Download biomedica product listVanin-1 ELISA Kit (Mouse/Rat VNN1) | BI-VAN1MR
-
Method
Sandwich ELISA, HRP/TMB, 12×8-well detachable strips
-
Sample type
Serum, plasma and urine
-
Sample volume
5 µl / well
-
Assay time
4 h / 30 min
-
Sensitivity
2.31 pmol/l
-
Standard range
0 – 200 pmol/l (= 0 – 10.6 ng/ml)
-
Conversion factor
1 pmol/l = 52.07 pg/ml (MW: 52.07 kDa)
-
Precision
In-between-run (n=5): ≤ 8 % CV
Within-run (n=7): ≤ 8 % CV
-
Specificity
Endogenous and recombinant mouse/rat Vanin-1.
-
Use
Research use only
-
Validation Data
See validation data tab for: precision, accuracy, dilution linearity, sample values
Mouse/Rat Vanin-1 ELISA Product Overview
This Vanin-1 ELISA kit is a 4.5 hour, 96-well sandwich ELISA for the quantitative determination of Vanin-1 in serum, plasma and urine.
Mouse/Rat Vanin-1 ELISA Assay Principle
The Vanin-1 mouse/rat ELISA kit is a sandwich enzyme immunoassay for the quantitative determination of Vanin-1 in mouse or rat samples. The kit utilizes recombinant mouse Vanin-1 as a calibrator. The VNN1 gene is conserved in chimpanzee, rhesus monkey, dog, cow, mouse, rat, and chicken https://www.ncbi.nlm.nih.gov/homologene/32130 .
The figure below explains the principle of the Vanin-1 sandwich ELISA:
Target antigen: mouse/rat Vanin-1
Target antigen: recombinant mouse Vanin-1
In a first step, assay buffer is pipetted into the wells of the microtiter strips. Thereafter, standard/control/sample and detection antibody (polyclonal sheep anti-mouse Vanin-1-HRPO) are pipetted into the wells, which are pre-coated with anti-mouse Vanin-1 antibody. Vanin-1 present in the standard/control/sample binds to the pre-coated antibody in the well and forms a sandwich with the detection antibody. In the washing step, all non-specific unbound material is removed. In a next step, the substrate (TMB, tetramethylbenzidine) is pipetted into the wells. The enzyme-catalyzed color change of the substrate is directly proportional to the amount of Vanin-1 present in the sample. This color change is detectable with a standard microplate reader. A dose response curve of the absorbance (optical density, OD at 450 nm) versus standard concentration is generated using the values obtained from the standards. The concentration of Vanin-1 in the sample is determined directly from the dose response curve.
Mouse/Rat Vanin-1 ELISA Typical Standard Curve
The figure below shows a typical standard curve for the Mouse Vanin-1 ELISA and Rat Vanin-1 ELISA. The Vanin ELISA kit is calibrated against recombinant mouse Vanin-1 peptide:
Mouse/Rat Vanin-1 ELISA Kit Components
|
Contents |
Description |
Quantity |
|
PLATE |
Anti-mouse Vanin-1 antibody pre-coated microtiter strips in stripholder packed in aluminium bag with desiccant |
12 x 8 tests |
|
WASHBUF |
Wash buffer concentrate 20x |
1 x 50 ml |
|
STD |
Stock standard (200 pmol/l) containing recombinant Mouse Vanin-1, (lyophilised) |
1 vial |
|
CTRL |
Control, lyophilised, exact concentration see label |
1 vial |
|
ASYBUF |
Assay buffer, ready to use |
1 x 7 ml |
|
CONJ |
Conjugate, polyclonal sheep anti-mouse Vanin-1 antibody - HRP |
1 x 6 ml |
|
SUB |
Substrate (TMB solution), ready to use |
1 x 13 ml |
|
STOP |
Stop solution, ready to use |
1 x 7 ml |
Storage instructions: All reagents of the Vanin-1 Mouse/Rat ELISA kit are stable at 4°C until the expiry date stated on the label of each reagent.
