Production and characterization of monoclonal antibody against recombinant human erythropoietin.

OBJECTIVE: To produce specific monoclonal antibody (mAb) against recombinant human erythropoietin (rHuEPO) for development of highly efficient methods for erythropoietin detection in biological fluids. METHODS: rHuEPO was covalently coupled with bovine serum albumin (BSA) and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology. The obtained F3-mAb was characterized by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE and Western blot. RESULTS: The isotype of F3-mAb was found to be IgM with an affinity constant of 2.1 x 10(8) L/mol. The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work. CONCLUSIONS: The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.

Development and application of a multi-target immunoaffinity column for the selective extraction of natural estrogens from pregnant women's urine samples by capillary electrophoresis.

In this paper, a methodology for the determination of three naturally occurring estrogens (estradiol, estrone and estriol) in pregnant women's urine has been described. The procedure included immunoaffinity column (IAC) extraction of 4 mL of urine sample and subsequent analysis of the extraction by micellar electrokinetic chromatography (MEKC). A multi-target polyclonal antibody that has high affinity to three estrogens was produced. Then the IAC was developed by coupling polyclonal antibody to CNBr-activated Sepharose 4B. The IAC showed high affinity for these estrogens. Recoveries of three estrogens from human serum matrix were greater than 92% with R.S.D. less than 4.5%. The final elute of urine sample was diluted with running buffer and then quantitated with MEKC. The experimental results demonstrated that IAC was a useful technique for extraction and concentration of estrogens from biological samples. Three estrogens levels in six pregnant women's urine were measured by both the present method and enzyme-linked immunoadsorbent assay (ELISA). The results of this method have been found to correlate well with those of ELISA.

Biotin-avidin amplified enzyme-linked immunosorbent assay for determination of isoflavone daidzein.

A biotin-avidin amplified enzyme-linked immunosorbent assay (BA-ELISA) method was developed and optimized for the determination of a weakly estrogenic isoflavone daidzein in serum, urine and Puerariae radix. Specific polyclonal antibody was produced against daidzein by immunization of rabbits with a conjugate of 7-O-(carboxymethyl)-daidzein and bovine serum albumin (BSA). The polyclonal antibody showed specific recognition of daidzein, while cross-reactivities to coumarin, 4-hydroxycoumarin, phenol, and other isoflavones such as puerarin and rutin were all lower than 1%. The linear range of daidzein calibration curve was 0.1-1000ngmL(-1). The detection limit was found to be 0.04ngmL(-1), and the intra-assay and inter-assay coefficients of variation were 7 and 16%, respectively. Human serum and urine samples were spiked with known amounts of daidzein and measured by the established BA-ELISA. Recoveries were between 91 and 107%. Daidzein in P. radix was determined by the BA-ELISA method and HPLC method, and the content of daidzein was determined to be 0.0219 and 0.0194%, respectively. The results indicated that there was a good agreement between the two methods. The established method is very useful for monitoring daidzein in biological samples and traditional Chinese medicine.

Development of an indirect competitive ELISA for the determination of papaverine.

Papaverine (1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline, PAP) is a member of the benzylisoquinoline sub-group of the opium alkaloids. It has been widely used for treating diseases like pulmonary arterial embolism and renal or biliary colic. In this paper, a specific conjugate of mono-demethylated papaverine-O-carboxylmethyl ether (MDMPAP-O-CME) and bovine serum albumin (BSA) was synthesized and used as the complete antigen (PAP-BSA), with which we successfully obtained a high-titer anti-PAP polyclonal antibody (pAb) by immunization of rabbits. The anti-PAP pAb showed high affinity to papaverine with an affinity constant (K(aff)) of 7.3x10(7)L/mol. With this antibody, we established a sensitive immunochemical method for the determination of papaverine based on indirect competitive enzyme-linked immunosorbent assay (ELISA). The optimal concentrations of the coated antigen (PAP-OVA) and purified pAb used in the ELISA were 5 and 1.2mug/mL, respectively. The cross reactivity of other benzylisoquinoline derived substances, including 1-(3,4-dihydroxybenzyl)-7-hydroxy-6-methoxy-isoquinoline (6-methoxy-papaveroline, MPAPO), morphine (MP) and codeine (CD) were all lower than 1%. The linear range of the calibration curve was 0.1-1000ng/mL. Normal human serum samples were spiked with known amount of papaverine and measured by the ELISA. Recoveries were between 102% and 105%. Papaverine content in a commercial papaverine hydrochloride injection sample was also determined using the established ELISA. Compared with the results given by the control experiment of HPLC, the recoveries of ELISA to detect injection samples were 102-110%. The limits of detection for synthetic serum samples and injection samples of papaverine hydrochloride were 0.25 and 0.06ng/mL, respectively.

Study on the interactions between anti-HIV-1 active compounds with trans-activation response RNA by affinity capillary electrophoresis.

