Enzyme labels include enzyme-labeled antigens, enzyme-labeled antibodies, and enzyme-labeled SPAs. The quality of the enzyme label is directly related to the success of the immunoenzyme technology, so it is called a key reagent. The most commonly used enzyme label is an enzyme-labeled antibody, which is obtained by linking an enzyme to a specific antibody by a suitable method. The quality of the enzyme-labeled antibody mainly depends on the enzymes and antibodies with good purity, high activity and high affinity, and secondly, there are good preparation methods. At present, high-quality enzymes (such as horseradish peroxidase, or HRP for short) are available in domestic products. High quality antibodies can be obtained by extraction and purification. In the preparation method, it is preferred to select a method which has high yield, does not affect the activity of the conjugate, and does not interfere with the interfering substance, and is easy to operate.
First, the choice of working concentration
In immunoenzyme technology, the first variable to be determined is the working concentration of the enzyme label. Because of the small changes in the concentration of the enzyme label, the test results can be greatly fluctuated. In addition, because the concentration is too high, the non-specific reaction can be increased, and the low concentration can affect the sensitivity of the assay. Therefore, the working concentration must be accurately titrated before the formal test.
The method of titrating the enzyme-labeled antibody is: physically adsorbing the antigen (or antibody) on the solid phase carrier, and then using a series of diluted enzyme-labeled antibodies (or anti-Ig antibodies) and the antigen (or antibody) adsorbed on the carrier The reaction determines the titer of the enzyme-labeled antibody, or the working concentration, by the degree of color reaction of the enzyme with the substrate.
The steps are as follows: firstly diluting the antigen (or antibody) with 0.05M PH9.6 coating buffer to about 10 μg/ml, adding 0.1 ml to the polystyrene plate well, overnight at 4 ° C, and washing with washing buffer the next day. 3 times. The enzyme-labeled antibody was sequentially diluted with 1:% BSA-PBS to 1:100, 1:200, 1:400, 1:800, 1:1600 (depending on the titer of the antibody), and added to the reaction well. Two wells per dilution, 0.1 ml per well, and washed at 37 ° C for 1 hour and then washed. Then add substrate solution, 0.1 ml per well, at 37 ° C for 10 to 30 minutes. The reaction was quenched with 2 M H 2 SO 4 0.05 ml.
As a result, it was judged that the OD value of each well was mainly read by an ELISA colorimeter. The titration curve is drawn by taking the OD as the ordinate and the concentration of the combination as the abscissa. The dilution of the enzyme-labeled antibody when the OD value is about 1.0 and the slope of the curve is the largest is the working concentration of the label.
The reagents and equipment for the test are shown in the ELISA section.
It should be noted that this method is a direct method of ELISA, and the measured working concentration can differ from the optimum concentration in practical applications by several titers. This requires the establishment of an ELISA experimental system, so the actual working concentration should be further determined (a square matrix method can be used) to achieve optimal experimental conditions.
Second, the enzyme preparation and its substrate
Any enzyme that is non-toxic and exhibits a colored chemical reaction can in principle be used as a marker. However, the enzyme used as the labeled antibody should satisfy the following requirements: (1) convenient source and easy purification; (2) high specific activity and stable nature; (3) enzyme activity and amount can be determined by a simple method. The enzymes commonly used in immunoenzyme technology are horseradish peroxidase (HRP) and alkaline phosphatase (AP), followed by glucose oxidase, β-galactosidase, lysozyme and malate dehydrogenase. Wait.
Horseradish peroxidase (HRP) is most commonly used because of its high specific activity, stability, small molecular weight and easy preparation of pure enzymes. HRP is widely distributed in the plant kingdom and has a high content in horseradish. It is a glycoprotein composed of a colorless enzyme protein and brown iron porphyrin with a sugar content of 18%. HRP consists of multiple isozymes with a molecular weight of 40,000 and an isoelectric point of pH 3-9. The optimum pH for enzyme catalysis is slightly different due to the hydrogen donor, but mostly at pH 5. The enzyme is dissolved in water and a solution of 58% or less saturated ammonium sulfate. The maximum absorption spectra of the prosthetic and enzymatic proteins of HRP are 403 nm and 275 nm, respectively, and the purity of the enzyme is generally represented by the ratio RZ (Reinheit Zahl) of OD403nm / OD275nm. The high purity enzyme RZ should be around 3.0 (up to 3.4). The smaller the RZ value, the more non-enzymatic proteins. It is worth noting that purity does not indicate enzyme activity. For example, when the enzyme is denatured, the RZ value can still be unchanged.
