CPS1 ELISA Kit

Catalog No. ABIN1125223

Product Details CPS1 ELISA Kit

Target: CPS1 (Carbamoyl-Phosphate Synthase 1, Mitochondrial (CPS1))

Binding Specificity: Mitochondrial

Reactivity: Human

Detection Method: Colorimetric

Application: ELISA

Characteristics: Homo sapiens,Human,Carbamoyl-phosphate synthase [ammonia], mitochondrial,Carbamoyl-phosphate synthetase I,CPSase I,CPS1,6.3.4.16

Components: Reagent (Quantity): Assay plate (1), Standard (2), Sample Diluent (1x20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A (1x120µl), Detection Reagent B (1x120µl), Wash Buffer(25 x concentrate) (1x30ml), Substrate (1x10ml), Stop Solution (1x10ml)

Application Details

Comment:

Gene Name: CPS1

Sample Volume: 100 μL

Plate: Pre-coated

Restrictions: For Research Use only

Independent Validation (ELISA)

Validation #103213 (ELISA)

by: Omary Lab, Department of Molecular & Integrative Physiology, University of Michigan Medical School

No.: #103213

Date: 06/11/2018

Antigen: CPS1

Lot Number: 7K295N

Method validated: ELISA

Positive Control: Human serum from patients with liver injury

Negative Control: Human serum from patients without liver injury

Notes:

Passed, the CPS1 ELISA kit ABIN1125223 specifically detects the antigen in human serum samples.

Validation Images

A. Standard curve and sample measurements using ABIN1125223. B. The standard curves for the assays reported were fitted with a 4-Parameter Logistic model, giving good quality fits (R2=0.999). C. CPS1 level comparison determined by western blotting and ABIN1125223. Normal serum from healthy humans was used undiluted. Serum from patient 1 was used undiluted, 1:5, and 1:25 diluted (middle panel). Sera from patient 2 and patient 3 were used at 1:5, 1:25, and 1:125 dilutions. 2µl of serum was used for Western blotting; presence of proteins was verified by Commassie staining (upper panel). The graph illustrates the ELISA results.

Full Methods

Full Protocol:

• Freeze human serum samples in aliquots until use. Dilute sera with PBS.
• Allow all reagents to reach RT and prepare all the reagents, standard solutions and samples as instructed by the manufacturer.
• Add 100µl of standard, blank or sample per well. Cover the plate. Incubate for 2h at 37°C.
• Remove the liquid.
• Add 100µl detection reagent A. Mix gently and cover the plate. Incubate for 1h at 37°C.
• Aspirate liquid and wash the wells 3x with 400µl wash buffer. Let it sit for 1-2min. Remove liquid Completely at each step.
• Add 100µl of detection reagent B. Cover the plate. Incubate for 1h at 37°C.
• Wash well 5x with 400µl wash buffer.
• Add 90µl substrate solution. Cover the plate. Incubate 15min at 37°C protected from light.
• Add 50µl stop solution. Gently tap the plate to ensure thorough mixing.
• Read absorbance at 450nm immediately.

Experimental Notes:

• The purpose of this report is to validate the measurement of human CPS1 in human serum samples, since it has been published that CPS1 is released into serum during liver injury, while it is not detected in healthy human serum (Weerasinghe et al. (2014)).

• To validate this ELISA, human serum samples previously validated to contain CPS1 by Western blotting were used. The signal in the positive controls samples (patients’ samples that contain high amount of CPS1 which was confirmed by Western blotting) was significantly higher than the blank whereas the signal from the negative control samples (healthy human sera in that CPS1 was not detected by Western blotting) was comparable to the baseline. 1:5, 1:25, or 1:125 diluted serum samples were tested as well.

• CPS1 was not detected in the sera of healthy human by both western blotting and ELISA method (lane 1 and 2). However, it is highly increased in the sera of patients with acute liver failure and the signals were in a dilution-dependent manner (lane 3 to 11).

• The standard curve for the assays reported were fitted with a 4-Parameter Logistic model. The calculated concentrations of standards were higher than expected, but it gave a good quality fits (R²=0.999). The results showed no CPS1 in the sera from healthy human, while it is highly increased in the sera from patients with liver injury, as expected. Although most of the samples were in the detection range, the calculated concentrations were highest in the most diluted samples.

Handling

Storage: 4 °C/-20 °C

Storage Comment: The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.

Target Details for CPS1

Target: CPS1 (Carbamoyl-Phosphate Synthase 1, Mitochondrial (CPS1))

Alternative Name: CPS1 (CPS1 Products)

Background: Synonyms: Carbamoyl-phosphate synthase [ammonia], mitochondrial,Carbamoyl-phosphate synthetase I,CPS1,CPSase I,Homo sapiens,Human

Pathways: Response to Growth Hormone Stimulus, Cellular Glucan Metabolic Process