[Immune functional changes in patients of acute Henoch-Schonlein purpura and regulatory effect of integrated Traditional Chinese and Western medicine on it].

OBJECTIVE: To observe the changes of immune function in children with acute Henoch-Schonlein purpura (AHSP) and the effects of integrated traditional Chinese and western medicine (TCM-WM) on it. METHODS: Immunological criteria in TCM-WM group (n = 35) treated by Yinfu Decoction (YFD) combined with transfer factor and 35 patients in the control group (n = 35) treated by conventional treatment were observed and compared before and after treatment. Also the criteria were compared with those of 40 healthy subjects. RESULTS: Before treatment, the levels of IgA, IgM, CD4, CD4/CD8 ratio, rosette forming rate of RBC-C3b receptor in patients were higher than those in the healthy subjects, but the levels of CD8 was obviously lower, the difference was significant (P < 0.01). The above-mentioned criteria were all improved in the two treated groups after treatment, and the improvement was more significant in the TCM-WM group than that in the control group (P < 0.05). Besides, the cure-markedly effective rate in the former was better than that in the latter significantly (P < 0.01). CONCLUSION: There exist multiple immune functional disturbance in AHSP patients. Combined treatment of YFD and transfer factor has obvious immune regulatory effect and is an effective therapy with few side-effects and low recurrence rate.

Antibody-mediated fluorescence enhancement based on shifting the intramolecular dimer<-->monomer equilibrium of fluorescent dyes.

A novel concept is described for directly coupling fluorescence emission to protein-ligand binding. It is based on shifting the intramolecular monomer<-->dimer equilibrium of two fluorescent dyes linked by a short spacer. A 13-residue peptide, recognized by a monoclonal antibody against human chorionic gonadotrophin (hCG), was labeled with fluorescein (F) and tetramethylrhodamine (T) at its N- and C-terminus, respectively. Spectral evidence suggests that when the conjugate is free in solution, F and T exist as an intramolecular dimer. Fluorescence quenching of fluorescein and rhodamine is approximately 98% and approximately 90%, respectively, due to dimerization. When the double-labeled peptide is bound to anti-hCG, however, the rhodamine fluorescence increases by up to 7.8-fold, depending upon the excitation wavelength. This is attributed to the dissociation of intramolecular dimers brought about by conformational changes of the conjugate upon binding. Fluorescein fluorescence, on the other hand, was still quenched because of excited-state energy transfer and residual ground-state interactions. Antibody binding also resulted in a approximately 3.4-fold increase in fluorescence anisotropy of the peptide. These changes in intensity and anisotropy allow direct measurement of antigen-antibody binding with a fluorescence plate reader or a polarization analyzer, without the need for separation steps and labeling antibodies. Because recent advances in peptide technology have allowed rapid and economical identification of antigen-mimicking peptides, the double-labeled peptide approach offers many opportunities for developing new diagnostic assays and screening new therapeutic drugs. It also has many potential applications to techniques involving recombinant antibodies, biosensors, cell sorting, and DNA probes.

Use of synthetic peptides as tracer antigens in fluorescence polarization immunoassays of high molecular weight analytes.

This paper describes a homogeneous immunoassay based on fluorescence polarization that enables subnanomolar detection of high molecular weight analytes. A monoclonal antibody (Mab) to human chorionic gonadotrophin (hCG) was screened against a panel of 221 synthetic peptides using the method of Geysen et al. (Geysen, H. M.; et al. J. Immunol. Methods 1987, 102, 259-274. Geysen, H. M.; et al. J. Mol. Immunol. 1986, 23, 709-715). One of these peptides, which was located near the C-terminus of the hCG beta chain, bound to the Mab with high affinity. It was labeled with tetramethylrhodamine (TMR) and used as the tracer antigen in a competitive fluorescence polarization immunoassay (FPIA) for hCG. The peptide-TMR conjugate binds specifically to the anti-hCG Mab with an antigen-binding affinity (Ka) of 1.5 x 10(7) M-1 at 6 degrees C. Its fluorescence intensity was enhanced by approximately 20% upon binding as a result of a prolonged excited-state lifetime. In a typical embodiment, hCG was determined at a level of 1 x 10(-9) M (95% confidence limit)--a 100-fold improvement over similar systems reported in the literature. This is mainly attributed to the large difference in hydrodynamic volume between the tracer and the antibody, which resulted in large changes in polarization of the peptide tracer upon binding. Issues related to sensitivity, specificity, and reversibility were also investigated. This method is believed to be of significant importance to rapid and economical measurements of high molecular weight antigens of clinical interest.