Co-fluorescence effect in time-resolved fluoroimmunoassays. A review.
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Biomedical subjects
Publications and source records attributed to Y Y Xu.
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This study was conducted in two parts. In the first part, 20 single-rooted teeth that had been scheduled for extraction were investigated. The electronic root canal lengths were measured in vivo with a Dental Sono-Explorer type Y-III, and the actual canal lengths were measured after extraction of the teeth. The rate of agreement of the two measurements was 77.5% within a range of +/- 0.5 mm, while it was 100% at +/- 2.0 mm, which is acceptable clinically. In the second part, there were 19 simulated canals whose lengths and apical foramen sizes were known beforehand. Experiments revealed a negative correlation between the areas of the apical foramina and the difference between the electronic and the actual root canal lengths. This relationship was shown by the linear regression equation: ŷ = 0.6-1.6x. With the exception of the smallest areas of foramina, electronic root canal length measurements were less than the actual lengths.
Using ConA-HRP and RCAI-HRP as probes, the distribution and changes of glycosides in mouse cornea were studied during pre- and postnatal development. Mannose residues were distributed mainly in stroma and endothelium, sialic acid residues in epithelium and galactose residues in both epithelium and stroma. Mannose residues in stroma showed an increased density toward endothelium before and after birth. Sialic acid and galactose residues were concentrated gradually at the corneal epithelial surface in accompanied with the development of cornea. The embryonic day 13 was the starting day to synthesize glycoconjugates from fibroblasts of mouse cornea.
We describe a quadruple-label fluorometric immunoassay for simultaneously measuring four analytes: thyroid-stimulating hormone (TSH), 17 alpha-hydroxyprogesterone (17 alpha-OHP), immunoreactive trypsin (IRT), and creatine kinase MM (CK-MM). The assay is based on immunoreagents labeled with four different lanthanide ions (Eu3+, Tb3+, Sm3+, and Dy3+), on dissociative fluorescence enhancement applying the principle of co-fluorescence, and on time-resolved fluorometry. The monoclonal anti-alpha-TSH and anti-IRT antibodies and the polyclonal anti-CK-MM antibody were labeled with Eu3+, Sm3+, and Dy3+, respectively; 17 alpha-OHP was labeled with Tb3+. The assay was performed in microtitration strip wells coated with a mixture of monoclonal antibodies against beta-TSH, IRT, and CK-MM and a polyclonal goat anti-rabbit IgG for capture of the rabbit anti-17 alpha-OHP antibodies. After completion of the immunoreactions, the bound fractions of the lanthanides were dissociated into the co-fluorescence enhancement solution, creating highly fluorescent chelates. The four lanthanide-specific signals were subsequently measured in a time-resolved fluorometer. The detection limits of the assay were 0.1 mIU/L for TSH, 2 nmol/L for 17 alpha-OHP, 2 micrograms/L for IRT, and 4 U/L for CK-MM.
Four-day old ducklings were infected with duck hepatitis B virus to simulate perinatal transmission of hepatitis B virus. Immune tolerance was characterized as persistent viremia, antigenemia, without detection of anti-DHBs or anti-DHBc. A synthetic peptide, P125-146, mimicking one of the epitopes of the native DHBV Pre-S protein was used to cross-link to tetanus toxoid or phytohemagglutinin. These 'novel' antigens were used to immunize immune tolerant ducks, aimed at bypassing T cell tolerance. After 5 injections, though no anti-DHBV Pre-S was detected, around 50% of the immunized ducks showed seroconversion to DHBV DNA negative.
To characterize the region on human IgG1 responsible for its high-affinity interaction with the human Fc gamma receptor class I (Fc gamma RI), we have analyzed the binding properties of a series of genetically engineered chimeric antidinitrophenyl antibodies with identical murine antibody combining sites and hybrid IgG1/IgG2 human constant (C) regions. In addition, we have investigated a panel of reciprocally point-mutated IgG1 and IgG2 chimeric antibodies to identify the amino acid residues that confer cytophilic properties to human IgG1. Our data unambiguously indicate that cytophilic activity of IgG1 is an intrinsic property of its heavy-chain C region 2 (CH2) domain. We report that the entire sequence spanning residues 234-237 (LLGG) is required to restore full binding activity to IgG2 and IgG4 and that individual amino acid substitutions failed to render IgG2 active. Nevertheless, the reciprocal single point mutations in IgG1 either significantly lowered its activity or abolished it completely. Finally, we observed that an IgG2 antibody containing the entire ELLGGP sequence (residues 233-238) was more active than wild-type IgG1. This finding suggests that in addition to the primary contact site identified in the N terminus of the gamma 1 CH2 domain, secondary sites involving residues from the C-terminal half of the domain may also contribute to the IgG1-Fc gamma RI interaction.
