LeukoComplete™ Gene Detection Kit for human ACTB

Cosmo Bio

Catalog No.: MIN-11150-HC-001

Primer and Enzyme for Real-Time PCR

LeukoComplete™ Cellular Immunity Detection Kit

This unique research-use test kit adds antigens (e.g., viral peptide fragments) to a small amount of whole blood and uses a dedicated plate to evaluate gene expression changes in stimulated T cells via qPCR. It is designed for human samples.

Features

This kit extracts and purifies mRNA from white blood cells (including T cells) in whole blood, followed by cDNA synthesis. The resulting cDNA can be analyzed by qPCR to evaluate relative gene expression changes. It supports the Ex vivo Activation of Gene in Leukocyte (EAGL) method and includes two proprietary plates—“Leukocyte Isolation Plate” and “mRNA Capture Plate”—for high throughput and reproducibility.

Specifications

Applications	Assessment of cellular immune responses Detection of cytokine-related genes
Work time	180 minutes + antigen stimulation time
Sample Types	Fresh blood (human, mouse) PBMC (human, mouse) Spleen (mouse)
Final Product	30 µL cDNA
Detection Targets	Control genes (human, mouse): ACTB, GAPDH, B2M Target genes (human): IFNG, IL-2, TNFSF2, IL-6, IL-10, GM-CSF, CXCL10, IL-4, IL-5 Target genes (mouse): IFNG, IL-10, IL-17
Measuring Equipment	Real-time PCR Digital PCR
Use Cases	T cell antigen screening Vaccine efficacy confirmation Allergy assessment

Kit Components

LeukoComplete™ Plate and RT Kit (Cat. No. 11130)

Component	Size
PT Buffer	15 mL × 1 bottle
Leukocyte Isolation Plate (Filter)	1 sheet
Deep Well Plate	1 sheet
Proteinase K	30 µL × 1
TCEP	300 µL × 1
Lysis Buffer	6 mL × 1 bottle
mRNA Capture Plate	1 sheet
Wash Buffer A	30 mL × 1 bottle
Wash Buffer B	50 mL × 1 bottle
Aluminum Seal	1 sheet
M-MLV Reverse Transcriptase	40 µL × 1
RNase Inhibitor	10 µL × 1
RT Buffer	1.5 mL × 2

LeukoComplete™ Antigen Coated Plate (Cat. No. 11140)

Component	Size
Antigen Coated Plate for SARS-CoV-2	1 sheet (24 tests)

* Overlapping peptides (15-mers with 11 aa overlap) from the SARS-CoV-2 spike protein are used as antigens. Each test includes four wells: a blank, two antigens, and a positive control.

LeukoComplete™ Gene Detection Kit

Component	Size
Primer Mix *	100 µL × 1
PCR Enzyme Mix	500 µL × 1

* Part number and Primer Mix content vary by target gene.

Product Data

Gene Extraction Performance

Figure 1: High correlation (R² > 0.9) was observed between sample concentration and expression levels for human blood, PBMC, mouse spleen, and mouse blood.

Target Gene Expression Over Time

Figure 2: Expression of IFNG, IL2, IL4, and GM-CSF genes in fresh human blood (n=4) exposed to antigen (CEF peptides) over time. log₂FC = ΔΔCt using control genes for normalization.

Comparison of Cellular Immunity Assays

Figure 3: Comparison between LeukoComplete™, ELISpot, and ICS in vaccinated subjects. LeukoComplete™ showed comparable or better sensitivity for IFNG detection.

Analysis Method

Figure 4: Relative gene expression evaluated using the Comparative Ct (ΔΔCt) method: ΔΔCt = (Ct_target - Ct_control) [with antigen] - (Ct_target - Ct_control) [without antigen]

Reagent Abbreviations

Reagent	Abbreviation
Phorbol 12-myristate 13-acetate	PMA
Ionomycin	IM
Phytohemagglutinin	PHA
Lipopolysaccharide	LPS
Concanavalin A	ConA
CEF control peptide pool	CEF

Expression Ranking

Rank	Mean Relative mRNA Expression (log₂FC, n=12)
+++	> 8
++	5 – 8
+	3 – 5
+/-	1.5 – 3
N.D.	< 1.5
-	Not evaluated

References

1. Mitsuhashi M, Tomozawa S, Endo K, Shinagawa A. Quantification of mRNA in whole blood by assessing recovery of RNA and efficiency of cDNA synthesis. Clin Chem. 2006;52(4):634–42.
2. Mitsuhashi M. Ex vivo simulation of leukocyte function: stimulation of specific subset of leukocytes in whole blood followed by the measurement of function-associated mRNAs. J Immunol Methods. 2010;363(1):95–100.
3. Saito et al. Rapid and high throughput assessment of cellular immunity against SARS-CoV-2 using the ex vivo gene activation method. Biochem Biophys Res Commun. 2023. doi: https://doi.org/10.1016/j.bbrc.2023.149398

Note

This product is a research reagent. Do not use for medical, diagnostic, or food-related purposes in humans or animals.

Product Specifications

• Reactivity: Human