Serum, plasma and urine samples are suitable for use in this Vanin-1 ELISA assay. Do not change sample type during studies. We recommend duplicate measurements for all samples, standards and controls. The listed sample collection and storage conditions listed are intended as general guidelines.
Serum & Plasma
Collect venous blood samples in standardized serum separator tubes (SST) or standardized blood collection tubes using EDTA, heparin or citrate as an anticoagulant. For serum samples, allow samples to clot for 30 minutes at room temperature. Perform separation by centrifugation according to the tube manufacturer’s instructions for use. Assay the acquired samples immediately or aliquot and store at -25°C or lower. Lipemic or haemolysed samples may give erroneous results. Samples can undergo at least 4 freeze-thaw cycles.
Urine
Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile container. Centrifuge to remove particulate matter, assay immediately or aliquot and store at -25°C or lower. Samples can undergo at least four freeze-thaw cycles.
Reagent Preparation
Wash Buffer
|
1. |
Bring the WASHBUF concentrate to room temperature. Crystals in the buffer concentrate will dissolve at room temperature. |
|
2. |
Dilute the WASHBUF concentrate 1:20, e.g. 50 ml WASHBUF + 950 ml distilled or deionized water. Only use diluted WASHBUF when performing the assay. |
The diluted WASHBUF is stable up to one month at 4°C (2-8°C).
Stock Standards & Control
|
1. |
Pipette 200 µl of distilled or deionized water into the stock standard (STOCK STD) and control (CTRL) vials. The exact concentration is printed on the label of each vial. |
|
2. |
Leave at room temperature (18-26°C) for 10 min. Vortex gently. |
Reconstituted STDs and the CTRL are stable for three hours at room temperature (18-26°C). STDs and CTRLs are stable -25°C or lower until expiry date stated on the label and can be subjected to up to three freeze-thaw cycles.
Preparation of the standard curve
|
1. |
Use polypropylene tubes and mark them as STD6 to STD1 as shown below (Graph 1). |
|
2. |
Mark STOCK STD as STD7. |
|
3. |
Pipette 50 µl of ASYBUF (assay buffer) into each tubes marked as STD6 to STD1. |
|
4. |
Prepare a two-fold serial dilution to obtain STD6 to STD2. Pipette 50 µl of the reconstituted STOCK STD = STD7 into the tube labelled STD6. Mix thoroughly. Continue serial dilutions for STD5, STD4, STD3, STD2 (see Graph1). |
|
5. |
ASYBUF serves as the zero standard (=STD1, 0 pmol/l). |
Preparation of STD7 to STD1
Sample Preparation
Bring samples to room temperature and mix samples gently to ensure the samples are homogenous. We recommend duplicate measurements for all samples.
Samples with values above STD7 (200 pmol/l) can be diluted with ASYBUF (Assay buffer).
Mouse samples
Mouse serum, plasma and urine samples must be diluted 1+7 with assay buffer (ASYBUF), eg. 5 µl sample + 35 µl ASYBUF. Diluted samples are stable at 4°C (2-8°C) overnight. Thus dilutions can be prepared one day before analysis.
Rat samples
Rat samples are used undiluted.
Mouse/Rat Vanin-1 ELISA Assay Protocol
Read the entire protocol before beginning the assay.