The catalytic reaction of HRP requires the substrate hydrogen peroxide (H 2 O 2 ) and the hydrogen donor (DH 2 ). The hydrogen donor is mostly a colorless reducing dye, and a colored oxidized dye (D) can be produced by the reaction. The process of enzymatic reaction is as follows:
HRP
DH 2 +H 2 O 2 ────→D+2H 2 O
There are many types of hydrogen donors, and the products formed have different characteristics. For example, the reaction product of DAB (3.3-diaminobenzidine) is an insoluble precipitate and has an electron density, so it is suitable for immunoenzymatic staining or electron microscopic observation. 5AS (5-aminosalicylic acid) was used in ELISA early, but its solubility is not large enough, and the blank pores are not easy to control to colorless, and it is rarely used. OT (o-tolylamine) is characterized by its ability to produce bright blue-green products with high sensitivity, but the reaction is greatly affected by temperature, and because of product instability, it needs to be measured in a short time. The hydrogen donors currently used more widely and satisfactorily are: OPD (o-phenylenediamine) and TMB (tetramethylbenzidine). The product formed by the former is dark orange or brown, the latter product is blue-green, the solubility of both is good, the color is stable in the dark, the blank can be nearly colorless, and the sensitivity is reported to be higher than the former. More than double. In addition, there is a hydrogen donor called ABTS [2, 2'- s-nitro- bis (3-ethylbenzothiapyrrolidin-6 sulfonic acid)], the reaction product is blue-green, sensitivity and stability All are good. Especially in terms of the potential for carcinogenesis, ABTS and TMB are both preferred hydrogen donors.
Since the substrate H 2 O 2 of HRP itself is an inhibitor of the enzyme, the H 2 O 2 used in the enzymatic reaction cannot be excessive. It should be controlled to reach a peak after the reaction in a short period of time (indicating that H2O2 has been depleted). This does not increase the color of the reaction product even if it is extended for a longer period of time.
Third, HRP labeled antibody method
There are many methods for cross-linking an enzyme with an antibody, and different methods can be employed depending on the structure of the enzyme. For the preparation of the HRP conjugate, a two-step glutaraldehyde method and a sodium periodate method can be used. Especially the simple sodium periodate method is more commonly used.
　　 Glutaraldehyde two-step method
1. Principle: Glutaraldehyde is a bifunctional reagent that is covalently bound to an enzyme and an amino group on an immunoglobulin by its aldehyde group to form an enzyme-glutaraldehyde-immunoglobulin conjugate.
2. Marking steps:
(1) 25 mg of HRP was weighed and dissolved in a 1.25% glutaraldehyde solution, and allowed to stand at room temperature overnight.
(2) The enzyme solution after the reaction was subjected to a Sephadex G-25 column and eluted with physiological saline. The flow rate was controlled at 1 ml / 1 minute and the brown effluent was collected. If the volume is greater than 5 ml, concentrate to 5 ml with PEG. Place in a small 25ml beaker and stir slowly.
(3) 12.5 mg of the antibody to be labeled was diluted to 5 ml with physiological saline, and added dropwise to the enzyme solution with stirring.
(4) With 0.25 ml of 1 M PH9.5 carbonate buffer, stirring was continued for 3 hours.
(5) Add 0.2 ml of 0.2 M lysine, mix and set at room temperature for 2 hours.
(6) An equal volume of saturated ammonium sulfate was added dropwise with stirring and allowed to stand at 4 ° C for 1 hour.
(7) Centrifuge at 3000 rpm for half an hour and discard the supernatant. The precipitate was washed twice with half-saturated ammonium sulfate and finally the precipitate was dissolved in a small amount of 0.15 M PBS, pH 7.4.
(8) The above solution was placed in a dialysis bag, dialyzed against 0.15 M P7.4 buffered saline of pH 7.4, and after removal of ammonium ions (detected by naphthalene reagent), the precipitate was removed by centrifugation at 10,000 rpm for 30 minutes, and the supernatant was an enzyme. The conjugate is stored frozen after being dispensed.
3. Result determination:
(1) Qualitative and potency titration: a specific antigen (or antibody) and an enzyme-labeled antibody (or anti-immunoglobulin antibody) are used as a two-way agar diffusion test or an immunoelectrophoresis test. The precipitation arc is then colored with the substrate of the enzyme to initially identify its activity. Finally, the enzyme conjugate is titrated by direct ELISA (or in a formal experimental system) (see selection of working concentrations in section (3)).
(2) Quantitative and molar ratio determination: measured by spectrophotometer (1 cm path length).
Enzyme amount (mg/ml) = OD403nm × 0.4
IgG amount (mg/ml) = OD280nm-OD403nm × 0.42) × 0.94 × 0.62
The amount of enzyme (mg / ml) IgG amount (mg / ml) amount of enzyme molar ratio = ──────── ÷ ─────── = ─── × 4
40,000 160,000 IgG amount
(3) The marking procedure of this law is relatively simple and reproducible. The disadvantage is that the enzyme utilization rate is low, and generally only 2 to 4% of the enzyme binds to the protein.
4. Reagents and equipment:
(1) 0.1 M PH6.8 phosphate buffered saline (PBS): 49 ml of 0.2 M Na 2 HPO 4 , 51 ml of 0.2 M NaH 2 PO 4 , 1.8 g of NaCl, and distilled water to 200 ml.
(2) 1.25% glutaraldehyde solution: 50 ml of 25% glutaraldehyde was mixed with 1 ml of PBS of pH 6.8.
