nucleic acid immunoassay
nucleic acid immunoassay
The ways to enhance the immune response with antigen-expressing DNA are described. Two methods of nucleic acid immunization are described: (i) injection of saline containing DNA expression vectors; and (ii) injection of DNA with a gene gun, and the preparation and preservation of DNA-encapsulated gold beads used in the gene gun method are also covered.
Author: J.E. Collier et al, Translator: Xuitao Cao et al. This experiment is from the "Compendium of Immunology Laboratory Guide".
Operation method
nucleic acid immunoassay Move Basic Option 1 Mouse Inoculation with DNA Animal handling (Appendix 2C) and inoculation (Appendix 2E) instructions should be read prior to immunization. Practice intramuscular and subcutaneous injections are required prior to the formal experiments; precise injection of DNA is critical to the success of the immunization. India ink (or the same dye) can be used to locate the correct injection site. 2 mg of purified expression plasmid DNA (see Experimental Strategy and CPI Unit 10. 3) Bacteriostatic 0.9% (m/V) NaCl 100 mg/m l ketamine hydrochloride 20 mg/ml Methylphenidate hydrochloride (xylazine.HCl) Suitable strain of mice 0.5 ml or Im l syringes with No. 27 or No. 30 needles Dissecting knife for intramuscular inoculation only (optional) Surgical staples or sutures, for intramuscular inoculation only Mouse fixation plates, for subcutaneous inoculation only (optional) 1. Precipitate 2 mg of purified expression plasmid DNA with ethanol in an I.5 ml microcentrifuge tube (Steps 4 to 8 in the Basic Scheme section of Unit 10.1). Fully dissolve the plasmid DNA with Iml of sterile 0.9 % NaCl. Fully solubilize the plasmid DNA with Iml of sterile 0.9 % NaCl to a final concentration of 2 ugDNA/Ml. Store briefly at 4°C or for several weeks at 1-20°C; avoid repeated freezing and thawing. 2. Transfer a sufficient volume of DNA solution (typically 50 ul) to a 0.5-ml or 1-ml syringe with a 27- or 30-gauge needle and remove all air. Intramuscular inoculation 3a. Prepare a mixture of 5m ll00 mg/m l of ketamine and lm l20 mg/m l of xylazine, and anesthetize mice by injecting 30-40W of the above mixture intraperitoneally (or dilute with 0-9% sodium chloride I:10 and inject 300-400M 1 ). The mice are laid flat on their backs with their stomachs facing upward, and an Icm-long incision is made in the medial thigh to expose the quadriceps muscle. In general, surgical exposure of the muscle increases the reliability of intramuscular injections. 4a. Insert the needle 0.5 cm above the knee into the quadriceps muscle at an angle of 30°, with the beveled side of the needle facing upward, to a depth of approximately 0.2 cm. Do not puncture the muscle. Of course, other muscle bundles such as the anterior tibiofibular, biceps, and scapular muscles can also be selected. A slow injection of 50/xl DN A saline solution is made over a period of at least 30 s. The incision is made with a surgical staple or suture. The incision is closed with surgical staples or sutures. An alternative protocol that does not require anesthesia of the mouse is to make a neck rest 0.1 to 0.2 cm from the head of the needle, which ensures that the needle does not puncture the muscle. This neck rest is made by cutting off a section of the end of the IOOul tip and passing a 30-gauge needle through the tip, with the wide end of the tip next to the syringe barrel and the narrow end about 9 mm above the needle, which ensures that it will not puncture the muscle, and using this device, the needle can be injected directly into the target muscle. Subcutaneous injection 3b. Anesthetized mice (as in step 3a) or with a mouse immobilization plate. The needle is inserted about 0.2 cm subcutaneously at the base of the tail, with the beveled side of the needle facing upward at an angle of 5° to 10°, almost parallel to the skin. The SOiLtl DNA salt solution was injected slowly. As the solution is injected, the blister rises. The later the injection, the greater the thrust required. Keep the hand still during injection to avoid piercing the skin or pulling the needle out. Compressed helium - purity level >4.5 ( 9 9 9 . 