Immune colloidal gold technique

(1) Principle

Immune colloidal gold technology is a new type of immunolabeling technology that uses colloidal gold as a tracer marker for antigens and antibodies. Colloidal gold is polymerized by chloroauric acid (HAuCl4) under the action of reducing agents such as white phosphorus, ascorbic acid, sodium citrate, tannic acid, etc. to form gold particles of a specific size, and becomes a stable colloidal state due to the electrostatic effect For colloidal gold. Colloidal gold is negatively charged in a weak alkaline environment and can form a strong bond with the positively charged groups of protein molecules. Because this bond is electrostatic, it does not affect the biological characteristics of the protein.

In addition to binding protein, colloidal gold can also be combined with many other biological macromolecules, such as SPA, PHA, ConA and so on. According to some physical properties of colloidal gold, such as high electron density, particle size, shape and color reaction, plus the immune and biological characteristics of the conjugate, colloidal gold is widely used in immunology, histology, pathology and cells Biology and other fields.

(2) Preparation of colloidal gold

Colloidal gold particles of different sizes can be prepared according to different reducing agents. The method commonly used to prepare colloidal gold particles is as follows.

1. Trisodium citrate reduction method

(1) Preparation of 10nm colloidal gold particles: Take 100ml of 0.01% HAuCl4 aqueous solution, add 3% of 1% trisodium citrate aqueous solution, heat and boil for 30min, cool to 4 ° C, the solution is red.

(2) Preparation of 15nm colloidal gold particles: take 100ml of 0.01% HAuCl4 aqueous solution, add 2% of 1% trisodium citrate aqueous solution, heat and boil for 15min-30min, until the color turns red. After cooling, add 0.1Mol / L K2CO30.5ml and mix well.

(3) Preparation of 15nm, 18nm-20nm, 30nm or 50nm colloidal gold particles: take 100ml of 0.01% HAuCl4 aqueous solution and heat to boil. Quickly add 4ml, 2.5ml, 1ml or 0.75ml of 1% trisodium citrate aqueous solution as needed, continue to boil for about 5min, and appear orange-red. The colloidal gold particles produced in this way are 15 nm, 18-20 nm, 30 nm and 50 nm, respectively.

2. Tannin-sodium citrate reduction method

Solution A: 1ml of 1% HAuCl4 aqueous solution was added to 79ml of double-distilled water and mixed well.

Liquid B: 4ml of 1% trisodium citrate, 0.7ml of 1% tannic acid, 0.2ml of 0.1Mol / L K2CO3 liquid, mix, and add double distilled water to 20ml.

Heat liquid A and liquid B to 60 ℃, add liquid B to liquid A quickly under electromagnetic stirring, the solution turns blue, continue heating and stirring until the solution turns bright red. The diameter of the gold particles produced by this method is 5 nm. If you need to prepare gold particles of other diameters, adjust the amount of tannic acid and K2CO3 according to the numbers listed in Table 15-1.

Table 15-1 Preparation of reagents for tannic acid-sodium citrate reduction method

Gold particle diameter

(Nm)

Liquid A

Liquid B

1% HAuCl4

Double distilled water

1% trisodium citrate

0.1Mol / L K2CO3

1% tannic acid

Double distilled water

5

1

79

4

0.20

0.70

15.10

10

1

79

4

0.025

0.10

15.875

15

1

79

4

0.0025

0.01

15.9875

3. Precautions for preparing high-quality colloidal gold

(1) Glassware must be thoroughly cleaned, preferably siliconized glassware, or glassware stabilized with colloidal gold prepared for the first time, and then rinsed with double distilled water before use. Otherwise, the stability of the gold particles after binding and activation of the biological macromolecules and the gold particles is affected, and the gold particles of the expected size cannot be obtained.

(2) Reagent preparation must be kept strictly pure, and all reagents must be prepared using de-ionized water or triple-distilled water and deionized, or the prepared reagents should be ultrafiltered or microporous filter membrane (0.45µm ) Filtration to remove the polymer and other impurities that may be mixed in.

(3) The pH of the prepared colloidal gold solution is preferably neutral (pH 7.2).

(4) The quality of chloroauric acid is high, and there are few impurities. It is best imported.

(5) Chloroauric acid mixed with 1% aqueous solution can keep stable for several months at 4 ℃. Since chloroauric acid is easy to deliquesce, it is best to dissolve the entire small package at one time when preparing.