In the presence of an excess of Y3+, the fluorescence intensities of Eu3+ and Sm3+, chelated with benzoyltrifluoroacetone (BTA) or thenoyltrifluoroacetone (TTA) in an aqueous solution containing 1,10-phenanthroline, were increased by factors ranging from 209- to 811-fold. This co-fluorescence phenomenon was used in a highly sensitive time-resolved fluorimetric detection of the lanthanides, Eu3+ and Sm3+. The detection limits of Eu3+ in the BTA- and TTA-based solutions were 4 and 15 fmol dm-3, respectively. The detection limits of Sm3+ were 0.11 and 0.12 pmol dm-3, respectively. The co-fluorescence enhancement systems were also applied in the double-label time-resolved fluorimetric immunoassay of luteinizing hormone and follicle stimulating hormone using specific antibodies labelled either with Eu3+ or Sm3+. The co-fluorescence enhancement solution was superior as compared with the commercial 'direct' fluorescence enhancement solution based on the acidic solution of beta-naphthoyltrifluoroacetone, trioctylphosphine oxide and Triton X-100, in respect to the signal level obtained and the sensitivity. It is suited to time-resolved fluorimetric immunoassays in which particularly high detection sensitivities are required, and it can also be used in double-label assays employing Eu3+ and Sm3+ chelate labels.
Synthetic peptides P37-49 and P63-79, derived from the pre-S region of duck hepatitis B virus (DHBV), have been synthesized. Only P37-49 was reactive with rabbit anti-DHBs/pre-S antibodies by radioimmunoprecipitation. Antiserum prepared against P37-47 reacted with a 35K polypeptide of native DHBs/pre-S by immunoblotting. It is concluded that P37-49 (MGQHPAKSMDVRR) mimics one of the epitopes of the DHBV pre-S antigen.
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Endomyocardial biopsy (EMB) was performed in 24 cases suspected of having myocarditis and 42 cases with cardiac symptoms and signs of unknown etiology. There was an obvious correlation between the clinical manifestations and the pathological findings in cases suspected of having myocarditis (r = 0.5296, P less than 0.005). The ratio of correspondence was 78.8%. It is shown that EMB is more sensitive than 201TI myocardial imaging in diagnosing myocarditis and serial EMB can follow its clinical process closely. Moreover, EMB can discriminate myocarditis from myocardiopathy.
To determine the risk of biopsy in different parts of heart and the artifacts of histomorphology, right heart endomyocardial biopsy in eight dogs were done. The result showed no tamponade occurred even 53 pieces of heart muscles were got from right ventricular free wall. In contrast, biopsies on right atrial free wall had higher risk in causing tamponade. It was confirmed by autopsy and histomorphology that endomyocardial biopsy could cause heart muscle damage. The artifacts caused by biopsy of normal beating heart muscle include: contraction bands (21.8%), widen of intracellular space (100%), interstitial edema (78%) and interstitial bleeding (53%). The results of the patients of right ventricular endomyocardial biopsy may provide practical value in the application of endomyocardial biopsy.
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Alpha-human atrial natriuretic polypeptide (alpha-hANP) was applied to 16 clinical patients, 6 patients with essential hypertension, 7 patients with congestive heart failure and 3 patients with cirrhosis. Following intravenous bolus injection of 400 micrograms of synthetic alpha-hANP, a hypotensive effect of very rapid onset was found, which was more potent in the hypertensive patients than in the normotensive cases. Cardiac functions were improved significantly with a similar time course as the depressor response in the cases of heart failure or hypertension. Hemodynamic observations showed a marked increase in cardiac output, cardiac index, stroke volume, ejection fraction and ejection rate, and a concomitant decrease of the pressure in the right side of the heart and pulmonary circulation in these subjects. In addition, the renal response to alpha-hANP induced obvious increases in urine volume, electrolytes and creatinine excretions in all the subjects. Finally, plasma levels of aldosterone, Arg-vasopressin and noradrenaline were also altered by alpha-hANP. No significant side effects were registered. The above result confirms the therapeutic actions of alpha-hANP in human subjects and opens the possibility to research alpha-hANP as a powerful pharmacological tool as well as potential new medicine for human disorders.
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