9 95 % ) ; maximum pressure, 2600 psi (1 psi = 6.894 76 XIO3Pa) 995%); maximum pressure, 2600psi (1psi = 6.894 76 XIO3Pa ) Suitable strains of mice Box for storing gold dust pellets encapsulating exogenous D N A (see Auxiliary Scheme 2 ; must be kept dry) Gene Gun and High-Pressure Helium Tank Regulator Valve, purchased separately or as part of the Helios Gene Gun Series (Bio-Rad). Clamps Ear muffs (e.g., earplugs) 1 . Connect the compressed helium to the regulator valve and the gene gun and use the gene gun according to the operating instructions. 2 . Anesthetize mice (see Basic Protocol 1, Step 3a; anesthesia is optional) and shave the abdomen. 3. Load the gun with the empty cartridge, open the helium tank and put on the ear muffs. Set the helium pressure gauge to the appropriate pressure (l Mm gold pellets at 300 to 500 psi; see Option 3). Clean the gun with helium by pulling the trigger 2 or 3 times rapidly. 4 . Remove the empty cartridge from the gun and load the cartridge encased in exogenous D N A gold dust pellets. The table summarizing the values required for the preparation of the particles in this operation can be consulted. Ultrasonic bath 1 . Connect the nitrogen tank to the regulating valve and sample tube preparation station according to the instructions. Also connect a 12-13in (30.5-33 cm) or shorter section of tubing to the IOml syringe through the l/8in Luer fitting. If a peristaltic pump is not available, prepare a second syringe with a coupling tube for ethanol removal. If using Powderject's Sample Tube Preparation Station, use a small piece of tubing (4in or 10 cm), connect it to the IOml syringe, and seal it with sealing film. 2 . Gently invert the DNA/gold powder/ethanol mixture at room temperature. Place the conical-bottomed centrifuge tube containing the pellet in an ultrasonic water bath for 5 to IOs until no chunks of gold dust are present. 3 . Remove water vapor from the empty PTFE tubing with nitrogen for at least 15 min, or as long as possible. Adjust the nitrogen pressure to 1 to 2 psi and control the airflow rate to 0.4 to 0.5 L/min by adjusting the pipe swivel. 4 . Cut a piece of dry tubing (~30in or 76.2 cm) equal to the rotating head of the tubing, connect this tubing to an IOml syringe, gently invert the DNA/gold powder suspension again, and use the syringe to quickly draw the suspension (no more than 3 ml) into the PTFE tubing. Leave 2 to 3 in (5 to 7.6 cm) of empty tubing at each end of the tubing; do not leave any liquid in this section of the tubing. Do not remove the syringe to prevent air bubbles in the tubing. 5 . Make sure that the nitrogen gas is completely turned off, connect the sampling tube to the tube adapter, and leave the DNA/gold powder suspension in the tube for 4 to 5 minutes. 6 . Disconnect the sample syringe and connect the worm pump (or a second syringe) to the sample tube. Remove the ethanol with the peristaltic pump (or syringe) at a rate of 0.5 to lin (1.27 to 2.54 cm) per second. It is not uncommon for a small portion of the gold dust to be removed with the ethanol. In order to remove ethanol uniformly at an accurate rate from the DNA/gold/dust/ethanol mixture, mark every Iin on the sample tube, time it with a timer, and calculate the rate. Do not use water during this process, as water can contaminate the Sample Tube Preparation Station. If using Powderject's Sample Tube Preparation Station, the loading syringe can be left on the sample tube to remove the ethanol once the gold has sunk. 7 . Remove the peristaltic pump and rotate the tubing at 20r/min with a tube swivel. Rotate the tube for 30~45s until the gold dust particles fall evenly on the tube. 8 . Control the flow rate of nitrogen at 0.35~0.4L/min through the adjusting valve, so that the nitrogen flows through the sample tube, and allow the tube to dry sufficiently for about 4 min until the gold powder changes from black color (wet) to bright brown color (dry). It is normal to see one or two drops of gold dust at the end of the tube, while too much gold dust indicates the presence of water in the tube. 9 . Stop turning the tube, turn off the nitrogen and remove the tube