Mouse Periostin ELISA Kit (Mouse POSTN) | BI-20433MS

Product details

Product overview
Principle of the Assay

Typical Standart Curve
ELISA kit Components

Mouse Periostin ELISA Product Overview

The Mouse Periostin ELISA kit is a 4.5 hour, 96-well sandwich ELISA for the quantitative determination of Periostin in mouse serum and plasma.

Mouse Periostin ELISA Assay Principle

This Periostin Assay is a sandwich enzyme immunoassay for the quantitative determination of Periostin in mouse serum and plasma samples.

The figure below explains the principle of the Mouse Periostin sandwich ELISA:

Capture antibody: monoclonal mouse anti-mouse Periostin antibody
Detection antibody: polyclonal goat anti-mouse Periostin antibody, biotin labeled
Target antigen: mouse Periostin

In a first step, standard/control/sample and detection antibody (biotinylated goat anti-mouse Periostin) are pipetted into the wells of the microtiter strips, which are pre-coated with monoclonal mouse anti-Periostin antibody. Periostin present in the sample binds to the pre-coated antibody in the well and forms a sandwich with the detection antibody. In a wash step non-specific unbound material is removed. In a next step, conjugate (strepatvidin-HRP) is added and reacts with the detection antibody. After another washing 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 mouse Periostin present in the sample. This color change is detectable with a standard microtiter plate reader. A dose response curve of the absorbance (optical density, OD at 450 nm) vs. standard concentration is generated, using the values obtained from the standard. The concentration of mouse Periostin in the sample is determined directly from the dose response curve.

Mouse Periostin ELISA Typical Standard Curve

The figure below shows a typical standard curve for the Periostin ELISA kit mouse:

Mouse Periostin ELISA Kit Components

Contents	Description	Quantity
PLATE	Mouse monoclonal anti-mouse Periostin antibody pre-coated microtiter strips in a strip holder, packed in an aluminum bag with desiccant	12 x 8 tests
WASHBUF	Wash buffer concentrate 20x, natural cap	1 x 50 ml
STOCK STD	Stock standard (16 nmol/l), recombinant mouse Periostin, red cap, lyophilized	1 vial
CTRL	Control, yellow cap, lyophilized, exact concentration see label	1 vial
ASYBUF	Assay buffer, red cap, ready to use	1 x 110 ml
AB	Goat polyclonal anti-mouse Periostin antibody, biotinylated, green cap, ready to use	1 x 7 ml
CONJ	Conjugate, (streptavidin-HRP), amber bottle, amber cap, ready to use	1 x 18 ml
SUB	Substrate (TMB solution), amber bottle, blue cap, ready to use	1 x 22 ml
STOP	Stop solution, white cap, ready to use	1 x 7 ml

Storage instructions: all reagents of the mouse Periostin ELISA kit are stable at 4°C until the expiry date stated on the label of each reagent.

Instructions for use

SAMPLE COLLECTION & STORAGE
REAGENT PREPARATION

SAMPLE PREPARATION
ASSAY PROTOCOL

Sample Collection & Storage

Serum and plasma are suitable for use in this Periostin kit. We recommend duplicate measurements for all samples, standards and controls. Do not change sample type during studies. The sample collection and storage conditions listed are intended as general guidelines.

Serum & Plasma

Collect venous blood samples by using standardized blood collection tubes for serum or plasma. Perform serum and plasma separation by centrifugation according to supplier’s instructions of the blood collection devices and measure the acquired serum or plasma samples as soon as possible. For longer storage aliquot and store samples at -25°C or lower. Samples are stable for at least four freeze-thaw cycles. Thawed samples should be assayed as soon as possible. Lipemic or haemolyzed samples may give erroneous results.

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).

Control

1.	Pipette 200 µl of distilled or deionized water into the control (CTRL) vial. The exact concentration is stated on the label of the vial.
2.	Leave at room temperature (18-26°C) for 15 min. Vortex gently.
3.	CTRL must be diluted 1+200 with ASYBUF (assay buffer) prior to the assay, e.g. 5 µl CTRL + 1000 µl ASYBUF (or 2.5 µl CTRL + 500 µl ASYBUF).

Note: 100 μl pre-diluted CTRL is required per well.

Reconstituted CTRL is stable at -25°C or lower until expiry date stated on the label and can undergo at least three freeze-thaw cycles. Pre-diluted CTRL should be measured as soon as possible (do not store or freeze).