|
1. |
Bring samples and reagents to room temperature (18-26°C). |
|
2. |
Mark positions for STD/CTRL/SAMPLE (standard/control/sample) on the protocol sheet. |
|
3. |
Take microtiter strips out of the aluminum bag. Store unused strips with desiccant at 4°C in the aluminum bag. Strips are stable until expiry date stated on the label. |
|
4. |
Pipette 50 µl ASYBUF (assay buffer, natural cap) into each well. |
|
5. |
Add 5 µl STD/CTRL/SAMPLE in duplicated into the respective wells. |
|
6. |
Add 50 µl CONJ (conjugate, amber cap) into each well. Swirl gently. |
|
7. |
Cover the plate tightly, swirl gently and incubate for 4 hours at room temperature (18-24°C). |
|
8. |
Aspirate and wash wells 5 x with 300 µl diluted WASHBUF (wash buffer). After the final wash, remove the remaining WASHBUF by strongly tapping the plate against a paper towel. |
|
12. |
Add 100 µl SUB (substrate, blue cap) into each well. |
|
13. |
Incubate for 30 min at room temperature in the dark. |
|
14. |
Add 50 µl STOP (stop solution, white cap) into each well. |
|
15. |
Measure absorbance immediately at 450 nm with reference 630 nm, if available. |
Calculation of Results
Read the optical density (OD) of all wells on a plate reader using 450 nm wavelength (reference wavelength 630 nm). Construct a standard curve from the absorbance read-outs of the standards using commercially available software capable of generating a four-parameter logistic (4-PL) fit. Alternatively, plot the standards’ concentration on the x-axis against the mean absorbance for each standard on the y-axis and draw a best fit curve through the points on the graph. Curve fitting algorithms other than 4-PL have not been validated and will need to be evaluated by the user.
Obtain sample concentrations from the standard curve. If required, pmol/l can be converted into pg/ml by applying a conversion factor (1 ng/ml = 18.87 pmol/l; Vanin-1 mouse/rat MW: 53 kDa).
Respective dilution factors have to be considered when calculating the final concentration of the sample.
Vanin-1 Protein
Vanin-1 is a GPI-anchored glycoprotein of 513 amino acids consisting of a base domain and an enzymatic nitrilase domain (Boersma et al., 2014). The ectoenzyme catalyzes the hydrolysis of pantetheine to pantothenic acid (vitamin B5) and cyteamine and thus, is involved in the regulation of oxidative stress and inflammation (Maras et al., 1999). Vanin-1 has a broad tissue expression with the highest levels being observed in kidney tubular epithelial cells (Pitari et al., 2000).The GPI anchor of Vanin-1 can be cleaved by a yet unknown mechanism, resulting in Vanin-1 being shed into the extracellular space.
|
Molecular weight |
52.07 kDa |
|
Cellular localization |
Extracellular, plasma membrane |
|
Post-translational modifications |
Glycosylation, lipidation (GPI-anchor) |
|
Sequence similarities |
Member of the Vanin family of proteins, sequence similarities with biotinidase family |
|
Alternative names |
Vascular non-inflammatory molecule 1, Tiff66, pantetheine hydrolase, pantetheinase, VNN1, HDLCQ8 |
|
Entrez/NCBI ID |
|
|
Genecards |
|
|
OMIM |
|
|
Protein Atlas |
|
|
Uniprot ID |
Vanin-1 Function
Vanin-1 is an epithelial ectoenzyme activating the conversion of pantetheine into pantothenic acid (vitamin B5) and cysteamine (Pitari et al., 2000). It has been suggested that the release of cysteamine by Vanin-1 promotes oxidative tissue damage and inflammation by inhibiting the activity of antioxidants like superoxide dismutase (SOD) and glutathione (GSH) (Hosohata et al., 2011; Saghaei et al., 2012). Indeed, Vanin-1 knockout mice have elevated stores of GSH and are more resistant to oxidative injury induced by whole-body gamma irradiation (Berruyer et al., 2004). On the other hand, several reports indicate that Vanin-1 might also act as tissue sensor for oxidative stress. In mice, antioxidant response-like elements could be identified in the promotor region of Vanin-1, which enhance the expression of Vanin-1 in the presence of oxidative stress (Berruyer et al., 2004). Similarly, Vanin-1 expression was shown to be upregulated in a human proximal tubular cell line after exposure to organic solvents (Hosohata et al., 2011). After renal ischemia-reperfusion in rats, a model involving oxidative tissue damage, renal Vanin-1 expression was also found to be upregulated (Yoshida et al., 2002).
