Mouse ACA-IgA (Anti-Cardiolipin IgA) ELISA Kit

SKU:
MOFI00147
€599

Description

ELISA Kit Technical ManualMSDS

Mouse ACA-IgA (anti-Cardiolipin Antibody IgA) ELISA Kit - Information

The Assay Genie Mouse ACA-IgA (anti-Cardiolipin Antibody IgA) ELISA Kit can assay for Mouse ACA-IgA in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How do our Mouse ACA-IgA (anti-Cardiolipin Antibody IgA) ELISA Kits Work?

This kit was based on sandwich enzyme-linked immune-sorbent assay technology. Antigen was pre-coated onto 96-well plates. And the Biotin- labelled antigen was used as detection antigen. The standards, test samples and Biotin-labelled antigen were added to the wells subsequently, and wash with wash buffer. HRP-Streptavidin Conjugate was added and unbound conjugates were washed away with wash buffer. TMB substrates were used to visualize HRP enzymatic reaction. TMB was catalysed by HRP to produce a blue colour product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the target amount of sample captured in plate. Read the O.D. absorbance at 450nm in a microplate reader, and then the concentration of target can be calculated.

Mouse ACA-IgA (anti-Cardiolipin Antibody IgA) ELISA Kit Data

Product Code

MOFI00147

Alias

ACA-IgA, anti-Cardiolipin Antibody IgA

Detection method

Sandwich ELISA, Double Antigen

Application

This immunoassay kit allows for the in vitro quantitative determination of Mouse ACA-IgA concentrations in serum plasma and other biological fluids.

Size

96T

Range

0.313-20ng/ml

Sensitivity

0.188ng/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of Mouse ACA-IgA and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse ACA-IgA in samples.

MatrixRecovery range(%)Average(%)
serum(n=5)88-10599
EDTA plasma(n=5)89-9995
UFH plasma(n=5)85-9892
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse ACA-IgA and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample1:21:41:8
serum(n=5)89-99%85-104%85-105%
EDTA plasma(n=5)82-101%83-100%83-98%
UFH plasma(n=5)83-97%83-91%81-93%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Mouse ACA-IgA (anti-Cardiolipin Antibody IgA) ELISA Kit Protocol

The below protocol is a sample protocol for Mouse ACA-IgA (anti-Cardiolipin Antibody IgA) ELISA Kit using a biotinylated detection antigen and streptavidin-HRP. Sandwich ELISA Kits allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Mouse ACA-IgA Antibody present in their sample.

Bring all reagents and samples to room temperature 30 minutes before use. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Assay Procedure:

1.Set standard, test sample (diluted at least ½ with Sample Dilution Buffer) and control (zero) wells on the pre-coated plate and record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (blank) wells.
2.Aliquot 100µl of standard solutions into the standard wells
3.Add 100µl of Sample / Standard dilution buffer into the control (blank) well.
4.Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and other biological fluids) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6.Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.Add 0.1 ml of Biotin-detection antigen working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9.Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

Mouse ACA-IgA (anti-Cardiolipin Antibody IgA) ELISA Kit components

96 Assays

Storage

ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antigen (Concentrated)120ul4°C (Protect from light)
Antigen Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5 -

Other materials and equipment required:

The Assay Genie Mouse ACA-IgA (anti-Cardiolipin Antibody IgA) ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir

Sample Preparation

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample TypeProtocol

Serum

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

Plasma

Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.

Urine & Cerebrospinal Fluid

Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.

Cell culture supernatant

Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.

Cell lysates

Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Tissue homogenates

The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.

Tissue lysates

Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Breast Milk

Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

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Additional Information

Product type:
ELISA
Reactivity:
Mouse
ELISA Type:
Sandwich
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