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Objective:The polyclonal antibody of aldosterone (ALD) for immunoassay was developed.And a chemiluminescence immunoassay (CLIA) for the determination of ALD in human blood was established.Methods:Aldosterone oxime was prepared by chemical modification and then conjugated with BSA to prepare immunogen.Rabbit anti ALD polyclonal antibody was prepared by immunizing rabbits with the ALD-BSA.The CLIA of ALD was performed using biotin streptavidin amplification system and competition method.Results:After identification,rabbit No.3 received the highest sensitivity to ALD antibody,and the 50％ binding inhibition (IC50) value for ALD concentration was 268 pg/ml.The measuring range of CLIA method using the antibody was 62.5-2 000 pg/ml.The assay sensitivity was 23.7 pg/ml.The intra-and inter-assay coefficients of variation were 6.9％-9.5％ and 8.5％-12.7％,respectively.Analytical recovery rate was in the range of 93.1％-104.1％.The correlation coefficient between measured and expected values were 0.996 after serial dilution.Compared with radioimmunoassay kit,the correlative equation was y =0.932x+4.596,the correlation coefficient was 0.948 (n =95).Conclusion:The result of methodological identification shows that it was in line with the basic requirements of clinical application.
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Objective:To develop a biotin-streptavidin system (BAS)-mediated folate receptor (FR)-targeted quantum dot (QD) fluorescent probe and preliminarily validate the targeting ability and signal amplification effect of the probe. Methods: Streptavidin (SA) was covalently coupled with QD through the active ester method;the physical characteristics of the prepared QD-SA were veri-fied. Biotinylated folate was synthesized through the carrier bovine serum albumin using the same method and then reacted with QD-SA to form the special probe. The probe was used to identify SKOV3 cells and FR-negative A549 cells to verify its targeting speci-ficity. QD-SA was used as the contrast. SKOV3 cells were imaged using the BAS-mediated FR-targeted QD probe with a biotinylated folate incubation time of 1 or 4 h. Various reaction times were also tested between the probe and the QD-FA that was formed without BAS mediation. Results:The BAS-mediated FR-targeted QD probe specifically recognized FR-positive SKOV3 cells. The probe ob-tained higher fluorescent intensity after 4 h than after 1 h of biotinylated folate incubation. The BAS-mediated FR-targeted QD probe al-so had a stronger fluorescent signal than the QD-FA probe. Conclusion:The proposed probe presents a great potential in the early diag-nosis of ovarian cancer because of its high specificity and sensitivity.
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Objective To explore a novel quantitative detection method for the concentration of specific IgG (sIgG) in food intolerance, taking egg sIgG detection for the example. Methods A total of 173 patients underwent food allergen sIgG detection were included in this study, and 78 healthy subjects were used as negative controls. The microtiter plates were coated with biotinylated bovine serum albumin (BSA) and linked with streptavidin. Then, the biotinylated egg antigen and specimen were successively added into the wells of plate.After washing, enzyme labeled anti-human IgG was added to establish an antigen indirectly coated liquid-phase reaction patterns of ELISA method. The concentration of the biotinylated allergens and enzyme labeled antibody were optimized and the reaction conditions were determined. This method was used to detect the sIgG in serum samples. Results The biotinylated egg white was selected as antigen, the optimal dilution rate was 1∶2 000,and the most suitable enzyme labeled antibody dilution ratio was 1∶12 000 in this method. The within-run and the between-run coefficients of variation were 4.83%-8.55%and 4.88%-7.93%respectively. The specificity is preferable, the species-crossed reaction rates with crab, cow milk and goat milk were＜10%. There was a good correlation between the assay developed in this study and the food intolerance detection kit provided by United States BIOMERICA Inc ( =0.977X+8.45, r=0.961, P＜0.05). Conclusion The detection method can be used to detect serum sIgG for egg intolerance patients with easy operation, highly accuracy and specificity. Furthermore, it showed the capacity of excellent repeatability and flexibility potential, which provided a good foundation for developing a kind of“personalized”random combination of food varieties ELISA kit.
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Objective To observe the expression of CD_(25),CD_(25) mRNA,interleukin-1?(IL-1?) in peripheral blood of newborns with hypoxic-ischemic encephalopathy(HIE) and its clinical significance.Methods The peripheral blood mononuclear cells(PBMC) of newborns with HIE and normal controls were isolated by routine Ficoll-Hypaque,and the level of CD_(25) induced and non-induced by phytohernagglutinin(PHA) were detected by biotin-streptavidin(BSA),CD_(25) mRNA was detected by real-time PCR and IL-1? in serum was detected by enzyme-linked immunosorbent assay on the first,third and seventh day after birth.The final expressive level of CD_(25) mRNA was based on rate of CD_(25) cDNA and gluceraldehyde phosphase dehydrogenase(GAPDH).Results On the first day,the positive rates of CD_(25) induced and non-induced by PHA and IL-1? were(3.6?1.1)%,(20.9?4.8)%,(17.7?8.2) ng/L,respectively.There were significant differences in HIE group and normal controls(P0.05).Conclusions There is obvious inflammatory(rea)ction in babies with HIE.The main character of PBMC of newborns is all infantile and undifferentiated,which surface sign is infantile,and lower level of CD_(25) is expressed.However,the level of CD_(25) mRNA can be expressed consistently during the course of disease.The immunity and immune regulative response in newborns is gradually built up during the growth and can participate in recondition of brain injury caused by HIE.
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The acticle describes a time—resolved fluoroimmunoassay for serum progesterone,based ona competitive solid—phase antigen technique。Progesterone—BSA conjugates are coated onto thewhite opaque microtitration wells。Anti—progesterone antibody is biotinylated.Eu~(3+) are labelledto streptavidin as the tracer.The assay standard curve covers a range of 0.1—100ng/ml.Thelowest detecting limit is 0.05ng/ml。The intra and inter C?V s are 6.45% and 8.93% respec-tively.The average recovery is 89.5%.40woman samples are measured with RIA and presentTRFIA and the results are satisfactory,giving a correlation coefficient of 0.9619.The perfor-mance charicter—istics of the assay are discussed and compared with other known immunoas-says.