The highest levels of Vanin-1 expression could be assigned to renal tubular epithelial cells, while no expression is detectable in glomeruli (Hosohata et al., 2011; Pitari et al., 2000). Hence, Vanin-1 released from renal cells could be detectable in urine. In a study aimed to identify biomarkers for renal tubular injury, Hosohata and colleagues could indeed show in a rat model of nephrotoxicant-induced injury that Vanin-1 is upregulated in renal tubules earlier than other markers and shed into urine (Hosohata et al., 2011). Subsequent studies further verified the validity of Vanin-1 as an early biomarker of renal tubular damage in drug-induced acute kidney injury (Hosohata et al., 2012, 2016a), obstructive nephropathy (Washino et al., 2019) and hydronephrosis (Hosohata et al., 2018), diabetic nephropathy (Fugmann et al., 2011), renal injury in experimental colitis (Hosohata et al., 2014) and spontaneously hypertensive rats under high salt intake (Hosohata et al., 2016b; Washino et al., 2018). Of note, Vanin-1 seems to have superior predictive value for acute kidney injury than established markers KIM-1, NGAL, or NAG (Fugmann et al., 2011; Hosohata, 2016; Hosohata et al., 2011).
-
Nephrology
- Acute kidney injury (Hosohata et al., 2016a)
- Diabetic nephropathy (Fugmann et al., 2011)
- Drug-induced acute kidney injury (Hosohata et al., 2016a)
- Hydronephrosis (Hosohata et al., 2018), obstructive nephropathy (Washino et al., 2019)
Literature
-
Vanin-1-/- mice exhibit a glutathione-mediated tissue resistance to oxidative stress.
Berruyer, C., Martin, F.M., Castellano, R., Macone, A., Malergue, F., Garrido-Urbani, S., Millet, V., Imbert, J., Duprè, S., Pitari, G., Naquet, P., Galland, F., 2004. Mol. Cell. Biol. 24, 7214–7224.
PMID:15282320; PMCID: PMC479710 -
The structure of vanin 1: a key enzyme linking metabolic disease and inflammation.
Boersma, Y.L., Newman, J., Adams, T.E., Cowieson, N., Krippner, G., Bozaoglu, K., Peat, T.S., 2014. Acta Crystallogr. D Biol. Crystallogr. 70, 3320–3329.
PMID:25478849 -
Fugmann, T., Borgia, B., Révész, C., Godó, M., Forsblom, C., Hamar, P., Holthöfer, H., Neri, D., Roesli, C., 2011. Kidney Int. 80, 272–281.
PMID:21544065 -
Vanin-1 in Renal Pelvic Urine Reflects Kidney Injury in a Rat Model of Hydronephrosis.
Hosohata, K., Jin, D., Takai, S., Iwanaga, K., 2018. Int J Mol Sci 19.
PMID:30332759; PMCID: PMC6214032 -
Role of Oxidative Stress in Drug-Induced Kidney Injury.
Hosohata, K., 2016. Int J Mol Sci 17.
PMID:27809280; PMCID: PMC5133827 -
Hosohata, K., Washino, S., Kubo, T., Natsui, S., Fujisaki, A., Kurokawa, S., Ando, H., Fujimura, A., Morita, T., 2016a. Toxicology 359–360, 71–75.
PMID:27317936 -
Hosohata, K., Yoshioka, D., Tanaka, A., Ando, H., Fujimura, A., 2016b. Hypertens. Res. 39, 19–26.
PMID:26376947 -
Early detection of renal injury using urinary vanin-1 in rats with experimental colitis.
Hosohata, K., Ando, H., Fujimura, A., 2014. J Appl Toxicol 34, 184–190.
PMID:23307618 -
Urinary Vanin-1 As a Novel Biomarker for Early Detection of Drug-Induced Acute Kidney Injury.