Standards

1.	Reconstitute the STOCK STD (stock standard) in 200 µl deionized water. Leave at room temperature (18-26°C) for 15 min and mix well prior to making dilutions.
2.	Mark tubes STD7 to STD1. Use polypropylene tubes.
3.	Pipette 1000 µl of ASYBUF (assay buffer) into tube marked as STD7.
4.	Pipette 300 µl of assay buffer into tubes marked as STD6 to STD1.
5.	Pipette 5 µl of the reconstituted STOCK STD into tube marked as STD7. Mix thoroughly.
6.	Prepare a two-fold serial dilution to obtain STD6 to STD2. Pipette 300 µl STD7 into the tube labeled STD6. Mix thoroughly. Continue serial dilutions for STD5, STD4, STD3, STD2.
7.	ASYBUF serves as STD1 (= 0 nmol/l mouse Periostin).

Note: 100 µl pre-diluted STD is required per well.

Established standard curve already includes the 1+200 assay pre-dilution, thus sample concentrations can be calculated directly from the standard curve without considering the 1+200 sample pre-dilution (see also chapter calculation of results).

Reconstituted STOCK STD is stable at -25°C or lower until expiry date stated on the label and can undergo at least three freeze-thaw cycles. Diluted STDs (standards) should be measured as soon as possible (do not store or freeze).

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 must be diluted 1+200 with ASYBUF (assay buffer) prior to the assay, e.g. 5 µl sample + 1000 µl ASYBUF.

Note: 100 μl pre-diluted sample is required per well.

Samples for which the OD value exceeds the highest point of the standard range can be further diluted with ASYBUF. Diluted samples should be measured as soon as possible (do not store or freeze).

Mouse Periostin 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.	Add 100 µl pre-diluted STD/CTRL/SAMPLE (standard/control/sample) into the respective wells. See chapters reagent preparation and sample preparation for details.
5.	Add 50 µl AB (biotinylated anti-Periostin antibody, green cap) into each well, swirl gently.
6.	Cover the plate tightly and incubate for 3 hours at room temperature (18-26°C).
7.	Aspirate and wash wells 5 x with 300 µl diluted WASHBUF. After the final wash, remove the remaining WASHBUF by strongly tapping plate against a paper towel.
8.	Add 150 µl CONJ (conjugate, amber cap) into each well. Swirl gently.
9.	Cover tightly and incubate for 1 hour at room temperature.
10.	Aspirate and wash wells 5 x with 300 µl diluted WASHBUF. After the final wash, remove the remaining WASHBUF by strongly tapping plate against a paper towel.
11.	Add 150 µl SUB (substrate, blue cap) into each well.
12.	Incubate for 30 min at room temperature in the dark.
13.	Add 50 µl STOP (stop solution, white cap) into each well. Swirl gently.
14.	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.

Samples, control, and standards are all diluted 1+200 prior to the assay. Hence, there is no need to take this dilution factor into account. Sample concentrations can be read directly from the standard curve.

Sample dilutions above 1+200 have to be considered when calculating the final sample concentration.

The quality control (QC) protocol supplied with the kit shows the results of the final release QC for each kit lot. Data for OD obtained by customers may differ due to various influences including the normal decrease of signal intensity throughout shelf life. However, this does not affect validity of results as long as an OD of 1.00 or more is obtained for the STD with the highest concentration and the value of the CTRL is within the target range (see label).

Background & Therapeutic Areas

Periostin Protein

Periostin (OSF-2) is secreted as a homodimeric soluble extracellular matrix protein expressed in collagen-rich fibrous connective tissues. Periostin is involved in osteoblast recruitment, attachment and spreading. It has been associated with the epithelial-mesenchymal transition in cancer and with the differentiation of mesenchyme in the developing heart. Periostin has functions in osteology, tissue repair, oncology, cardiovascular and respiratory diseases, and in various inflammatory settings. There are at least 5 isoforms of mouse Periostin, caused by alternative splicing (http://www.uniprot.org/uniprot/Q15063).

Molecular weight	93 kDa
Cellular localisation	Extracellular or secreted
Post-translational modifications	Glycosylation, disulphide bonds
Sequence similarities	FAS1 superfamily
Alternative names	POSTN, OSF-2, OSF2, PDLPN, PN
OMIM	608777
Uniprot ID	Q15063

Periostin Function

Periostin is involved in osteoblast recruitment, attachment and spreading. It has been associated with the epithelial-mesenchymal transition in cancer and with the differentiation of mesenchyme in the developing heart. Periostin has functions in osteology, tissue repair, oncology, cardiovascular and respiratory diseases, and in various inflammatory settings.

