The innovative anti-tumor medication paclitaxel offers a broad spectrum of anti-tumor properties and is effective against a number of malignancies. Ovarian cancer and breast cancer in particular have a higher curative impact1. The unpleasant reactions of the natural immune system known as hypersensitivity reactions, which include allergies and autoimmune diseases, can be brought on by excessive dosages of paclitaxel. These reactions, which are also known as immune system overreactions, can be hazardous and uncomfortable2. With a compound annual growth rate of 3.0%, the number of new cases of breast cancer climbed from 2.045 million in 2017 to 2.301 million in 2021 .It's predicted that there will be 2.342 million new cases of breast cancer worldwide in 2022.
So far, there are many paclitaxel drugs in various stages of development3, of which antibody-drug conjugate and albumin paclitaxel are two of the most highly regarded4,5. They both use the hydrophilic properties of proteins to improve the hydrophobicity of paclitaxel itself, greatly improving its bioavailability and reducing the side effects of paclitaxel itself. This type of drug, however, is a combination of proteins and compounds. Trying to detect its stability in plasma using conventional mass spectrometry is difficult to develop. Therefore, there is a need for simpler, more convenient, easily scalable, and affordable methods to detect the fallout of its small molecules.
To develop the most effective and safest PTX medications, our PACLISA team employs ELISA. This technique aids in determining the PTX levels within antibody-drug conjugates.
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Figure1. Rate of different cancers（https://www.who.int/）
Our team is working towards developing an ELISA kit specifically designed to detect small PTX molecules in pharmaceuticals. This tool aims to aid companies and research institutes in formulating drugs with the optimal PTX molecule concentration, striking a balance between minimizing paclitaxel's toxicity and maximizing its therapeutic efficacy.
Many different methods have been developed to detect the concentration of small molecules of PTX in drugs, such as mass spectrometry. However, we believe that ELISA method is more sensitive and cheaper to identify the concentration of small PTX molecules of antibody-drug conjugate.Our team constructed plasmids containing light chains (PEE12.4-33D9L) and heavy chains (PEE6.4-32D9H) by homologous recombination. Then the two plasmids are transformed into a bacterium for amplification, and the cell containing the ideal plasmid finally expresses the antibody(33D9) that we want. We coated the 33D9 on the 96-well plate, then added the drug to be detected (the primary antibody) and adsorbed on the antibody we made, and finally added the second antibody for color development. From our experiment, we determined a linear relationship between the antibody-coupled drug and its fluorescence value, allowing us to establish a standard curve for the antibody-drug conjugate. By drawing blood at various time intervals as the drug metabolizes, we can ascertain the concentration of the antibody-bound drug at each of these points. This process provides insights into the rate at which small molecules dissociate from the antibody-drug conjugate, further aiding in assessing its stability within blood circulation.
Figure2. Work flow of our design
1. Singla, A. K., Garg, A. & Aggarwal, D. Paclitaxel and its formulations. Int. J. Pharm. 235, 179–192 (2002).
2. Kloover, J. S., Bakker, M. A. den, Gelderblom, H. & Meerbeeck, J. P. van. Fatal outcome of a hypersensitivity reaction to paclitaxel: a critical review of premedication regimens. Br. J. Cancer 90, 304–305 (2004).
3. Marupudi, N. I. et al. Paclitaxel: a review of adverse toxicities and novel delivery strategies. Expert Opin. Drug Saf. 6, 609–621 (2007).
4. Shao, T. et al. Construction of paclitaxel-based antibody–drug conjugates with a PEGylated linker to achieve superior therapeutic index. Signal Transduct. Target. Ther. 5, 1–3 (2020).
5. Karmali, P. P. et al. Targeting of albumin-embedded paclitaxel nanoparticles to tumors. Nanomedicine Nanotechnol. Biol. Med. 5, 73–82 (2009).