from the rotor. Observe the distribution of the gold dust in the tube in the light and mark the two ends of the sample tube where the gold dust is uniformly distributed by folding the slits or using the marker method. Use only tubes that have a uniform coating or where the majority of the gold coating is in the form of fine gold lines. If the gold powder does not form a coating in the tube, but forms a thick gold line, it is because the ethanol is removed too slowly. If the gold coating appears in swirls or bands, it is because the ethanol is being removed too quickly. If the gold dust is sparsely distributed or patchy, it is possible that the DNA/gold dust/ethanol preparation process contaminated the water and further drying is required (Step 10). 10. Contamination with water during preparation: note down the volume of the prepared DNA/gold powder/ethanol mixture, centrifuge the mixture, and wash the precipitate with fresh anhydrous ethanol 3 to 5 times. Resuspend the pellet with equal volumes of fresh anhydrous ethanol. Prepare sample tubes with this DNA/gold powder/ethanol mixture (Steps 3 to 9) and purge empty tubes under nitrogen for a longer time (Steps 3 to 9). If necessary, prepare the tubes when the weather is not humid. 11. Use a razor blade to cut off the marked tubing and discard the unwanted section. Cut the remaining tubing into 0.5in (1.27 cm) pieces, preferably using a sample tube cutter or razor blade to cut the tubing. Place the tubing in a glass tube with desiccant. Seal the cap with sealing film. The tubes can be stored at 4°C for 8 to 12 months. 12. Optional: Quantification of DNA content in sample tubes. Place 5 sample tubes in a 1.5 ml microcentrifuge tube with 500 ul of TE buffer at pH 8.O. Sonicate until the gold dust is dislodged from the tube walls and the DNA is re-solubilized. Centrifuge at maximum speed for approximately 5 min. measure the supernatant A26 with a spectrophotometer to calculate the amount of DNA. Theoretically, if 100 % of the DNA is encapsulated in the gold dust pellet, then l Mg of DNA can be obtained per tube, which corresponds to an A260 value of 0.2 %. 1 . Narcotic mice (see Basic Option 1, step 3a) are shaved on the abdomen with an electric razor. DNA-coated gold particles were inoculated at different points with a gene gun at pressures in the range of 100-500 psi in increments of IOOpsi (see Basic Option 2). Each condition was inoculated twice, and it was determined that there was no overlap of inoculation sites on the abdominal skin, and that the distance between the points and the outer ring of points was about 0.5 cm, with each mouse inoculated with four points. 2 . Biopsy the inoculated spots with a skin punch, this can be done within minutes of inoculation. Place the biopsied skin on the IinXlin's card in the direction of the skin. Because the perforated area heals quickly, no stitches are needed. Mice that have had multiple biopsies can be euthanized (Appendix 2 G). 3 . Place the biopsies from the cards in a sectioning box and fix the tissue with 4 % paraformaldehyde. Embed and section in paraffin (CPI module 21.4). Stain with eosin and observe the depth of the granules in the sections under the microscope. In all sections, particles were found in the stratum corneum, epidermis and dermis. Counting the particles in each layer makes it possible to determine under which conditions the particles in the epidermis are the highest percentage of the total (usually 40% to 60%). Generally, a pressure variation of at least IOOpsi is used to cause a significant difference in the depth of particle inoculation. For more product details, please visit Aladdin Scientific website.
(Figure 1)
5. - Hold the mouse flat with one hand (or pinch the neck of the mouse with one hand, Appendix 2C) so that the scraped skin is exposed and stretched. Point the gun directly at the scraped abdominal skin at an angle of 90° and pull the trigger. Look for a brown spot in the middle of the target area. Return the mouse to its cage and observe its awakening from anesthesia. The hit area will be moderately red within IOmin after Gene Gun inoculation.
Spectrophotometer (optional)