Mouse ANA ELISA Kit (MOFI01271)- High Sensitivity

Description

Description

Mouse ANA(Anti-nuclear Antibody) ELISA Kit

Anti-nuclear antibodies (ANAs) are autoantibodies that target components within the cell nucleus and are associated with autoimmune diseases. The Assay Genie Mouse ANA ELISA Kit serves as a valuable tool for studying ANAs in mouse models of autoimmune diseases, enabling researchers to investigate immune system dysfunctions, evaluate treatment effectiveness, and deepen our understanding of autoimmune phenomena in mouse models

Datasheet MSDS

Key Features

	Save Time	Pre-coated 96 well plate
	Quick Start	Kit includes all necessary reagents
	Publication Ready	Reproducible and reliable results

Overview


Product Name:	Mouse ANA(Anti-nuclear Antibody) ELISA Kit
Product Code:	MOFI01271
Size:	96 Assays
Alias:	Anti-nuclear Antibody
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse ANA concentrations in serum plasma and other biological fluids.
Sensitivity:	0.281ng/ml
Range:	0.469-30ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Additional Information


Recovery	Matrices listed below were spiked with certain level of Mouse ANA and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse ANA in samples. (Not availabe)
Linearity:	The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse ANA and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. (Not Available)
CV(%)	Intra Assay <8 Inter Assay <10

Kit Components

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8x12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/ -20°C
Sample/Standard Dlution Buffer	20ml	4°C
Biotin-labeled Antibody (Concentrated)	120ul	4°C (Protection from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate (SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protection from light)
Stop Solution	10ml	4°C
Wash Buffer (25X)	30ml	4°C
Plate Sealer	5	-

Other materials required:

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

Protocol

*Note: Protocols are specific to each batch/lot. For the exact instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human 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 antibody 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.

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 Type	Protocol
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.

Anti nuclear Antibodies (ANA) - Background

What is an Anti-nuclear Antibody?

Anti-nuclear antibodies (ANAs) are a type of autoantibody that target various components within the cell nucleus such as DNA, RNA, or proteins associated with chromatin. These antibodies are produced by the immune system and can be detected in the blood. ANAs are associated with autoimmune diseases, which are conditions in which the immune system mistakenly attacks the body's own cells and tissues.

When are ANAs produced?

In healthy individuals, the immune system typically maintains a state of self-tolerance, which means it recognizes and tolerates the body's own cells and tissues as "self" and does not mount an immune response against them. However, in autoimmune diseases, this self-tolerance breaks down, and the immune system mistakenly recognizes certain components of the body as foreign or abnormal. As a result, autoantibodies, including anti-nuclear antibodies (ANAs), may be produced.

In the absence of autoimmune diseases or other conditions that disrupt immune tolerance, healthy individuals do not produce significant levels of ANAs.

Clinical Significance of ANAs

ANAs are some of the only specific disease markers for systemic autoimmune disorders, which affect around 3-5% of the general population. Thus, testing for presence of ANAs is the first step towards getting a diagnosis for any autoimmune connective tissue disorders.

A disease that generally produced a positive ANA test is systemic lupus Erythematosus (SLE). Other diseases that produce ANAs are Rheumatoid Arthritis, Scleroderma, Myositis,Sjogren's and Psoriatic Arthritis.

Some specific inflammatory conditions such as ankylosing spondylitis, however, yield negative ANA titers.

ANA Tests for Autoimmune Disorders

The detection of ANAs is typically done through a laboratory test called an ANA test. This test involves taking a blood sample from a patient and analyzing it for the presence of ANAs. The most common method used is indirect immunofluorescence assay (IFA), where the patient's serum is mixed with cells that have been fixed to a microscope slide and stained with a fluorescent dye. If ANAs are present in the serum, they will bind to the cell nuclei, and the pattern of fluorescence can be observed under a microscope.

A positive ANA test is usually reported as both a ratio (called a titer) and a pattern. Titers higher than 1:640 indicates a greater possibility of autoimmune disorders.

ANA Test Patterns

The following are some common patterns that can be observed in anti-nuclear antibody (ANA) tests:

Homogeneous pattern: This pattern appears as a diffuse and uniform staining of the nucleus. It can be associated with various autoimmune diseases, including systemic lupus erythematosus (SLE), but it can also be found in individuals without autoimmune conditions. THis is the most common type of pattern.

Speckled pattern: The speckled pattern appears as fine speckles or dots distributed throughout the nucleus. It can be seen in autoimmune diseases such as SLE and Sjögren's syndrome.

Nucleolar pattern: This pattern shows dense, round structures within the nucleus, around the nucleoles. It is associated with autoimmune diseases like scleroderma, as well as with certain medications and viral infections (False positives).

Centromere pattern: The centromere pattern appears as a distinct staining of the centromeric region of the chromosomes. It is strongly associated with limited cutaneous systemic sclerosis (also known as limited scleroderma).

Peripheral or rim pattern: This pattern involves staining along the outer edges of the nucleus, creating a rim-like appearance. It can be seen in SLE.

Cytoplasmic pattern: This pattern involves staining in the cytoplasm of the cells rather than the nucleus. It can be associated with various autoimmune diseases, including SLE and Sjögren's syndrome, as well as with viral infections and other conditions.

Positive ANA tests

It's important to note that while the presence of ANAs is associated with autoimmune diseases, not all individuals with positive ANA tests will develop these conditions. ANA tests are useful as screening tools, but a positive result should be followed up with further testing and evaluation by a healthcare professional to determine the underlying cause.

Mouse ANA ELISA Kit FAQs

Q: What is the purpose of the Mouse ANA Elisa Kit?

This Mouse ANA Elisa Kit is a research tool designed for the detection and quantification of anti-nuclear antibodies (ANAs) in mouse serum samples.This kit can be used to investigate the presence of ANAs in mouse models of autoimmune diseases, evaluate the effectiveness of treatments or interventions, and study immune system function in various research fields.

Q: What type of samples can be used with the Mouse ANA Elisa kit?

The Mouse ANA Elisa kit is suitable for biological fluids such as serum samples

Q: Can the Mouse ANA ELISA kit be used for diagnostic purposes?

No, the Mouse ANA Elisa Kit is strictly intended for research purposes only and is not suitable for diagnostic use in clinical settings. It is important to use validated diagnostic assays specifically approved for clinical diagnostics.

Q: Is the Mouse ANA kit compatible with other species?

This Mouse ANA kit has been developed specifically for mouse samples.

Q: Where can I find additional technical support or assistance with the Mouse ANA ELISA kit?

For any technical inquiries or assistance regarding the Mouse ANA ELISA kit, you can reach out to our team. They will be available to answer your questions and provide the necessary guidance to ensure a successful experiment.

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