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 IC 50 value of 0.68 µg/ml with a detection limit of 0.19 µg/ml, belotecan showed an IC 50 value of 0.87 µg/ml with a detection limit of 0.22 µg/ml and irinotecan showed an IC 50 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.

Progress in Immunoassays of Toxic Alkaloids in Plant-Derived Medicines: A Review

“…Kiko et al [ 52 ] prepared 3-succinylaconitine by succinylation at the C-3 hydroxyl position of aconitine. The same method has also been used to prepare haptens such as camptothecin [ 53 ], morphine [ 54 ], retrorsine [ 55 ], and tropane [ 56 ]. Wang et al [ 57 ] prepared a hapten of methamphetamine by introducing a carboxyl group on the second amino group.…”

“…Designation at different sites in the compound also exposes different fragments of the molecule to immune B cell epitopes, generating antibodies with different recognition properties. Yan et al [ 53 ] designed two haptens based on B- and E-ring modifications to investigate the effects of haptens’ analogs on the sensitivity of immunization. One case is to introduce an active carboxyl group at the C-7 site, and the other case is to introduce a four-carbon spacer arm containing a carboxyl group to connect to the hydroxyl group at the C-20 site.…”

“…Currently, the carriers for hapten coupling are mainly proteins, including BSA, ovalbumin (OVA), keyhole limpet hemocyanin (KLH), human serum albumin (HSA), etc. BSA is most commonly used in the toxic alkaloids field due to its stable physicochemical properties, non- dewaterability, low cost, and easy availability, as well as its high lysine content (i.e., high coupling density of hapten and carrier) [ 52 , 53 , 57 , 58 , 64 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 ]. Similarly, HSA also has a large amount of lysine and more free amino acids, which are widely used [ 59 , 75 ].…”

“…Similarly, HSA also has a large amount of lysine and more free amino acids, which are widely used [ 59 , 75 ]. KLH is also commonly used as an immunogen protein [ 53 , 70 , 76 , 77 ]. It is more expensive than BSA and has poor water solubility, but it has higher molecular weight and better immunogenicity than BSA.…”

“…Yan et al screened various carrier proteins to select the most suitable immunogen for the production of mAbs against camptothecin, and KLH showed better immunogenicity than BSA. OVA is characterized by poor solubility, instability, and variability, and contains fewer active groups than BSA, but it is often used as an unrelated carrier protein for antibody screening and immunoassays [ 53 , 61 , 64 , 73 , 78 , 79 ].…”