Hosohata, K., Ando, H., Fujimura, A., 2012. Journal of Pharmacology and Experimental Therapeutics 341, 656–662.
-
Vanin-1: a potential biomarker for nephrotoxicant-induced renal injury.
Hosohata, K., Ando, H., Fujiwara, Y., Fujimura, A., 2011. Toxicology 290, 82–88.
PMID:21907259 -
Is pantetheinase the actual identity of mouse and human vanin-1 proteins?
Maras, B., Barra, D., Duprè, S., Pitari, G., 1999. FEBS Letters 461, 149–152.
-
Pitari, G., Malergue, F., Martin, F., Philippe, J.M., Massucci, M.T., Chabret, C., Maras, B., Duprè, S., Naquet, P., Galland, F., 2000. FEBS Letters 483, 149–154.
-
Effects of captopril on the cysteamine-induced duodenal ulcer in the rat.
Saghaei, F., Karimi, I., Jouyban, A., Samini, M., 2012. Exp. Toxicol. Pathol. 64, 373–377.
PMID:21036019 -
Washino, S., Hosohata, K., Oshima, M., Okochi, T., Konishi, T., Nakamura, Y., Saito, K., Miyagawa, T., 2019. Int J Mol Sci 20.
PMID:30791405; PMCID: PMC6412925 -
Washino, S., Hosohata, K., Jin, D., Takai, S., Miyagawa, T., 2018. Clin. Exp. Pharmacol. Physiol. 45, 261–268.
PMID:29027259 -
Monitoring changes in gene expression in renal ischemia-reperfusion in the rat.
Yoshida, T., Kurella, M., Beato, F., Min, H., Ingelfinger, J.R., Stears, R.L., Swinford, R.D., Gullans, S.R., Tang, S.-S., 2002. Kidney Int. 61, 1646–1654.
PMID:11967014
PMID:15282320; PMCID: PMC479710Calibration
The Vanin-1 Mouse/Rat immunoassay is calibrated against Mouse Vanin-1 protein.
Mouse/Rat Vanin-1 ELISA Detection Limit & Sensitivity
To determine the sensitivity of the Vanin-1 mouse/rat ELISA, experiments measuring the Lower Limit of Detection (LOD) and the Lower Limit of Quantification (LLOQ) were conducted.
The LOD, also called the detection limit, is the lowest point at which a signal can be distinguished above the background signal, i.e. the signal that is measured in the absence of Vanin-1, with a confidence level of 99%. It is defined as the mean back calculated concentration of standard 1 (0 pmol/l of Vanin-1, five independent measurements) plus three times the standard deviation of the measurements.
The LLOQ, or sensitivity of an assay, is the lowest concentration at which an analyte can be accurately quantified. The criteria for accurate quantification at the LLOQ are an analyte recovery between 75 and 125% and a coefficient of variation (CV) of less than 25%. To determine the LLOQ, standard 2, i.e. the lowest standard containing Vanin-1, is diluted, measured five times and its concentration is back calculated. The lowest dilution, which meets both criteria, is reported as the LLOQ.
The following values were determined for the Vanin-1 mouse/rat ELISA:
|
LOD |
2.31 pmol/l |
|
LLOQ |
6.25 pmol/l |
Mouse/Rat Vanin-1 ELISA Precision
The precision of an ELISA is defined as its ability to measure the same concentration consistently within the same experiments carried out by one operator (within-run precision or repeatability) and across several experiments using the same samples but conducted by several operators using different ELISA lots (in-between-run precision or reproducibility).
