Our colloid products include instructions for use. The experimental method is outlined below.
1. Immunochromatography kit: Structure
(1) Sample pad
Receives and distributes samples uniformly onto a strip.
Operates as a filter.

(2) Conjugate pad
Maintains the gold colloid, antibody conjugate and the detection reagent in a dry state. Releases the detection reagent quickly and uniformly to the membrane.

(3) Membrane
Holds reagents (antibodies etc.) on the surface through the formation of an immuno-complex. Controls the flow rate of the system.

(4) Absorbent pad
Absorbs the sample.

2. Swabbing of antibody on membrane

Materials

Membranes: Membranes for immunochromatography may be purchased from manufacturers such as Millipore. There are several types with different flow rates. The flow rate affects detection sensitivity, measuring time, and the appearing of the unspecific reaction

Antibody for test lines: Antibody for the antigen to be detected. The operating concentration is 0.01?2 mg/mL (0.01?2mg/test). If the antibody concentration is low, proteins such as BSA, which do not affect the antigen?antibody reaction, are added to give a protein concentration of about 0.5?1 mg/mL. The antibody solution is diluted with phosphate buffer(s) (PB) (e.g., 10 mM PB (pH 7)).
Antibody for control lines: A secondary antibody to that conjugated with the gold colloid. Example: anti-rabbit IgG antibody, anti-mouse IgG antibody.

Blocking solution: Liquid in which proteins or polymers, such as gelatin, casein, BSA, IgG, PVP, or PVA, are dissolved in buffer solution. Example: PBS solution containing 1% BSA.

Method

1. The antibody solution is applied to the membrane in the form of a line.
(The position of the line depends on the sensitivity of the reaction.)
2. The membrane is dried at room temperature or at 37°C for 30 to 60 minutes.
3. The membrane is dipped in blocking solution for 10 to 30 minutes and shaken gently.
4. The membrane is then dipped in weak buffer solution or distilled water, shaken, and washed (2 to 3 times).
5. Any excessive moisture on the membrane surface is removed, and the membrane is dried at room temperature or under vacuum.

Items to note

- Use of the membrane
- Amount of antibody solution to be used
- Use of blocking solution
- Dilution of the antibody solution
- Position of application of the antibody solution

3. Preparation ofgold-antibody conjugate

Materials

Gold colloid: A colloid with a particle size of 50 nm is generally used. This may be purchased from Winered Chemical Corporation.
The colloid is diluted with buffer solution of a specific pH. On combining with an antibody, the pH may be adjusted.
Antibody: Antibody for the antigen to be detected. The antibody concentration after addition to the gold colloid (OD₅₂₅ = 1) is 50?500 ng/mL.
Blocking solution: Contains proteins or polymers, such as gelatin, casein, BSA, IgG, PVP, or PVA, in buffer.
Fiberglass: Required for the conjugate pad. May be purchased from manufacturers such as Millipore.
Stabilizing solution for gold colloid: Low-salt buffer solution containing gold colloid stabilizers such as BSA or PEG, along with added sugars such as sucrose and/or trehalose.
Sugars are soaked in the fiberglass, which increases the stability of the dried antibody.
Siliconized tube: To prevent the gold colloid from sticking to the receptacle, preparation of the gold-antibody conjugate is carried out in a siliconized tube.

Items to note

- Amount of gold colloid to be used
- Amount of antibody to be added 
- pH at the time of antibody conjugation
- Composition of the blocking solution
- Composition of the gold colloid suspension

This is known as the “sandwich” system. Haptens can be detected by a competitive reaction system.

4. Example: Preparation of gold-antibody conjugate

Materials

Tris buffer: The optimal pH varies depending on the antibody to be used; e.g., 5 mM or 10 mM, pH 9.2.
Gold colloid: WRGH1 (OD₅₂₅ = 12) from Winered Chemical Corporation.
Antibody:Diluted with Tris of appropriate concentration and pH (e.g., 5 mM, pH 9.2). Final concentration 0.1~0.2 mg/mL or 30~60mg/mL.
1% BSA solution: Prepared using 5mM Tris, pH 9.2.
BSA?PEG mixed solution: Containing 1% BSA solution and 1% PEG solution, mixed at a ratio of 9:1.
Stabilizing solution for gold colloid conjugate: Tris buffer of appropriate concentration and pH (e.g., 5 mM, pH 9.2) is used. pH adjustment may not be necessary. Because during conjugation reaction, the gold colloid may aggregate in buffer solution in the presence of high concentration of salt, becoming a purple-blue color, dialysis of salt may be required.

Method

The gold colloid solution and the antibody solution are mixed in a 1:1 ratio and the mixture is left to stand for 15 minutes.Example
For 100µL each of gold colloid and antibody solutions:

1. BSAPEG mixed solution (100µL) is added, the mixture is centrifuged at 5000 rpm for 5 minutes, and the supernatant is removed.

2.BSA?PEG mixed solution (200µL) is again added and mixing is carried out ultrasonically (about 30 seconds), followed by addition of another 200µL of BSA?PEG mixed solution. The mixture is again centrifuged and the supernatant is removed.

3. Stabilizing solution (100µL) is added and mixing is carried out by Voltex or ultrasound (10 seconds). Depending on the antibody used, the antibody concentration and the pH of the buffer solution and stabilizing solution may vary.

Bibliography
1. Masatoshi Watabe, Shigeaki Furukawa, Suguru Akamatsu, and Tetsuya Oda, “Simple diagnostic technical development”, Bio-industry, No. 10, Vol. 22, p60-65 (2005).
2. “Method of manufacture of precious-metal colloids, precious-metals particles, composites, and precious-metal particulates.” Masatoshi Watabe, Patent No. 4368855, Japan.
Patent international publication number: WO 2005/023468 A1
3. Julian E. Beesley, “Colloidal Gold: A new perspective for cytochemical marking”, Royal Microscopical Society Handbook No. l7, Oxford Science Publications, Oxford University Press (l989).
4. Sadanori Yokota and Osamu Fujimori, “Immunogold method: Immunohistochemistry with colloid gold”, Soft Science Company, 1992.

Manufacturing and selling company:  Winered Chemical Corporation
246-4 Inume-machi, Hachioji, Tokyo 193-0802, Japan
TEL: (81)42-624-3755 FAX: (81)42-624-3732 URL: www.winered.jp e-mail: mawatabe@nifty.com