Validation Data

Calibration
Detection Limit & Sensitivity
Precision
Accuracy

Dilution Linearity & Paralellism
Specificity
Sample Stability
Sample Values

All Biomedica ELISAs are validated according to international FDA/ICH/EMEA guidelines. For more information about our validation guidelines, please refer to our quality page and published validation guidelines and literature.

Show validation literature

CPMP/ICH/381/95
ICH Topic Q2 (R1) „Validation of Analytical Procedures: Text and Methodology“
including:
ICH Q2A “Text on Validation of Analytical Procedures”
ICH Q2B “Validation of Analytical Procedures: Methodology”
Food and Drug Administration
Guidance for Industry, Bioanalytical Method Validation, Draft Guidance, September 2013

Calibration

The mouse Periostin immunoassay is calibrated against recombinant mouse Periostin isoform 1 of Q62009 (Uniprot ID).

Mouse Periostin ELISA Detection Limit & Sensitivity

To determine the sensitivity of the mouse Periostin 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 Periostin, with a confidence level of 99%. It is defined as the mean back calculated concentration a Periostin-free sample (three 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%. The lowest concentration of Periostin, which meets both criteria, is reported as the LLOQ.

The following values were determined for the mouse Periostin ELISA:

LOD	0.003 nmol/l
LLOQ	0.125 nmol/l

Mouse Periostin 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 at different locations using different ELISA lots (in-between-run precision or reproducibility).

Within-Run Precision

Within-run precision was tested by measuring the same samples five times within one mouse Periostin ELISA lot. The experiment was conducted by one operator.

ID	n	Mean mouse Periostin [nmol/l]	SD [pmol/l]	CV (%)
Sample 1	5	1.0	0.06	6
Sample 2	5	7.8	0.14	2

In-Between-Run Precision

In-between-run precision was assessed by measuring the same samples 15 times within multiple mouse Periostin ELISA lots. The measurements were carried out by multiple operators.

ID	n	Mean mouse Periostin [nmol/l]	SD [pmol/l]	CV (%)
Sample 1	15	1.0	0.06	6
Sample 2	15	7.9	0.22	3

Mouse Periostin 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 mouse Periostin ELISA was measured by adding recombinant mouse Periostin to mouse samples containing a known concentration endogenous Periostin. The % recovery of the spiked concentration was calculated as the percentage of measured compared over the expected value.

This table shows the summary of the recovery experiments in the mouse Periostin ELISA in different sample matrices:

		% Recovery
		+1.6 nmol/l		+8 nmol/l
Sample matrix	n	Mean	Range	Mean	Range
Mouse serum	4	72	60 - 86	97	88 - 107
Mouse plasma	4	86	67 - 96	88	70 - 100

Show individual measurements

Data showing recovery of Periostin in mouse serum samples:

		Mouse Periostin [nmol/l]			% Recovery
Sample matrix	ID	Reference	+1.6 nmol/l	+8 nmol/l	+1.6 nmol/l	+8 nmol/l
Mouse serum	ms1	1.7	2.7	7.8	79	88
Mouse serum	ms2	4.1	2.7	9.2	60	89
Mouse serum	ms3	3.4	4.0	10.2	64	107
Mouse serum	ms4	2.5	3.6	9.7	86	105
				Mean	72	97
				Min	60	88
				Max	86	107

Data showing recovery of Periostin in mouse plasma samples:

		Mouse Periostin [nmol/l]			% Recovery
Sample matrix	ID	Reference	+1.6 nmol/l	+8 nmol/l	+1.6 nmol/l	+8 nmol/l
Mouse plasma	mp1	2.9	3.6	67	67	70
Mouse plasma	mp2	3.2	4.4	96	96	85
Mouse plasma	mp3	2.6	3.7	85	85	98
Mouse plasma	mp4	3.2	4.4	96	96	100
				Mean	86	88
				Min	67	70
				Max	96	100

Mouse Periostin ELISA Dilution Linearity and Parallelism

Tests of dilution linearity and parallelism ensure that both endogenous and recombinant samples containing mouse Periostin behave in a dose dependent manner and are not affected by matrix effects. Dilution linearity assesses the accuracy of measurements in diluted mouse samples spiked with known concentrations of recombinant analyte. By contrast, parallelism refers to dilution linearity in mouse samples and provides evidence that the endogenous analyte behaves the same way as the recombinant one. For dilution linearity and parallelism are assessed for each sample type and are considered acceptable if the results are within 20% of the expected concentration.

Dilution Lineartiy

Dilution linearity was assessed by serially diluting mouse samples spiked with 8 nmol/l recombinant mouse Periostin.