The study on the interactions between two anti-human immunodeficiency virus type 1 (anti-HIV-1) active compounds with trans-activation response (TAR) RNA by affinity capillary electrophoresis (ACE) with UV absorbance detection is presented. The results showed that the novel active molecules could interact with TAR RNA and inhibit the reproduce process of HIV-1. The binding constants were estimated by the change of migration time of the analytes through the change of concentrations of TAR RNA in the buffer solution. The yielded binding constants of 8.87 x 10(3)M(-1) for active compound C(3) and 8.42 x 10(3)M(-1) for MC(3) at 20.0 degrees C, 0.626 x 10(3)M(-1) and 0.644 x 10(3)M(-1) at 37.0 degrees C, respectively. The thermodynamic parameters Delta H and DeltaS were obtained and shown that both hydrophobic and electrostatic interaction played roles in the binding processes. The results showed that the presented method was an easy and simple method to evaluate the interaction of small molecules with some bioactive materials.

Determination of morphine by capillary zone electrophoresis immunoassay combined with laser-induced fluorescence detection.

A competitive immunoassay for detecting morphine in bio-samples was established by capillary zone electrophoresis combined with laser-induced fluorescence detection (CZE-LIF). The antigen of morphine was labeled with isothiocyano-fluorescein (FITC) and then incubated with morphine monoclonal antibody and samples. The linear range was 50-1000 ng/mL, which was suitable for clinical and forensic applications. The detection limit can reach 40 ng/mL, based on S/N = 2. The recoveries of morphine from serum were satisfactory.

Production and characterization of anti-recombinant human erythropoietin (rhEPO) monoclonal antibody.

Anti-rhEPO McAb is valuable for the determination of recombinant human erythropoietin (rhEPO) levels for diagnosis of renal anemia and for doping control analysis. In this paper, anti-rhEPO hybridoma was prepared by fusion of SP2/0 murine myeloma cells with spleen cells isolated from immunized BALB/c mouse, using an enzyme-linked immunosorbent assay (ELISA) method to screen the positive hybridoma. The purified McAb was characterized by ELISA, SDS-PAGE, and Western-blotting. Experimental results showed that the subclass and the light chain of anti-rhEPO McAb was IgG1 and kappa light chain. The molecular weight of anti-rhEPO McAb was 166,000 Daltons. The affinity constant (K(aff)) of anti-rhEPO McAb with coated antigen was 5.0 x 10(5)L/mol.

Direct immunoassay of estrone by capillary electrophoresis with laser-induced fluorescence detection.

An immunoassay for estrone (E(1)) in women's serum, based on the competitive reaction between fluorescein-labeled complete antigen and E(1) with limited amount of anti-estrone monoclonal antibody is described. A thermally reversible hydrogel, poly-N-isopropylacrylamide (pNIPA), was added to the buffer to improve the reproducibility. With a laser-induced fluorescence (LIF) detector, the capillary electrophoretic immunoassay (CEIA) can be applied to determine E(1) at a concentration lower than 19.6 pg/mL. The E(1) levels in ten normal women's serum were measured at the range of 118.6-222.0 pg/mL.

Pharmacokinetics and tissue distribution of iv injection of polyphase liposome-encapsulated cisplatin (KM-1) in rats.

AIM: The pharmacokinetics and biodistribution of cisplatin encapsulated in polyphase liposome (KM-1) were compared with those of free drug in rats. METHODS: The platinum levels in serum and normal organs, after a single dose of iv injection of free or encapsulated cisplatin to rats, were determined by induced coupled plasma atomic emission spectrometry. RESULTS: Serum platinum concentration-time curve after a single iv dose of KM-1 4.5 mg/kg in rats was fitted with an open three-compartment model. The pharmacokinetic parameters were as follows: Vc=0.10 L/kg, T1/2pai=0.3 h, T1/2alpha=3.5 h, T1/2beta=2.7 h, AUC=265 mg.h.L(-1), and CL(s) =0.02 g.L.h(-1). KM-1 was cleared from the circulation much more slowly than free cisplatin. Liver and spleen had the highest concentration of platinum after KM-1 treatment. CONCLUSION: KM-1 remained in the bloodstream longer than its free drug, and was taken mainly by the reticuloendothelial system.

Direct immunoassay of estriol in pregnancy serum by capillary electrophoresis with laser-induced fluorescence detector.

A simple and sensitive capillary electrophoretic immunoassay (CEIA) was described for the determination of estriol (E(3)) in pregnant women's serum. The method was based on the competitive reaction of fluorescein-labeled E(3) antigen and E(3) with limited amounts of monoclonal antibody. The addition of the thermally reversible hydrogel, poly-N-iso propylacrylamide (pNIPA) in the buffer serving as a replaceable packing material, improved the reproducibility of the method. With laser-induced fluorescence detector (LIF), this method can be applied to determine E(3) at concentrations lower to 31.6 pg ml(-1). Recoveries from human steroid-free serum matrix were greater than 94% with relative standard deviation (R.S.D.) values less than 3.5%. Serum E(3) levels of ten normal pregnant women were measured at the range of 10.2-15.6 ng ml(-1).

