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Signosis

[Signosis] Mouse Anti-SmD1 ELISA Kit

Cat-No. EA-5209

Signosis는 생명 과학 연구 및 생물학적 데이터 분석을 위한 혁신적인 솔루션을 제공하는 글로벌 생명과학 기업입니다.

제품 설명

Mouse Anti-SmD1 ELISA Kit

제품 번호

EA-5209

제품 특징

Introduction

Antibodies against the Sm antigens of the small nuclear ribonucleoproteins (snRNPs) are a very specific marker for the diagnosis of systemic lupus erythematosus (SLE). Antibodies against the Sm proteins are found in 20-30% of SLE patients. Anti-Sm antibodies are observed at a high titer in the active period of SLE and at a low titer in the nonactive period. Antibodies directed against Sm proteins as well as DNA found in sera from patients with SLE have been included as one of the American College of Rheumatology classification criteria for SLE. Sm antigen is now known to be consisted of at least nine different polypeptides. Anti-Sm antibodies are predominantly directed against the SmD1 protein of the snRNP complex.

Principle of the assay

Anti-SmD1 ELISA kit measures anti-Sm antibodies in the serum. It is based on the principle of a solid phase enzymelinked immunosorbent assay. The assay utilizes the SmD1 protein for immobilization on the microtiter wells and antimouse IgG antibody conjugated to horseradish peroxidase (HRP) for detection. The test sample is allowed to react simultaneously with the two components, resulting in AntiSm antibodies being sandwiched between the solid phase and enzyme-linked antibodies. After incubation, the wells are washed to remove unbound-labeled antibodies. A HRP substrate, TMB, is added to result in the development of a blue color. The color development is then stopped with the addition of Stop Solution changing the color to yellow. The concentration of anti-Sm antibody is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm.

Materials provided with the kit

• 8x12 96-well plate coated with SmD1 (4oC).

• Anti-mouse IgG antibody conjugated to HRP (4oC).

• Mouse SmD1 Positive Control (4oC).

• 1X Diluent buffer (4oC).

• 5X Assay wash buffer (4oC).

• Substrate (4oC).

• Stop Solution (4oC)

Material required but not provided

• Microplate reader capable of measuring absorbance at 450 nm

• Shaker

Reagent preparation before starting experiment

• Dilute the 5x Assay wash buffer to 1x buffer 40ml 5x Assay wash buffer 160ml ddH2O

• Dilute 1000 times of anti-mouse IgG antibody conjugated to HRP with 1X Diluent buffer.

Storage and Preparation

Store all reagents at 2-8°C. All reagents must be brought to room temperature (20- 25°C) prior to use.

When stored at 2-8°C, the diluted Assay wash buffer is stable until the kit expiration date.

SAMPLE COLLECTION AND STORAGE

Serum

Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove serum and assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles.

Plasma

Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles.

Assay procedure

1. Take the desired number of well strips from the plate. Make sure the rest of strips are well sealed.

2. Add 100 µl of diluted samples (1:100 diluted with 1X Diluent Buffer) or positive control (1:50 dilution) per well and incubate for 1 hour at room temperature with gentle shaking.

*Note: We recommend having a blank condition. For the blank, add only 1x Diluent buffer to the well.

3. Aspirate each well and wash by adding 200µl of 1X Assay wash buffer. Repeat the process twice for a total of three washes. Completely remove liquid at each wash by firmly tapping the plate against clean paper towels.

4. Add 100µl of diluted anti-mouse IgG antibody conjugated to HRP to each well and incubate for 30 minutes at room temperature with gentle shaking.

5. Repeat the aspiration/wash as in step 3.

6. Add 100µl of Substrate to each well and incubate for 5-15 minutes.

*Note: Positive control will turn blue. Samples should be stopped when blue color begins to appear in blank.

7. Add 50µl of Stop solution to each well. The color in the wells should change from blue to yellow.

8. Determine the optical density of each well with a microplate reader at 450 nm within 30 minutes.