Immunological assay of sisomycin by fluorescence polarization

Fluorescence polarization immunoassays (FPIA) of aminoglycoside antibiotics (gentamicin, tobramycin and amikacin) in plasma have been described. The use of sisomicin, a structural analogue of a C1a gentamicin fraction, is an alternative to gentamicin therapy for economical reasons. We have tested a therapeutic monitoring of this drug bases on FPIA using its croos reaction with gentamicin. Tracer (gentamicin labelled with fluorescein) and antisera were purchased from Abbott S.A. (gentamicin kit). Standard curves were obtained with sisomicin standard plasma samples. Within run and run-to-run precisions expressed as coefficient of variation were lower than 9.2 p. cent. The measurement of concentrations as low as 0.15 mg/l are possible. In order to evaluate specificity of this assay in clinical situation, we compared FPIA and liquid chromatography results.     

BSA免疫动物过程中抗原抗体亲和动力学特性的研究

目的 本课题使用牛血清白蛋白(BSA)作为抗原,通过对小鼠和家兔进行连续多次免疫和间隔长久免疫,探讨不同免疫周期下抗体产生及亲和力成熟的动力学变化规律.方法 采用ELISA和毛细管电泳测定抗体抗原的亲和常数.结果 伴随免疫次数的增加,特异性抗体效价持续增强.抗体亲和力水平在二次免疫应答时存在一个明显的突变过程,小鼠的抗体亲和常数从1.6×108 L/mol上升到6.9×108 L/mol.随后免疫次数虽然继续增加,但亲和力变化趋于稳定,最终稳定于7.2×108 L/mol,而B细胞对抗原的亲和力会持续增强.结论 抗体抗原亲和力随免疫次数的增加趋向饱和,BSA的线性表位为优势表位,随免疫次数的增加比重逐渐增强.     

SNP genotyping with fluorescence polarization detection

When a fluorescent molecule is excited by plane polarized light, the fluorescence emitted is also polarized. The degree of fluorescence polarization (FP) detected, under constant temperature and solvent viscosity, is proportional to the molecular weight of the dye molecule. By monitoring the FP of a fluorescent dye, one can detect significant changes in the molecular weight of the molecule without separation or purification. Because the size of the probe is altered in the course of a number of single nucleotide polymorphism (SNP) genotyping reactions, FP is therefore an excellent detection mechanism for these assays. Indeed, FP detection can be used in SNP genotyping with the primer extension TaqMan((R)) and Invader((R)) assays. Use of FP detection makes it possible to reduce the cost of TaqMan((R)) and Invader((R)) probes by abrogating the need for a fluorescence quencher. Moreover, inexpensive, unpurified, and unlabeled probes are used in the primer extension reaction with FP detection. As an end-point detection mechanism, FP detection is suitable for high-throughput SNP genotyping.     

Investigation of long-term traces and memory mechanisms by polarization

In experiments on cats alternate direct electrical stimulation of the motor and temporal cortex, together with polarization (2–6a) of these regions in the intervals between combination of these stimuli, caused the fastest possible formation of connections between these regions, as reflected by a motor response to polarization alone. The character of the motor response and the frequency of its appearance during polarization correspond fully with observations during the action of the combined stimuli. No motor response occurs to stimulation of the temporal region alone or to the action of afferent stimuli.     

Value of total thyroxine determination by fluorescence polarization in examination for dysthyroidism

Total T4 has been measured by fluorescence polarization (Abbott TDX). The intra-assay variation coefficient is three fold smaller than that obtained by radioimmunoassay (T4 RIA). The interassay variation coefficient of T4 TDX (7%) is better than that of T4 RIA (9%). There is a strong correlation between the two methods (r = 0,95). The mean value of T4 TDX is significantly lower than that of T4 RIA in 218 euthyroid patients without treatment as well as in 92 euthyroid patients under T4 treatment and in 14 hypothyroid patients. In 30 hyperthyroid patients the mean values were identical. The automatization and the short time of manipulation (0,5 hour) allow to obtain the result during the consultation.     

Field trial of the brucellosis fluorescence polarization assay

Fluorescence polarization assay (FPA) is a homogeneous technique which was applied to the serological diagnosis of bovine brucellosis. Because of its simplicity and because it may be performed very rapidly, it was an ideal test to adapt to field use. The FPA was used to test cattle on six dairy farms in Baja California, Mexico. Anticoagulated blood, serum, and milk were collected from each animal. The anticoagulated blood was tested immediately on the farm while serum and milk were tested subsequently in the laboratory. Cattle on one farm (n = 140) were thought not to be infected with Brucella abortus and the other farms were thought to have high prevalence of the infection. The whole blood FPA (FPA(bld)) did not detect antibody in any of the cattle on the first premise. This finding was confirmed using a number of other serological tests, including the buffered antigen plate agglutination test, the complement fixation test, the indirect and competitive enzyme immunoassays, and the FPA using serum and milk. Cattle on the other premises (n = 1122) were tested in a similar fashion. The sensitivity of the FPA(bld), relative to the serum FPA (considered the definitive test), was 99.1% and the relative specificity of the FPA(bld) was 99.6%. These results compared favourably with those obtained using the other serological tests.     

