Background[1-3]
Lecithin cholesterol acyltransferase (LCAT) ELISA kit is used for the in vitro quantitative detection of human lecithin cholesterol acyltransferase (LCAT) content in serum, plasma, tissue, cell supernatant and related liquid samples.
Experimental principle
The kit uses a double-antibody one-step sandwich saturated polyester heart enzyme-linked immunosorbent assay (ELISA). To the coated microwells pre-coated with human lecithin cholesterol acyltransferase (LCAT) capture antibody, add the specimen, standard, and HRP-labeled detection antibody in sequence, incubate and wash thoroughly. The substrate TMB is used to develop color. TMB is converted into blue under the catalysis of peroxidase, and converted into the final yellow under the action of acid. The depth of color is positively correlated with the human lecithin cholesterol acyltransferase (LCAT) in the sample. Use a microplate reader to measure the absorbance (OD value) at a wavelength of 450 nm and calculate the sample concentration.
Operation steps
1. Take out the required slats from the aluminum foil bag that has been equilibrated at room temperature for 20 minutes, and seal the remaining slats in a ziplock bag and return it to 4°C.
2. Set up standard wells and sample wells, and add 50 μL of standards of different concentrations to each standard well;
3. Add 50 μL of the sample to be tested into the sample well; do not add it to the blank well.
4. Except for the blank well, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard well and sample well, seal the reaction well with a sealing film, and place in a 37°C water bath or constant temperature Incubate for 60 minutes.
5. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μL), let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat washing the plate 5 times (you can also use washing plate machine washing).
6. Add 50 μL each of substrates A and B to each well, and incubate at 37°C in the dark for 15 minutes.
7. Add 50 μL of stop solution to each well, and within 15 minutes, measure the OD value of each well at a wavelength of 450 nm.
Apply[4][5]
For research on the association between lecithin cholesterol acyltransferase gene single nucleotide polymorphisms and susceptibility to coronary heart disease
In mammals, lecithin:cholesterol acyltransferase (LCAT) is a key enzyme in the reverse transport of cholesterol back to liver metabolism. It is involved in maintaining the homeostasis of plasma cholesterol and regulating high density lipoprotein (high density lipoprotein). Lipoprotein HDL) and other lipoprotein metabolism have anti-atherosclerotic functions and are one of the susceptibility genes for CAD.
With the completion of human genome sequencing, studying sequence variations associated with disease phenotypes has become a major topic in medical genetics.
By studying the known cSNPs of the LCAT gene and screening unknown cSNPs in Chinese Han patients with D and normal individuals, and comparing the distribution of these cSNPs in patients and normal controls, it is revealed that the cSNPs of the LCAT gene are associated with lipid metabolism disorders and coronary disease. relationship with heart disease, in order to elucidate its role in the occurrence and development of CAD.
First, the polymerase chain reaction-restriction fragment length polymorphism method was used to study the three known eSNp sites (6o8C>T, 91 IT>C and 1188C>T) in Chinese normal people and CAD patients. distribution frequencies, and preliminarily explore their relationship with lipid metabolism and coronary heart disease.
Secondly, DHPLC technology was used to screen for unknown csNPs in the exon region of the AT gene. After further verification through sequencing and PCR-RFLP methods, statistical analysis was performed on the correlation between these cSNPs and lipid metabolism and D.
Research results on three known cSNP sites showed that only the 6O diethanolamine SC>T polymorphic site exists in the Chinese population. In the control group, the frequencies of the two alleles were 97.13% (608C) and 2.87% (608T), respectively, while in the CAD patient group they were 99.26% (608C) and 0.7 Qian (608T). Allele frequency distribution Meet Hardy which einberg balance. After the chi-square test, the distribution of alleles (p = 0.034) and genotype frequency (p = 0.032) of this locus was significantly different between the control group and the patient group. The frequency of 6OST in group D was significantly lower than that in the control group, suggesting that L. The 608C>T polymorphic site of the T gene may be related to D.