(3) 1 M PH9.5 carbonate buffer: 3 ml of 1 M sodium carbonate was mixed with 7 ml of 1 M sodium hydrogencarbonate.
(4) 0.2 M lysine solution: 29.2 mg of lysine was dissolved in 1 ml of 0.01 M PH9.5 carbonate buffer.
(5) 0.15 M PH7.4 PBS and saline.
(6) PH7.8 saturated ammonium sulfate solution and semi-saturated ammonium sulfate solution.
(7) Naphthalene reagent and polyethylene glycol (PEG, MW2000).
(8) Purified specific antibody or anti-Ig antibody.
(9) HRP (RZ>3.0).
(10) Sephadex G-25 column (2 cm × 50 cm).
(11) Stirrer, spectrophotometer, centrifuge.
(12) Dialysis bags, large and small beakers, test tubes, straws, etc.
Simple sodium periodate method
This method uses NaIO4 to first oxidize the sugar molecules on the surface of HRP to aldehyde groups, and then combines with the amino groups on Ig. The yield of the enzyme-labeled antibody is high, and nearly 70% of HRP and Ig are combined, 99% of Ig. In combination with enzymes, there is no significant loss of enzyme and Ig activity, which is currently the most commonly used method.
1. Principle: The classic sodium periodate method requires dinitrofluorobenzene to block the residual α- and epsilon amino groups on HRP to avoid cross-linking between enzyme molecules. Later, Wilson changed the use of NaIO 4 to oxidize HRP at a low pH, thereby eliminating the dinitrofluorobenzene blocking HRP step. The hydroformylase formed by the oxidation of HRP by NaIO 4 can be linked to the amino group of the antibody molecule to form a Schiff's sputum, which can be further reduced with NaBH 4 (or ethanolamine) to form a stable enzyme-labeled antibody.
2. Marking steps:
(1) Weigh 5 mg of HRP dissolved in 1 ml of distilled water.
(2) 0.2 ml of a freshly prepared 0.1 M NaIO4 solution was added to the supernatant, and the mixture was stirred at room temperature for 20 minutes in the dark.
(3) The above solution was placed in a dialysis bag, dialyzed against 1 mM sodium acetate buffer of pH 4.4, and allowed to stand overnight at 4 °C.
(4) Add 20μl of 0.2M PH9.5 carbonate buffer to raise the pH of the above hydroformin RP to 9.0-9.5, then immediately add 10mg IgG (antibody, or SPA5mg) in 1ml 0.01M carbonate buffer In a solution, gently stir at room temperature for 2 hours.
(5) Add 0.1 ml of freshly prepared 4 mg/ml NaBH4 solution, mix and store at 4 °C for 2 hours.
(6) The above solution was placed in a dialysis bag, dialyzed against 0.15 M PH7.4 PBS, and allowed to stand overnight at 4 °C.
The remaining steps (purification) are the same as (6), (7), and (8) of the glutaraldehyde labeling step.
3. Result determination:
The calculation was the same as the glutaraldehyde method except that the amount of the marker IgG was slightly different.
IgG amount (mg/ml) = (OD280nm-OD403nm × 0.3) × 0.62
4. Reagents and equipment:
(1) 0.1 M NaIO4: 241 mg of sodium periodate (Guangzhou Chemical Reagent Factory, batch No. 830602) was weighed and dissolved in 10 ml of distilled water.
(2) 1mM PH4.4 sodium acetate buffer:
0.2M NaAc (1.361 g / 50 ml) 3.7 ml
0.2M HAc (0.601ml/50ml) 6.3ml
Add distilled water to 2,000 ml.
(3) 0.2M PH9.5 carbonate buffer:
Na2CO3 0.32g
NaHCO3 0.586g
Add distilled water to 50ml
Distilled water was used for 20-fold dilution to form a 0.01 M PH9.5 carbonate buffer.
(4) NaBH4 solution (4mg/ml):
When used, weighed 44 mg of NaBH and dissolved it in 1 ml of distilled water.
(5) For other reagents and equipment, see the glutaraldehyde labeling method.
Fourth, matters needing attention
1. Under the condition of high quality HRP, the antibody to be labeled should also have high activity, high titer (minimum 1:16), high purity and good affinity, which is to ensure the high titer of the marker and the immune activity is good. condition.
2. The pH and concentration of the reagents used must be strictly controlled. The reagents used are preferably (or necessary) freshly prepared. For example, the glutaraldehyde used in the glutaraldehyde labeling method should be fresh and pure, and the condensed body (impurities) can be formed because the glutaraldehyde is stored for a long time. Otherwise, it affects the effect of the mark.
3. It should be protected from light when stirring at room temperature. The indoor temperature is generally 25 °C. The markers should be carefully checked before each dialysis to prevent leakage.
4. Concentrated markers are quite stable, often added with 30-40% glycerol and stored at -10 °C. It can be stored for 1 to 2 years at 4 ° C, but diluted to 1: 10 and can only be stored for several weeks. The formulated use solution should be used within 12 hours. Avoid repeated freezing and thawing.
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