Dissociation kinetics of antigen-antibody interactions: studies on a panel of anti-albumin monoclonal antibodies

Kinetic parameters and equilibrium association constants (K) are reported for a panel of anti-bovine serum albumin (BSA) monoclonal antibodies (MAb) immobilized onto agarose particles. For 12 covalently immobilized MAb of moderate affinity (K = 0.25 x 10(8)-1.2 x 10(8) M-1) measured dissociation time constants varied two orders of magnitude, from 2.1 to 410 min. Directly measured association rate parameters agree with values calculated from measured equilibrium and dissociation rate parameters. Dissociation time constants and equilibrium association constants were also determined for eight MAb immobilized biospecifically (via their Fc regions). A significantly lower K was observed with those MAb which were covalently immobilized as opposed to biospecifically immobilized. These decreases in K appear to reflect decreased association rates rather than increased dissociation rates. The data suggest that, for the MAb described herein, dissociation rates do not correlate with equilibrium association constants.     

Development of a rapid method of immunoassay of C reactive protein by polarization of fluorescence

Increased level of C reactive protein in the serum of neonates indicates the presence of infection, whereas the classic signs (hyperthermia, neutrophilia...) may often fail. So the determination of C reactive protein is useful and has to be rapidly performed on a small quantity of blood. The authors evaluate a fluorescence-polarization immunoassay (TDX Abbott System), and compare this method to the radial immunodiffusion (RID) and the laser immunonephelemetry. Sensitivity (detection limit = 10 mg/l), reproducibility (cv less than 5 p. cent), linearity (until 243 mg/l), are satisfactory. The assay correlates well with RID (r = 0.98, p less than 0.001) and laser immunonephelometry (r = 0.93, p less than 0.001). The proposed method is rapid (10 minutes), requires small quantities of blood (less than 100 microliters), and is not influenced by important amounts of haemoglobin, bilirubin or triglycerides. Thus, this method provides a good means for C reactive protein determination, especially in neonates.     

Determination of relative antigen-antibody avidities by radial immunodiffusion

Radial immunodiffusion can be used to determine relative antigen-antibody avidities in exactly the same way as demonstrated previously for quantitative immunoelectrophoresis (Birkmeyer et al., 1981). Antigen-antibody interactions of greater avidity result in a greater value of (delta Area/delta [Antigen]) in plots of immunoprecipitin circle area versus antigen concentration while interactions of equal avidity will yield equal values of (delta Area/delta [Antigen]). This was demonstrated using antigens of different weight ranging from 8000 to 66,000.     

Comparison of fluorescence polarization immunoassay and bioassay of vancomycin.

Human serum samples were analyzed for vancomycin concentrations by two different methods: the fluorescence polarization immunoassay and the disk plate bioassay. Each assay method offered acceptable precision. The correlation between both assay methods was excellent (correlation coefficient = 0.985). Excluding technical time, the bioassay was the least expensive method to perform but was more labor intensive than the fluorescence polarization immunoassay.     

Fluorescence resonance energy transfer imaging via fluorescence polarization measurement

Fluorescence resonance energy transfer (FRET) has become a widely used spectroscopic tool for detecting molecular interactions and molecular proximity in solution, as well as in membranes. On the other hand, fluorescence polarization (FP) is a convenient measure: ratiometric and simple to execute. This work presents a novel methodology for determining energy transfer efficiency (E) via FP measurement. The methodology is based on the fact that a donor's fluorescence lifetime is shortened due to FRET and, consequently, its FP increases. As a model, the present work evaluates the E between fluorescein and rhodamine conjugated ConA attached to the receptors in the lymphocyte membrane. It shows not only that FRET imaging via FP is possible, but also that it is inexpensive, simple to perform, conveniently adaptable to the commonly used fluorescent microscopy, and readily interpretable.     

Determination of relative antigen-antibody affinities by quantitative immunoelectrophoresis

The relative affinities of an antibody population for different antigens or of different antibody populations for one antigen can be determined by quantitative immunoelectrophoresis. Antigen-antibody interactions of greater average affinity result in a greater increase in rocket area as a function of the amount of antigen applied to the wells. This is measured as the slope of the line in plots of rocket area versus antigen amount. Quantitative immunoelectrophoresis of different antigens, which nevertheless have the same affinity for antibody, produces plots with the same slopes. The relative magnitudes of the slopes of these lines reflect the relative average affinities of different antibody populations for an antigen.     

Use of fluorescence polarization to monitor MHC-peptide interactions in solution

We describe here fluorescence polarization-based methods to investigate class I MHC-peptide interactions in solution. Fluorescein-labelled peptides were used to determine MHC/peptide complex association and dissociation constants as well as the equilibrium binding constant (KD). Furthermore, we developed a competition assay for the determination of IC50 values of nonlabelled compounds. Both kinetic and equilibrium parameters are of prime importance for the development of immunomodulating compounds. The assays described here show a good reproducibility and require only picomolar amounts of labelled tracers. A high ratio between the experimental values obtained for bound and free labelled ligand as well as a low standard deviation, permits the detection of class I MHC ligands with low affinity. Fluorescence polarization allows the direct measurement of the ratio between free and bound labelled ligand in solution without any separation step. Thus, in combination with microtiter-plates, the time for analysis is significantly decreased to 10 s per sample. Our assays represent versatile tools for characterizing the binding of single ligands as well as for rapid screening of large numbers of compounds.