DESCRIPTION

CTLA-4, also known as CD152, belonging to the immunoglobulin superfamily, is primarily found on activated T cells and regulatory T cells (Tregs). CTLA-4 is closely related to the T-cell costimulatory CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 acts as a negative regulatory molecule of T-cell responses. Besides the full-length transmembrane form, CTLA-4 also exists in a truncated soluble form (sCTLA-4).

PRINCIPLE OF THE ASSAY

An anti-tag antibody is pre-coated onto the bottom of wells. After adding antigen or samples, Tagged-Capture antibody and HRP-conjugated detection antibody, a sandwich complex is formed in the solution. TMB acts as a HRP substrate, and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns the solution yellow. The color intensity is proportional to the quantity of bound protein, which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 8 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 16 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	8	725.7	23.3	3.2
2	8	184.8	4.0	2.2
3	8	101.8	3.0	2.9

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	703.0	55.2	7.9
2	16	175.6	10.2	5.8
3	16	98.2	4.6	4.7

RECOVERY

The recovery of human CTLA-4/CD152 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:2	82	79-85
	1:4	107	103-109
Cell culture supernatant	1:2	87	79-92
	1:4	89	80-97
Urine	1:2	75	70-80
	1:4	91	84-100
Cell lysate	1:2	83	72-92
	1:4	94	90-96

LINEARITY

To assess the linearity of the assay, human plasma, urine and cell lysate samples were spiked with high concenthumanions of human CTLA-4/CD152 and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Cell culture supernatant samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay.

		Human plasma(Sample Diluent PT1)	Cell culturesupernatant(Sample Diluent PT4B1)	Urine(Sample Diluent PT4B1)	Cell lysate(Sample Diluent PT4B1)
1:2	Average% of Expected	83	100	78	71
	Range (%)	81-85	-	76-79	70-73
1:4	Average% of Expected	107	87	89	78
	Range (%)	105-109	86-88	88-90	78-79
1:8	Average% of Expected	108	89	96	85
	Range (%)	101-114	88-91	96-97	84-86
1:16	Average% of Expected	101	-	101	89
	Range (%)	90-112	-	99-102	88-91

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human CTLA-4/CD152 One-Step ELISA Kit produced at Sunlong Medical™.