The table below shows the mean recovery and range of serially diluted recombinant mouse Periostin in mouse serum and plasma:

		% Recovery of recombinant mouse Periostin in diluted samples
		1 + 1		1 + 3		1 + 7
Sample matrix	n	Mean	Range	Mean	Range	Mean	Range
Mouse serum	4	120	111-135	117	110 - 130	111	97 - 118
Mouse plasma	4	113	109 - 120	102	99 - 106	93	89 - 96

Show individual measurements

Data showing dilution linearity of recombinant mouse Periostin in mouse serum samples. Reference values indicate sample concentrations after the 8 nmol/l spike:

		Mouse Periostin [nmol/l]				% Recovery
Sample matrix	ID	Reference	1+1	1+3	1+7	1+1	1+3	1+7
Mouse serum	ms1	10.1	6.8	3.3	1.5	135	130	118
Mouse serum	ms2	10.1	6.0	2.8	1.2	118	110	97
Mouse serum	ms3	11.5	6.6	3.3	1.6	115	114	112
Mouse serum	ms4	11.7	6.5	3.4	1.7	111	115	115
					Mean	120	117	111
					Min	111	110	97
					Max	135	130	118

Data showing dilution linearity of recombinant mouse Periostin in mouse plasma samples. Reference values indicate sample concentrations after the 8 nmol/l spike:

		Mouse Periostin [nmol/l]				% Recovery
Sample matrix	ID	Reference	1+1	1+3	1+7	1+1	1+3	1+7
Mouse plasma	mp1	7.4	4.2	2.0	0.9	113	106	96
Mouse plasma	mp2	8.2	4.5	2.1	0.9	110	99	89
Mouse plasma	mp3	7.6	4.6	1.9	0.9	120	99	96
Mouse plasma	mp4	8.1	4.4	2.1	0.9	109	106	91
					Mean	113	102	93
					Min	109	99	89
					Max	120	106	96

Parallelism

Parallelism was assessed by serially diluting mouse samples containing endogenous mouse Periostin with assay buffer.

The table below shows the mean recovery and range of serially diluted endogenous mouse Periostin in mouse serum and plasma samples:

		%Recovery of endogenous mouse Periostin in diluted samples
		1+1		1+3
Sample matrix	ID	Mean	Range	Mean	Range
Mouse serum	4	116	114 - 119	113	110 - 116
Mouse plasma	4	128	121 - 147	130	108 - 163

Show individual measurements

Data showing dilution linearity of endogenous mouse Periostin in mouse serum samples:

		Mouse Periostin [nmol/l]			% Recovery
Sample matrix	ID	Reference	1+1	1+3	1+1	1+3
Mouse serum	ms1	1.7	0.9	0.5	115	116
Mouse serum	ms2	4.1	2.5	1.1	119	111
Mouse serum	ms3	3.4	1.9	0.9	114	110
Mouse serum	ms4	2.5	1.4	0.7	116	116
				Mean	128	130
				Min	121	108
				Max	147	163

Data showing dilution linearity of endogenous mouse Periostin in mouse plasma samples:

		Mouse Periostin [nmol/l]			% Recovery
Sample matrix	ID	Reference	1+1	1+3	1+1	1+3
Mouse plasma	mp1	2.9	2.1	1.2	147	163
Mouse plasma	mp2	3.2	1.9	0.9	121	108
Mouse plasma	mp3	2.6	1.6	0.7	122	114
Mouse plasma	mp4	3.2	2.0	1.1	123	134
				Mean	128	130
				Min	121	108
				Max	147	163

Mouse Periostin ELISA Specificity

The specificity of an ELISA is defined as its ability to exclusively recognize the analyte of interest.

The specificity of the mouse Periostin ELISA was shown by characterizing both the capture and the detection antibody through epitope mapping and affinity measurements. In addition, the specificity of the ELISA was established through competition experiments, which measure the ability of the antibodies to exclusively bind mouse Periostin.

This assay recognizes endogenous and recombinant mouse Periostin.

Competition of Signal

Competition experiments were carried out by pre-incubating mouse samples with an excess of capture antibody. The concentration measured in this mixture was then compared to a reference value, which was obtained from the same sample without the pre-incubation step.

The table below shows the competition of endogenous concentrations of mouse Periostin in mouse samples:

		Mouse Periostin [nmol/l]		% Competition
Sample matrix	ID	Reference	Reference + antibody	% Competition
Serum	ms1	9.2	0.0	100
Serum	ms2	8.6	0.0	100
Serum	ms3	10.1	0.0	100
Serum	ms4	10.0	0.0	100
Plasma	mp1	2.9	0.0	100
Plasma	mp2	3.0	0.0	100
Plasma	mp3	2.8	0.0	100
Plasma	mp4	3.4	0.0	100

Isoforms of Mouse Periostin

Currently, five different isoforms of mouse Periostin (http://www.uniprot.org/uniprot/Q62009) have been identified. They are generated by alternative splicing.

