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CAR Expression Test Services

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All products and services are For Research Use Only and CANNOT be used in the treatment or diagnosis of disease.

Methods of CAR Detection

Evaluation of CAR expression in CAR-T cells is a critical step in the development of CAR-T therapy. Creative Biolabs utilizes several techniques for the detection and quantification of CAR expression in CAR-T cell therapy, including:

• Flow cytometry

Flow cytometry is a widely used method to analyze CAR expression on the surface of CAR-T cells. A specific fluorescently labeled anti-CAR antibody is used to bind to the CAR protein on the cell surface, and flow cytometry is used to measure the fluorescence intensity, indicating the level of CAR expression.

• Real-time quantitative PCR (qPCR)

qPCR can be used to quantify the expression of CAR mRNA in CAR-T cells. This method involves isolation of total RNA, reverse transcription of RNA into cDNA, followed by PCR amplification for the CAR gene. The amount of CAR mRNA can be quantified based on the amplification data.

• Western Blot (WB)

WB technique uses CAR-specific antibodies such as anti-idiotype monoclonal antibodies to detect the presence of CAR expression in a sample. The expression level of the CAR can be quantified using WB analysis by comparing the protein band intensity of the CAR protein to a known standard or control.

Fig.1 Current CAR detection methods.¹

Our Services

By utilizing our comprehensive range of detection methods and expertise in CAR-T cell therapy, Creative Biolabs aims to aid researchers in evaluating CAR expression. Our scientists offer accurate and reliable results about the percentage of cells expressing CAR and the intensity of CAR expression. One commonly used method is flow cytometry, which allows for the measurement of CAR expression levels on the cell surface. To further validate these results, other techniques such as WB, qPCR, or ELISA can also be employed. It is crucial to note that the selection of the appropriate antibody for CAR detection should be based on the specific design of the CAR construct and the goals of the study. In addition, we provide CAR-specific monoclonal antibody development services for CAR expression detection. Our team of scientists has a deep understanding of the advantages and limitations associated with each method and can assist you in selecting the most suitable approach for your research requirements.

Support Data

Here are some representative data for CAR expression tests.

Case 1: Analysis of CAR expression by WB.

Fig.2 Analysis of CAR expression levels in CAR Jurkat cells. After transducing the different CAR constructs into Jurkat cells, CAR expressions were confirmed by WB.²

Case 2: Analysis of CAR expression by flow cytometry.

Fig.3 Analysis of CAR expression levels in BCMA-CAR-T cells. CAR expressions were confirmed by flow cytometry with different reagents (BCMA antibody, anti-human-IgG-antibody and BCMA peptide).³

Case 3: Analysis of CAR expression by flow cytometry and qPCR.

Fig.4 Comparison of CAR detection methods. The quantification results of CAR expressions from flow cytometry and qPCR were compared. The results assessed by qPCR are similar to the results from flow cytometry.³

References

1. Hu, Yifei, and Jun Huang. "The chimeric antigen receptor detection toolkit." Frontiers in immunology 11 (2020): 1770.
2. Kang, Chung Hyo, et al. "C-Met-Specific chimeric antigen receptor T cells demonstrate anti-tumor effect in c-met positive gastric cancer." Cancers 13.22 (2021): 5738.
3. Reichman, Avinoam, et al. "Comparison of FACS and PCR for detection of BCMA-CAR-T cells." International Journal of Molecular Sciences 23.2 (2022): 903.