an enzyme‑linked immunosorbent assay for camptothecin

Abstract

The use of camptothecin and its analogues has increased in clinical settings and in agriculture. Therefore, camptothecins and their derivatives, metabolites and degradation products are frequently found in the environment. Therefore, it is important to develop an ELISA for the quantification of camptothecins in human plasma, plants, animal tissues and other matrices. The present study developed a novel competitive indirect ELISA for camptothecin using a monoclonal antibody (MAb). In total, two haptens and various carrier proteins were tested to select the most suitable immunogen for the production of MAbs against camptothecin. Hapten 1 conjugated with keyhole limpet hemocyanin was selected for the preparation of MAb 5A3, and was used to establish a competitive indirect ELISA for camptothecin. A total of three derivatives of camptothecin used in clinical practice were examined. Topotecan showed an IC50 value of 0.68 µg/ml with a detection limit of 0.19 µg/ml, belotecan showed an IC50 value of 0.87 µg/ml with a detection limit of 0.22 µg/ml and irinotecan showed an IC50 value of 2.85 µg/ml with a detection limit of 0.47 µg/ml. The cross‑reactivity results suggested that the assay developed in the present study possessed a high sensitivity to camptothecin. Therefore, this immunoassay technique may be suitable for monitoring the levels of camptothecin in compound analysis, clinical applications, and analyses of food and environmental samples.

Figure 1.

Structures and synthetic routes of haptens and hapten-protein conjugates. (A) Structure of camptothecin and synthetic route of hapten 1 and hapten 2. Red group in hapten 1 indicates the added succinic functional group. Red group in hapten 2 indicates the substituted and oxidized functional group. (B) Synthetic route of the active ester method. Red groups of haptens, carriers and N-hydroxysuccinimide indicate the functional groups involved in the chemical reaction. (C) Synthetic route of the mixed anhydride method. Red groups in haptens, carriers and isobutylchlorocarbonate indicate the functional groups involved in the chemical reaction. (D) Structures of three camptothecins. Con c.H₂SO₄, high concentration of H₂SO₄; DMAP, dimethylaminopyridine; DCC, N,N-dicyclohexylcarbodiimide; DMF, dimethyl formamide; EDCI, 1-(3-dimethylaminopropyl)-3-ethyl carbon diimide hydrochloride; RT, room temperature.

Figure 2.

UV absorbance spectrum of haptens, carrier proteins and hapten-protein conjugates solutions in PBS. Concentration of all analytes was 100 µg/ml. Haptens, carrier proteins and hapten-protein conjugates presented in different panels were measured under the same conditions. (A) Hapten-BSA conjugates. Hapten 1 profile is shown in blue. Hapten 1-BSA profile is presented in black. BSA profile is presented in red. (B) Hapten-KLH conjugates. Hapten 1-KLH profile is presented in black. KLH profile is presented in red. (C) Hapten 1-OVA conjugates. Hapten 1-OVA profile is presented in black. OVA profile is presented in red. OVA, ovalbumin; KLH, keyhole limpet hemocyanin.

Figure 3.

Indirect ELISA results of antisera and four camptothecins. (A) Non-immunized serum collected 1 week before immunization from every mouse was used as control serum. (B) Inhibition rate for four camptothecins at various concentrations by indirect ELISA. MAb 5A3 was diluted 1:128,000, with a final concentration of 0.014 µg/ml. (C) Inhibition curve and linear regression analysis of camptothecin by indirect ELISA. MAb 5A3 was diluted 1:128,000, with a final concentration of 0.014 µg/ml. Data are presented as the mean ± standard deviation (n=3). *P<0.05. R², angular coefficient; KLH, keyhole limpet hemocyanin; MAb, monoclonal antibody.