Double-stranded DNA antigen, preparation method thereof, reagent containing antigen, kit and application
A kit and DNA molecule technology, applied in the field of immunodetection, can solve the problems of affected test results and low purity, and achieve the effects of high specificity, high purity and accurate test results.
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[0041] As mentioned in the background technology, the antigens used in the detection of anti-double-stranded DNA antibodies in the prior art have poor specificity and are difficult to accurately detect the defects of double-stranded DNA antibodies in samples. In order to improve this situation, in this application In a typical embodiment, a method for preparing a double-stranded DNA antigen is provided, the preparation method comprising: extracting the double-stranded DNA; using protease and RNase to remove the histone and RNA components in the double-stranded DNA to obtain the first Purify DNA; shear the first purified DNA into 500-2000bp fragments to obtain sheared DNA; use nuclease S1, exonuclease I and demethylase to eliminate single strand or methylation in the sheared DNA The modified DNA molecule is obtained to obtain a second purified DNA; and the second purified DNA is purified and recovered to obtain a double-stranded DNA antigen.
[0042] The preparation method of t...
Embodiment 1
[0131] 1. Antigen preparation:
[0132] Sigma's DNA extraction kit was used to extract double-stranded DNA from calf thymus tissue, proteinase K and RNase A were added to remove histone and RNA components, and then purified with phenol-chloroform to obtain the first purified DNA; 2-butanol was used for the first purification The DNA was concentrated to obtain double-stranded DNA at a concentration of 2 mg / mL. Then use a 4.5-gauge hypodermic needle to pipette the solution about 100 times at a frequency of 20 times / min to obtain a DNA fragment of about 500 bp, and then add nuclease S1 and DpnI demethylase to eliminate the formation of the extraction and shearing process. The single-stranded structure and the DNA molecules modified by methylation are then purified by electrophoresis gel and purified by cutting the gel through a column to recover and prepare a highly purified double-stranded DNA antigen.
[0133] 2. Magnetic microsphere coating and suspension:
[0134] Take 10-3...
Embodiment 2
[0169] 1. Antigen preparation:
[0170] Sigma's DNA extraction kit was used to extract salmon sperm double-stranded DNA, proteinase K and RNase A were added to remove histone and RNA components to obtain the first purified DNA, and then 1 butanol was used to concentrate to obtain 1.8 mg / mL of concentrated DNA , and then use a 4.5-gauge hypodermic needle to pipette the solution about 90 times at a frequency of 10 times / min to obtain a DNA fragment of about 2000 bp, and then add nuclease S1, exonuclease I and demethylase DpnI to eliminate extraction and The single-stranded structure formed during the shearing process and the DNA molecules modified by methylation are then purified by electrophoresis gel and gel-cutting through a column to recover and prepare a highly purified double-stranded DNA antigen.
[0171] 2. Magnetic microsphere coating and suspension:
[0172] Take 10-30mg containing -NH 2 The naked magnetic microspheres were washed 3-5 times with phosphate buffer, 1 m...
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