Within-Run Precision
Within-run / intra-assay precision was assessed by measuring 2 samples of known concentrations 6 times within one Vanin-1 mouse/rat ELISA kit lot by one operator.
|
ID |
n |
Mean Vanin-1 [pmol/l] |
SD [pmol/l] |
CV (%) |
|
Sample 1 |
6 |
8 |
0.6 |
8 |
|
Sample 2 |
6 |
20 |
1.6 |
8 |
In-Between-Run Precision
In-between-run /intra-assay precision was assessed by measuring 2 samples 5 times within Vanin-1 mouse/rat ELISA kit lots by 2 different operators.
|
ID |
n |
Mean Vanin-1 [pmol/l] |
SD [pmol/l] |
CV (%) |
|
Sample 1 |
5 |
63 |
4.0 |
6 |
|
Sample 2 |
5 |
41 |
3.1 |
8 |
Mouse/Rat Vanin-1 ELISA Accuracy
The accuracy of an ELISA is defined as the precision with which it can recover samples of known concentrations.
The recovery of the Vanin-1 Mouse/Rat ELISA was measured by adding recombinant Mouse Vanin-1 to mouse or rat samples containing a known concentration endogenous Vanin-1. The %recovery of the spiked concentration was calculated as the percentage of measured compared over the expected value. All our ELISAs are expected to have %recovery rates within 15% of the nominal value of the sample.
This table shows the summary of the recovery experiments in the Vanin-1 mouse/rat ELISA in different sample matrices:
|
|
% Recovery |
||||
|
+25 pmol/l |
+100 pmol/l |
||||
|
Sample matrix |
n |
Mean |
Range |
Mean |
Range |
|
Mouse |
7 |
93 |
86-124 |
90 |
78-101 |
|
Rat |
4 |
94 |
68-103 |
87 |
78-95 |
Data showing recovery (R [%]) of recombinant Mouse Vanin-1 in mouse serum samples:
|
Mouse Vanin-1 [pmol/l] |
% Recovery |
|||||
|
Sample matrix |
ID |
Reference |
+ 25 pmol/l |
+ 100 pmol/l |
+ 25 pmol/l |
+ 100 pmol/l |
|
Mouse |
m1 |
10 | 27 | 84 | 86 | 78 |
|
Mouse |
m2 |
25 | 36 | 106 | 93 | 93 |
|
Mouse |
m3 |
43 | 44 | 107 | 90 | 85 |
|
Mouse |
m4 |
27 | 37 | 103 | 94 | 88 |
|
Mean |
92 | 87 | ||||
|
Min |
86 | 78 | ||||
|
Max |
94 | 93 | ||||
Data showing recovery of recombinant Mouse Vanin-1 in a mouse urine samples:
|
Mouse Vanin-1 [pmol/l] |
% Recovery |
|||||
|
Sample matrix |
ID |
Reference |
+ 25 pmol/l |
+ 100 pmol/l |
+ 25 pmol/l |
+ 100 pmol/l |
|
Mouse |
m5 |
32 |
47 |
107 |
124 |
90 |
|
Mouse |
m6 |
22 |
34 |
101 |
92 |
89 |
|
Mouse |
m7 |
37 |
41 |
121 |
89 |
101 |
|
Mean |
92 |
90 |
||||
|
Min |
89 |
89 |
||||
|
Max |
124 |
101 |
||||
Data showing recovery of recombinant Mouse Vanin-1 in rat plasma samples:
|
Mouse Vanin-1 [pmol/l] |
% Recovery |
|||||
|
Sample matrix |
ID |
Reference |
+ 25 pmol/l |
+ 100 pmol/l |
+ 25 pmol/l |
+ 100 pmol/l |
|
Rat |
r1 |
6 |
26 |
81 |
91 |
78 |
|
Rat |
r2 |
3 |
19 |
85 |
68 |
82 |
|
Rat |
r3 |
8 |
28 |
100 |
95 |
95 |
|
Rat |
r4 |
10 |
31 |
96 |
103 |
90 |
|
Mean |
93 |
86 |
||||
|
Min |
68 |
78 |
||||
|
Max |
103 |
95 |
||||
Mouse/Rat Vanin-1 ELISA Dilution Linearity & Parallelism
Tests of dilution linearity and parallelism ensure that both endogenous and recombinant samples containing Vanin-1 behave in a dose dependent manner and are not affected by matrix effects. Dilution linearity assesses the accuracy of measurements in diluted samples spiked with known concentrations of recombinant analyte. By contrast, parallelism refers to dilution linearity in samples and provides evidence that the endogenous analyte behaves in same way as the recombinant one.