Flow injection determination of gaseous sulfur dioxide with gas permeation denuder-based online sampling and preconcentration.

A simple and novel flow injection method for the determination of gaseous SO(2) is described based on gas permeation denuder (GPD) online sampling and preconcentration. The GPD is easily prepared with poly(vinylidene) difluoride microporous membrane as gas permeable material and two Perspex blocks with smooth and flat interface and rectangular engraved channels of mirror image. The sample gas is on one side of the membrane and phosphate buffer of pH 7.0 as the absorbing solution is on the other side. Gaseous SO(2) permeates partially through the gas permeable membrane and dissolves in the absorbing solution. After preconcentration for 5.0 or 8.0 min, the solution is injected into the flow of 5.0 x 10(-4) mol L(-1) 5,5'-dithiobis(2,2'-dinitrobenzoic acid) (DTNB) in 0.025 mol L(-1) phosphate buffer. The resulting product formed between DTNB and absorbed SO(2) is spectrophotometrically monitored at 410 nm with a charge coupled device (CCD) fiber optic spectrometer. The calibration graphs for preconcentration of 5.0 and 8.0 min are linear up to 4.0 and 3.2 mg m(-3) with detection limits of 50 and 35 micro g m(-3), respectively. The corresponding analysis speeds are 8.5 and 6 samples h(-1). The method is selective and just suffer from interference of hydrogen sulfide at higher than 1% of SO(2) level (in m/V) with an error >+10%. The assay just uses cheap and common membrane and reagents and shows potential application in the monitoring of atmospheric SO(2).

Cyclic voltammetry characterization of metal complex imprinted polymer.

Polymer capable of specific binding to Cu(2+)-2, 2'-dipyridyl complex was prepared by molecular imprinting technology. The binding specificity of the polymer to the template (Cu(2+)-2, 2'-dipyridyl complex) was investigated by cyclic voltammetric scanning using the carbon paste electrode modified by polymer particles in phosphate buffer solution. Factors that influence rebinding of the imprinted polymer were explored. The results demonstrated that cyclic voltammetry was an efficient approach to explore interactions between template and imprinted polymers.

Production and characterization of anti-estrone monoclonal antibody.

OBJECTIVE: Determination of estrone (E1) levels has a significant meaning in evaluating physiological effect and diagnosing some diseases. In order to detect free E1 in biological fluids, a monoclonal antibody specific for E1 was prepared after the complete antigen of E1 was synthesized. The purified monoclonal antibody was fully characterized for later immunoassay. METHODS: 3-O-carboxymethyl ether derivative of E1 was synthesized and in turn coupled to bovine serum albumin (BSA) to form complete antigen E1-BSA. A monoclonal antibody (McAb) specific for E1 was produced both in vitro and in vivo by a hybridoma anti-E1. Anti-E1 was prepared by fusion of SP2/0 murine myeloma cells with spleen cells isolated from immunized BALB/c mouse. The McAb was characterized by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE and Western-blotting. The specificity of the immunoassay was investigated by determining the cross-reactions of E1 analogs when free E1 was detected by competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). RESULTS: Analysis revealed that anti-E1 McAb (E1-McAb) was of the IgG1 type, the molecular weight of E1-McAb was 164,000 daltons. The affinity constant of E1-McAb with coated complete antigen was 8.2 x 10(8) L/mol. The linear range for free E1 determined by CI-ELISA was 10 pg/mL-10 ng/mL. The detection limit was 21.4 pg/mL (defined as twice the standard deviation of the blank). CONCLUSION: The CI-ELISA developed with E1-McAb was both sensitive and specific. The prepared E1-McAb can be used in some immunoassays.

A new competitive enzyme-linked immunosorbent assay (ELISA) for determination of estrogenic bisphenols.

Bisphenol A and other hydroxylated diphenylalkanes (generally known as bisphenols) have been identified as potential estrogenic substances. In this paper, a specific polyclonal antibody was produced against these compounds by immunization of rabbits with a conjugate of 4,4-bis (4-hydroxyphenyl) valeric acid and bovine serum albumin (BHPVA-BSA). The polyclonal antibody showed specific recognition of the bisphenol structure, while the cross reactions of other common phenolic compounds such as phenol, hydroquinol and p-hydroxybenzoic acid were all lower than 1%. The linear range of bisphenol A calibration curve was 1-10 000 ng ml(-1). Real water samples and mouse serum samples were spiked with known amount of bisphenol A and measured by the competitive ELISA. Recoveries were between 92 and 105%. The detection limits were found to be 0.1 ng ml(-1) for real water samples and 2 ng ml(-1) for serum samples. The method is very useful for monitoring bisphenol compounds in environmental and biological samples.