PAB-Exatecan IgG Labeling Kit

PAB protein (Protein of Antibody Binding, MW 25 kDa) contains the IgG Fc-binding domain derived from Protein G and includes a cysteine residue at its C-terminus. Under reducing conditions, the C-terminal cysteine of PAB is converted into a reduced thiol group. Through appropriate coupling reactions, toxins or dyes, including pH-sensitive dyes, can be specifically attached to this reduced thiol group. Such site-specific conjugation does not interfere with the specific binding between PAB and the IgG Fc region. PAB conjugated with toxins or dyes, including pH-sensitive dyes, can serve as a universal binding reagent for human or mouse IgG antibodies, enabling the study of antibody internalization or phagocytosis in target cells.

Exatecan (also known as irinotecan or irinotecan hydrochloride) is a water-soluble derivative of camptothecin (CPT). It exhibits potent antitumor activity and, in tumor cells, induces apoptosis by inhibiting DNA topoisomerase I. It is one of the commonly used cytotoxic payloads in antibody–drug conjugates (ADCs).

The PAB-Exatecan IgG labeling kit includes the PAB-Exatecan reagent, which is a complex where MC-Val-Cit-PAB-Exatecan is site-specifically conjugated to PAB via a reductive thiol group at the C-terminus of PAB. The conjugation between PAB and Exatecan is achieved using the widely used cleavable linker MC-VC-PABC, which contains a maleimide spacer, the Val-Cit dipeptide sequence as a substrate for cathepsin, and the self-cleaving PABC spacer. PAB-Exatecan can serve as a universal binding reagent for human or mouse IgG antibodies, and by detecting the cytotoxic effect of the antibody-PAB-Exatecan complex on target cells, it reflects the endocytic or phagocytic activity of the antibody. The PAB-Exatecan IgG labeling reagent employs a unique method to remove unlabeled free MC-Val-Cit-PAB-Exatecan, making the process simple and rapid—antibody labeling can be completed within 30 minutes. The labeled antibody does not require purification and can be directly used in subsequent endocytosis or phagocytosis assays. Compared with commonly used conjugated toxins such as DT3C, the PAB-Exatecan reagent has a smaller molecular weight, thereby reducing the potential impact of PAB-IgG conjugation on antibody function. Additionally, the VC-PABC linker better mimics the actual application of ADCs.

PAB-ExatecanIgG Labeling KitSpecifications are as follows. The amount of labeled IgG is calculated based on a molar ratio of 3:1, using PAB with a molecular weight of 25 kDa and IgG antibody with a molecular weight of 150 kDa..

Specifications	ReagentVolume	ReagentConcentration (PAB)	Labeled IgG
50 μg	2x50μL	250μg/mL	50μg

According toPAB MW 25KDaandIgGantibodyMW 150KDacalculation,IgGandPAB-Exatecanlabeled in a molar ratio of3:1can meet the needs of most experiments, and the molar ratio can also be optimized according to specific experimental requirements, up to a maximum of6:1, which can enhance the detection signal intensity. The following experiment usesTrastuzumab andPAB-Exatecancombined in a molar ratio of3:1to form a complex, and assesses its cytotoxic effect onHER-2expressingSK-BR-3cells to reflect the antibody's internalization efficiency.

1)In a96well white transparent cell culture plate, seedMcCoy's 5A (10% FBS)culture medium at100μlper well,4,000SK-BR-3cells,incubate overnight in a37℃ 5% CO₂ incubator

2)Prepare10xtest concentrationsTrastuzumab-PAB-ExatecanComplex: As required by the experimentTake an appropriate amountOpti-MEM IDilute in culture mediumTrastuzumabto50μg/mL, then according to10:1add250μg/mL ofPAB-Exatecanto a final concentration of22.7μg/mL, for example,30μLof50μg/mL Trastuzumabadd3μL250μg/mLof thePAB-Exatecan, thusthe concentrations ofTrastuzumabandPAB-Exatecan(10x)are approximately300 nMand900 nM, mix well by pipetting, and incubate at room temperaturefor 30 minutes.It is recommended that after the first use ofPAB-Exatecan, it be stored short-term(2 weeks) at4℃, and for long-term storage, aliquot according to single-use amounts and store protected from lightat -20℃or lower

3)usingOpti-MEM IThe prepared medium was used to perform a 3X serial dilution of the above-preparedTrastuzumab andPAB-Exatecancomplex, resulting in a total of8concentration levels.4)Remove the

SK-BR-3experimental cell plate, and add10μL of the gradient concentrations of the above-preparedTrastuzumab-PAB-Exatecancomplex to each well. Based onTrastuzumab, the highest final concentration is calculated to be30 nM.,3xdilutions, totaling8concentration levels.5)Incubate at

37℃and 5%CO2in an incubator for4days; it is recommended to replenish with20μL McCoy's 5A(10% FBS) culture medium after3days.

6)Performcell viabilityassays; for example, you can use theUA-Glo® Luminescent Cell Viability Assay(UA070103) to measure cell viability.

Do not mix different batches.
Do not alter the volume of the test reagent without rigorous validation.
For research use only.