(3) Preparation of colloidal gold-labeled protein

The adsorption of colloidal gold on protein mainly depends on the pH value. Under the conditions close to the isoelectric point of the protein or partial alkali, the two easily form a strong conjugate. If the pH value of colloidal gold is lower than the isoelectric point of protein, it will aggregate and lose its binding ability. In addition, the size, ionic strength and molecular weight of the colloidal gold particles all affect the binding of the colloidal gold to the protein.

1. Preparation of protein solution to be labeled The protein to be labeled was dialyzed against 0.005 Mol / L pH 7.0 NaCl solution at 4 ° C overnight to remove excess salt ions, and then centrifuged at 100 000g at 4 ° C for 1 h to remove the polymer.

2. Preparation of standard colloidal gold solution Adjust the pH value of colloidal gold solution with 0.1Mol / L K2CO3 or 0.1Mol / L HCl. When labeling IgG, adjust to 9.0; when labeling McAb, adjust to 8.2; when labeling affinity chromatography antibody, adjust to 7.6; when labeling SPA, adjust to 5.9-6.2; when labeling ConA, adjust to 8.0; label avidin Time, adjust to 9 ～ 10.

Because the colloidal gold solution may damage the electric plate of the pH meter, it is appropriate to use precision pH test paper for measurement when adjusting the pH.

3. Determination of the ratio of colloidal gold to labeled protein

(1) According to the requirements of the protein to be labeled, after adjusting the pH of the colloidal gold, dispense into 10 tubes, 1 ml per tube.

(2) Serially dilute the labeled protein (IgG as an example) with 0.005Mol / L pH9.0 borate buffer to 5µg / ml ～ 50µg / ml, take 1ml respectively, add it to the gold gel solution listed above, and mix uniform. Add only 1ml of diluent to the control tube.

(3) After 5 minutes, add 0.1 ml of 10% NaCl solution to the above tubes, mix well and let stand for 2 hours. Observe the results.

(4) Observation of the results. The control tubes (without added protein) and the tubes with insufficient amount of added protein showed a red-to-blue aggregation phenomenon; while the amount of added protein reached or exceeded the minimum stable amount The tubes remain red. The minimum amount of protein used to stabilize the red color of 1 ml of colloidal gold solution is the minimum amount of the labeled protein. In actual work, it can be appropriately increased by 10% to 20%.

4. Combination of colloidal gold and protein (IgG) Adjust the pH of the colloidal gold and IgG solution to 9.0 with 0.1 Mol / L K2CO3 respectively, stir the IgG solution electromagnetically, add the colloidal gold solution, continue to stir for 10 min, add a certain amount of stabilizer to prevent antibodies Precipitation occurs when the protein and colloidal gold polymerize. Commonly used stabilizers are 5% fetal bovine serum (BSA) and 1% polyethylene glycol (molecular weight 20KD). Amount added: 5% BSA makes the final concentration of the solution 1%; 1% polyethylene glycol is added to 1/10 of the total solution.

5. Purification of colloidal gold-labeled protein

(1) Ultracentrifugation method: according to the size of colloidal gold particles, the type of labeled protein and the different stabilizers, different centrifugation speeds and centrifugation times are selected.

The colloidal gold-goat anti-rabbit IgG conjugate with BSA as a stabilizer can be centrifuged at low speed (1 200 r / min for 20 nm gold colloids and 1 800 r / min for 5 nm gold colloids) for 20 min, and the aggregated precipitate is discarded. Then, the 5 nm colloidal gold conjugate was centrifuged at 6,000 g for 4 h at 4 ° C; the 20 nm to 40 nm colloidal gold conjugate was centrifuged at 14 000 g for 1 h at 4 ° C. Aspirate the supernatant carefully, use PB solution containing 1% BSA (containing 0.02% NaN3) to resuspend the precipitate to 1/10 of the original volume, and store at 4 ° C. If 50% glycerol is added to the conjugate, it can be stored at -18 ℃ for more than one year.

In order to obtain an immunogold reagent with uniform particles, the above-mentioned preliminarily purified conjugate can be further subjected to density gradient centrifugation with 10% -30% sucrose or glycerol, and the conjugate of colloidal gold and protein with different gradients can be collected by banding.