Recombinant mouse Periostin used in this assay is identical to Q62009-1 (isoform 1): 838AA, mass (Da): 93,144.

This mouse Periostin ELISA assay detects:

Isoform 1 (mouse Periostin recombinant standard/calibrator utilized in this assay)
Isoform 2 (due to sequence homology)
Isoform 3 (due to sequence homology)
Isoform 5 (experimentally analyzed with recombinant protein).

Isoform 4 shows a change of sequence in the respective epitope of the capture antibody. Thus, it is not certain if this assay detects Isoform 4.

Epitop Mapping of Utilized Antibodies

Epitop mapping has been performed by Pepperprint.

Capture Antibody:

The capture antibody which is a monoclonal mouse antibody binds to m_e1: KGMTSEER (AA544-551) of Q62009-1 (isoform 1).

Detection Antibody:

p_e1: TQHYSDVSK (AA115-123)

p_e2: EIEGKGSY (AA127-134)

p_e3: EDDLSSF (AA246-252)

p_e4: PAMT (AA672-676)

p_e5: RISTGGGE (AA738-746)

Summary:

Sample Stability

Sample Preparation

We recommend separating plasma or serum by centrifugation as soon as possible, e.g. 20 min at 2,000 x g, preferably at 4°C (2-8°C). Samples can be stored at 4°C (2-8°C) overnight. For long term storage, aliquot the acquired plasma or serum samples and store at -25°C or lower.

Freeze-Thaw Stability

The stability of endogenous mouse Periostin was tested by comparing mouse Periostin measurements in samples that had undergone five freeze-thaw (F/T) cycles. The mean recovery of sample concentration after five freeze-thaw cycles is 97%.

		Mouse Periostin [pmol/l]			Mean Recovery [%]
Sample matrix	ID	Reference	2x F/T	5x F/T	2x F/T	5 x F/T
Serum	s1	5.5	5.8	5.0	106	92
Serum	s2	6.3	6.7	6.2	107	100
Serum	s3	6.6	7.3	6.5	110	98
Serum	s4	5.7	5.4	5.6	95	99
				Mean	108	97

Undiluted samples can be subjected to five freeze-thaw cycles.

Sample Values

Periostin Values in Healthy Control Mice

To provide expected values for circulating mouse Periostin, a panel of samples from healthy mice was tested.

A summary of the results is shown below:

			Mouse Periostin [nmol/l]
Sample type	Sample matrix	n	Median	Range
Adult mice	Serum	28	3.4	1.6 – 5.4
Adult mice	Plasma	5	2.5	1.3 – 2.9
4 week old mice, female	Serum	13	8.0	5.3 – 10.0
4 week old mice, male	Serum	11	8.4	7.6 – 10.1

We recommended establishing the normal range for each laboratory.

Matrix Comparison

Comparison of mouse serum and plasma samples

		Mouse periostn [nmol/l]
Sample matrix	n	Mean	Median	Min	Max	SD
Mouse serum	28	3.47	3.35	1.6	5.4	0.93
Mouse plasma	5	2.4	2.5	1.3	2.9	0.64

Citations

Age- and Strain-Related Differences in Bone Microstructure and Body Composition During Development in Inbred Male Mouse Strains. Papageorgiou M., Föger-Samwald U., Wahl K., Kerschan-Schindl K., Pietschmann P., 2020. Calcif Tissue Int;106(4):431-443. PMID: 31901965.
Age- and Strain-Related Differences in Bone Microstructure and Body Composition During Development in Inbred Male Mouse Strains. Papageorgiou, M., Föger-Samwald, U., Wahl, K., Kerschan-Schindl, K., Pietschmann, P., 2020. Calcif Tissue Int.
Validated and in-depth characterized sandwich ELISA for the quantification of mouse periostin. Gadermaier E., Wallwitz J., Berg G., Himmler G. ASBMR 2018.

Reviews

Sort by:

Submit a Review

There are no reviews yet.

Be the first to review “Mouse Periostin ELISA | BI-20433MS”

Downloads

Mouse Periostin ELISA | BI-20433MS

Mouse Periostin ELISA Product Overview

Mouse Periostin ELISA Assay Principle

Mouse Periostin ELISA Typical Standard Curve

Mouse Periostin ELISA Kit Components