Dilution Linearity
Dilution linearity was assessed by serially diluting samples spiked with 100 pmol/l recombinant Mouse Vanin-1 with assay buffer.
The figure and table below show the mean recovery and range of serially diluted recombinant Mouse Vanin-1 in several sample matrices:
|
% Recovery of recombinant Mouse Vanin-1 in diluted samples |
|||||||
|
|
1+1 |
1+3 |
1+7 |
||||
|
Sample matrix |
n |
Mean |
Range |
Mean |
Range |
Mean |
Range |
|
Mouse |
4 |
85 |
80-97 |
80 |
73-83 |
75 |
62-96 |
|
Rat |
3 |
93 |
88-105 |
110 |
106-119 |
126 |
115-136 |
Data showing dilution linearity of 100 pmol/l recombinant Mouse Vanin-1 spiked into mouse serum samples (ref) containing endogenous Vanin-1:
|
Mouse Vanin-1 [pmol/l] |
Recovery (%) |
|||||||
|
Sample matrix |
ID |
Ref + 100 pmol/l |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
Mouse |
m1 |
104 |
42 |
19 |
9 |
80 |
73 |
71 |
|
Mouse |
m2 |
121 |
49 |
24 |
15 |
81 |
80 |
96 |
|
Mouse |
m3 |
102 |
46 |
20 |
8 |
90 |
80 |
62 |
|
Mouse |
m4 |
112 |
55 |
23 |
11 |
97 |
83 |
80 |
|
|
|
|
|
|
Mean |
85 |
80 |
75 |
|
|
|
|
|
|
Min |
80 |
73 |
62 |
|
|
|
|
|
|
Max |
97 |
83 |
96 |
Data showing dilution linearity of 100 pmol/l recombinant Mouse Vanin-1 spiked into rat plasma samples (ref) containing endogenous Vanin-1:
|
Vanin-1 [pmol/l] |
Recovery (%) |
|||||||
|
Sample matrix |
ID |
Ref + 100 pmol/l |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
Rat |
r1 |
82 |
43 |
22 |
12 |
105 |
106 |
115 |
|
Rat |
r2 |
92 |
43 |
25 |
15 |
93 |
110 |
126 |
|
Rat |
r3 |
89 |
39 |
27 |
15 |
88 |
119 |
136 |
|
|
|
|
|
|
Mean |
93 |
110 |
126 |
|
|
|
|
|
|
Min |
88 |
106 |
115 |
|
|
|
|
|
|
Max |
105 |
119 |
136 |
Parallelism
Parallelism was assessed by serially diluting samples containing endogenous Vanin-1 with assaybuffer.