(2) Gel filtration method: This method is only applicable to the purification of colloidal gold protein conjugates using BSA as a stabilizer. The colloidal gold protein conjugate was put into a dialysis bag, dehydrated and concentrated in silica gel to 1/5 to 1/10 of the original volume. Then centrifuge at 1500r / min for 20min. Take the supernatant and add to Sephacryl S-400 (Propylene Dextran Gel S-400) chromatography column for purification. The chromatographic column is 0.8 cm × 20 cm, the sample volume is 1/10 of the bed volume, and it is eluted with 0.02 Mol / L PBS solution (containing 0.1% BSA, 0.05% NaN3, pH 8.2 with IgG marker), The flow rate is 8 ml / h. Collect the eluent according to the red and dark tubes. Generally, the liquid filtered first is slightly yellow, sometimes turbid, and contains impurities such as large particles of polymer. Followed by the purified colloidal gold protein conjugate, the red gradually deepened with the increase of concentration, clear and transparent, and finally eluted a yellowish labeled protein component. The purified colloidal gold protein conjugate was filtered, sterilized, divided and stored at 4 ℃. Finally, 70% to 80% of the output can be obtained.

6. Quality identification of colloidal gold protein conjugate

(1) Measurement of the average diameter of colloidal gold particles: dip the gold-labeled protein reagent with a nickel mesh (copper mesh is also available) that supports the membrane, and observe it directly under a transmission electron microscope after natural drying. Or observe after counterstaining with uranium acetate. Calculate the average diameter of 100 gold particles.

(2) Determination of OD520nm value of colloidal gold solution: The maximum absorption value peak of colloidal gold particles appears between the wavelength of 510nm and 550nm. Dilute the colloidal gold protein reagent 1:20 with 0.02Mol / L pH8.2 PBS solution (containing 1% BSA, 0.02% NaN3), OD520 = 0.25. The OD520 of the general application solution should be 0.2 to 0.4.

(3) Determination of specificity and sensitivity of gold-labeled protein: Microporous membrane immunogold-silver staining method (MF-IGSSA) was used. The soluble antigen (or antibody) is adsorbed on the carrier (filter paper, nitrocellulose membrane, microporous filter membrane), and the antibody (or antigen) labeled with colloidal gold is directly or indirectly stained with silver imaging to detect the corresponding antigen or Antibodies to identify the specificity and sensitivity of gold-labeled proteins.

(4) Application of colloidal gold labeling technology in immunology

Colloidal gold labeling technology is easy to prepare, sensitive and specific, and does not require the use of radioactive isotopes or enzyme-chromogenic substrates with potential carcinogens, nor fluorescent microscopes. It has a wide range of applications, except for light microscopes. In addition to the immunohistochemical method of electron microscopy, it is more widely used in various liquid-phase immunoassays, solid-phase immunoassays, and flow cytometry.

1. Liquid-phase immunoassay: combining colloidal gold with antibodies to establish a micro-agglutination test to detect the corresponding antigen, as with indirect hemagglutination, agglutination particles can be directly observed with the naked eye. Based on the principle that gold particles agglomerate during the immunological reaction and cause color loss, a homogeneous sol particle immunoassay (SPIA) has been successfully applied to the detection of PCG, and a spectrophotometer is directly used for quantitative analysis.

2. Gold-labeled flow cytometry: Colloidal gold can significantly change the scattering angle of the red laser. The sheep anti-mouse Ig antibody labeled with colloidal gold was used in flow cytometry to analyze the surface antigens of different types of cells. At a wavelength of 632nm, the 90-degree scattering angle can be amplified more than 10 times without affecting cell activity. Furthermore, it is co-labeled with fluorescein and does not interfere with each other.

3. Colloidal gold solid phase immunoassay

(1) Dot-IGS / IGSS is a method of combining dot ELISA and immunocolloidal gold. The protein antigen was directly spotted on the nitrocellulose membrane. After reacting with the specific antibody, the secondary antibody labeled with colloidal gold was added dropwise. As a result, gold particles aggregated at the reaction site of the antigen and antibody, forming red spots visible to the naked eye. Dot-IGS. This reaction can be enhanced by silver developing solution, namely spot gold and silver staining (Dot-IGS / IGSS).

(2) Dot immuno-gold filtration assay (DIGFA) The principle of this method is exactly the same as that of dot immuno-gold filtration assay, except that there is strong absorbent material under the nitrocellulose membrane, which is diafiltration. Device. After adding the antigen (antibody), quickly add the antibody (antigen), and then add gold to label the second antibody. Due to the diafiltration device, the reaction is very fast, and the color reaction can be displayed within a few minutes. This method has been successfully applied to the detection of human immunodeficiency virus (HIV) and the detection of alpha-fetoprotein in human serum.