Spot immunofiltration assay

["Collaborating Expert | Dr. Jinfei Li", "Oncology Peking Union Medical College"], ["Reviewed by | Dr. Yifeng Nie", "Nanobiomedicine University of Chinese Academy of Sciences"]

Summary

A detection test that utilizes antigens or antibodies to carry out a corresponding specific immune response, and is one of the main methods used in immediate testing (POCT) today.

Principle

After the antigen-antibody reaction, macromolecular colloidal gold complexes are formed, resulting in a positive result, i.e. a red spot on the membrane. When the liquid passes through the microporous filtration membrane, the antigen or antibody in the filtrate comes into contact with the corresponding antibody or antigen on the membrane, which plays the role of concentration by affinity chromatography, so as to achieve the purpose of rapid detection, and at the same time, the infiltration of the washing liquid can achieve the purpose of thorough washing in a short period of time, which simplifies the operation steps.

Appliance

Antibodies and tumor markers for the detection of various infectious diseases, as well as early pregnancy diagnostic reagents for the detection of the urine HCG "gold standard".

Operation method

Speckle immunofiltration test

Principle

The antigen or antibody spot is added to the solid-phase carrier nitrocellulose film, the film is attached to the absorbent material, and reagents such as specimen, immunocolloid gold and washing solution are added dropwise to the film for antibody or antigen reaction, and the excess reagent quickly penetrates into the absorbent material, and the reaction is gradually carried out through percolation. After the antigen-antibody reaction, macromolecular colloidal gold complexes are formed, resulting in a positive result, i.e. a red spot on the membrane. When the liquid passes through the microporous filtration membrane, the antigen or antibody in the filtrate comes into contact with the corresponding antibody or antigen on the membrane, which plays the role of concentration by affinity chromatography, so as to achieve the purpose of rapid detection, and at the same time, the infiltration of the washing liquid can achieve the purpose of thorough washing in a short period of time, which simplifies the operation steps.

Materials and Instruments

① Protein standards or proteins to be tested

② Non-specific protein binding agent (e.g., bovine serum albumin)

③ Wash buffer

① Protein standard or protein to be tested ② Non-specific protein conjugate (e.g., bovine serum albumin) ③ Wash buffer

⑤ Detection secondary antibody

⑥ Diafiltration device

⑦ NC membrane

⑧ Blocking solution (0.2% BSA and 0.05% Tween 20 washing solution)

⑨ Gold standard antibody

Move

1. Filtering device: a small plastic box (4×3×0.6 cm), divided into two layers: bottom and lid, with a 0.5 cm diameter hole in the center of the lid. The bottom of the box is filled with highly absorbent bedding material, and a piece of NC membrane is placed immediately under the hole in the lid. When the lid is closed tightly, it is a filtration device. Add 1~2 ml of specific antibody (primary antibody) or antigen to the NC membrane in the hole, dry naturally at room temperature, and store in a sealed plastic bag with desiccant.

2. Take out the filter and add 2 drops (about 100 ml) of sealing solution on the NC membrane of the small hole;

3, wait until it penetrates into the box, in the NC membrane, add 1 drop (about 50 ml) of the specimen to be examined;

4、After it penetrates into the cassette, add 2 drops (about 100 ml) of washing solution on the NC membrane;

5. When it penetrates into the cassette, add 1 drop (about 50 ml) of gold-labeled antibody to the NC membrane, so as to make it react with the sample adsorbed on the NC membrane;

6. When it penetrates into the cartridge, add 2 drops (about 100 ml) of washing solution on the NC membrane;

7. Until the washing solution completely penetrates into the cassette.

Expected results: Positive reaction when there is a red reaction on the small hole NC membrane, negative reaction without coloring.

Caveat

1. The amount of specific antibody (primary antibody) or antigen, the specimen to be examined and the gold standard antibody must be determined in advance by applying the square array method to determine the most appropriate amount.

2. The padding at the bottom of the cassette should be highly absorbent, otherwise the results may be affected.

3、Spot gold immunofiltration test indirect method to measure the antibody in serum specimen, the main reason for false positive results is the interference of non-purpose IgG in the serum specimen.

Common Problems

Quality control: The quality control of droplet gold methods is often performed by spotting a QC spot on a nitrocellulose membrane. The QC spot for the sandwich method is preferably the corresponding antigen, and can also be filled with SPA or an anti-antibody against the gold standard antibody. Indirect QC points are most conveniently made from crude human IgG by salting out.

For more product details, please visit Aladdin Scientific website.https://www.aladdinsci.com/

Tags:

Immunological experiments