The figure and table below show the mean recovery and range of serially diluted endogenous Vanin-1 in several sample matrices:
|
% Recovery of endogenous Vanin-1 in diluted mouse and rat samples |
|||||||
|
|
1+1 |
1+3 |
1+7 |
||||
|
Sample matrix |
n |
Mean |
Range |
Mean |
Range |
Mean |
Range |
|
Mouse |
4 |
97 |
84-103 |
84 |
71-94 |
95 |
85-105 |
|
Rat |
3 |
92 |
87-106 |
- |
- |
- |
- |
Data showing dilution linearity of endogenous Vanin-1 in mouse serum samples:
|
Mouse Vanin-1 [pmol/l] |
Recovery (%) |
|||||||
|
Sample matrix |
ID |
Ref + 100 pmol/l |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
Mouse |
m1 |
43 |
20 |
9 |
5 |
92 |
81 |
92 |
|
Mouse |
m2 |
27 |
11 |
5 |
3 |
84 |
71 |
85 |
|
Mouse |
m3 |
32 |
17 |
7 |
4 |
103 |
87 |
105 |
|
Mouse |
m4 |
37 |
19 |
9 |
5 |
102 |
94 |
98 |
|
|
|
|
|
|
Mean |
97 |
84 |
95 |
|
|
|
|
|
|
Min |
84 |
71 |
85 |
|
|
|
|
|
|
Max |
103 |
94 |
105 |
Data showing dilution linearity of endogenous Vanin-1 in rat plasma samples:
|
Vanin-1 [pmol/l] |
Recovery (%) |
|||||||
|
Sample matrix |
ID |
Ref + 100 pmol/l |
1+1 |
1+3 |
1+7 |
1+1 |
1+3 |
1+7 |
|
Rat |
r1 |
14 |
7 |
- |
- |
92 |
- |
- |
|
Rat |
r2 |
9 |
3 |
- |
- |
87 |
- |
- |
|
Rat |
r3 |
10 |
5 |
- |
- |
106 |
- |
- |
|
|
|
|
|
|
Mean |
92 |
- |
- |
|
|
|
|
|
|
Min |
87 |
- |
- |
|
|
|
|
|
|
Max |
106 |
- |
- |
Mouse/Rat Vanin-1 ELISA Specificity
The specificity of an ELISA is defined as its ability to exclusively recognize the analyte of interest.
Competition of Signal
Competition experiments were carried out by pre-incubating mouse and rat samples with an excess of coating antibody. The concentration measured in this mixture was then compared to a reference value, which was obtained from the same sample but without the pre-incubation step. Mean competition was 99%.
|
Vanin-1 mouse rat[pmol/l] |
|
|||
|
Sample matrix |
ID |
Reference |
Reference + 3.5 ng/well Vanin-1 antibody |
% Competition |
|
Mouse |
m1 |
31 |
0 |
100 |
|
Mouse |
m2 |
49 |
0 |
100 |
|
Mouse |
m3 |
36 |
0 |
100 |
|
Mouse |
m1 |
14 |
0 |
100 |
|
Rat |
r1 |
11 |
1 |
95 |
|
Rat |
r2 |
14 |
0 |
97 |
|
Rat |
r3 |
13 |
0 |
100 |
|
Rat |
r4 |
11 |
0 |
100 |
Cross Reactivity
Potentially cross-reactive with Vanin-1 from various monkey species. No cross-reactivity with mouse Vanin-1.
Sample Stability
Standard Freeze-thaw Stability
The stability of recombinant Mouse Vanin-1 was tested by comparing 3 measurements in standards spiked to different values that had undergone 5 freeze-thaw cycles.
The mean recovery of standard concentration after 5 freeze-thaw cycles is 117%.
Vanin-1 concentrations of samples after freeze-thaw (F/T) cycles:
|
Mouse Vanin-1 [pmol/l] |
Recovery (%) 5x F/T vs Reference |
|||||
|
Sample matrix |
ID |
Reference |
1x |
3x |
5x |
|
|
Standard |
s1 |
34 |
42 |
40 |
47 |
137 |
|
Standard |
s2 |
75 |
84 |
87 |
88 |
117 |
|
Standard |
s3 |
166 |
180 |
176 |
183 |
110 |
| Mean |
117 |
|||||
Sample Values
Vanin-1 Values
To provide reference values for circulating mouse and rat Vanin-1, a panel of samples was tested.
A summary of the results is shown below:
| Vanin-1 [pmol/l] | |||||
|
Sample matrix |
n |
Mean |
Median |
Minimum |
Maximum |
|
Mouse serum |
5 |
24 |
22 |
9 |
39 |
|
Mouse plasma |
5 |
25 |
24 |
19 |
34 |
|
Mouse urine |
6 |
26 |
21 |
3 |
62 |
|
Rat serum |
8 |
7 |
7 |
6 |
11 |
|
Rat plasma |
8 |
8 |
7 |
5 |
16 |
It is recommended to establish the normal range for each laboratory.
