Extraction, preliminary characterization and immunostimulatory activity in vitro of a polysaccharide isolated from Strongylocentrotus nudus eggs.

A new water-soluble polysaccharide (SEP-2), with a molecular weight of 6.78 × 10(5)Da, was isolated from Strongylocentrotus nudus eggs under the extraction conditions optimized by response surface methodology (RSM). Preliminary characterization of SEP-2 was performed by HPSEC and GC-MS, indicating that SEP-2 could be a D-glucan containing a (1 → 4)-linked D-Glcp backbone with (1 → 3)-linked D-Glcp side chains. In subsequent immunostimulatory studies, significantly enhanced ROS level, NO production and inflammatory cytokines secretion (IL-1ß, IL-6, and TNF-α) were observed in SEP-2 treated murine macrophage cell line RAW264.7. These results suggest that SEP-2 might have the capability to enhance the microbicidal activity and killing function of macrophages through the polarization toward the pro-inflammatory M1 state.

[Polyclonal antibody production and immunoassay development for detection of beta-lactamase in milk].

OBJECTIVE: To produce polyclonal antibody with affinity against several types of beta-lactamase and develop an indirect competitive ELISA for detection of beta-lactamase in milk. METHODS: A mixture of beta-lactamase was used as immunogen for animal immunization to produce polyclonal antibody. After purification polyclonal produce animal immunization to characterization, the obtained antibody was employed to establish an ELISA method for beta-lactamase determination. RESULTS: The polyclonal antibody was acquired, with the concentration of 17.9-18.9 mg/ml in antiserum. The molecular weights were 55, 25 and 160 kD for heavy chain, light chain and the whole antibody, respectively. The developed ELISA method showed the LOD 0.2 ng/ml and the linear range of 0.2-200 ng/ml. The recoveries ranged from 80% to 115%, with RSD bellow 20%. CONCLUSION: With the polyclonal antibody, the proposed indirect competitive ELISA method was successfully applied to determine beta-lactamase in milk.

Development of a sensitive biotin-avidin amplified enzyme-linked immunosorbent assay for the determination of ketamine in biological samples.

An effective biotin-avidin amplified enzyme-linked immunosorbent assay (BA-ELISA) was developed to determine ketamine in biological samples. A conjugate of ketamine and ovalbumin (OVA) was used for immunization to produce polyclonal antibody. The conjugate of ketamine and bovine serum albumin (BSA) with polyclonal antibody was calculated to have an affinity constant (K(aff)) of 3.30 x 10(8)(mol/L)(-1). The linear range of ketamine was 0.1-1000 microg/L with recoveries from 89.6% to 99.9% in spiked sample analysis. The detection limit of ketamine was 0.03 microg/L, which is more sensitive than that of the traditional ELISA. The results obtained by BA-ELISA agreed well with that of the traditional ELISA, with a correlation coefficient of 0.98.

Efficient purification of GP-1D8 glycoprotein from human breast carcinoma tissue by immunoaffinity chromatography.

Anti-GP-1D8 monoclonal antibody was produced in our laboratory. Immunoaffinity purification of GP-1D8 glycoprotein from human breast carcinoma tissues, on column with the monospecific antibody, is developed. The procedure permits purification of GP-1D8 to a highly purified state. It appeared as a single band in sodium dodcyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of GP-1D8 was determined to be 66 kDa by mass spectrometry. Its antigenicity was stable between 0 and 70 degrees C. It was breast cancer specific, as determined by immunohistochemistry and immunocytochemistry. The preparation of anti-GP-1D8 monoclonal antibody will facilitate further detection of, and functional study on, GP-1D8. The study also provides a simple, economical, and efficient method for affinity purification of target proteins from human or animal tissues.

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.

Purification and characterization of 66-kDa glycoprotein from human breast carcinoma.

We extracted a 66-kDa glycoprotein (GP-1D8) from breast invasive ductal carcinoma tissues. The monoclonal antibody (mAb) against GP-1D8 was prepared in our laboratory. Western blotting with the purified protein using the mAb demonstrated a single band of 66 kDa. Immunocytochemical and immunohistochemical analysis revealed strong expression of GP-1D8 protein in the cytoplasm of MCF-7 cells and different types of breast carcinoma tissues, but GP-1D8 is absent in normal breast and benign breast tumor tissues. Glycosylation analysis showed GP-1D8 contained methylated salic acid. GP-1D8 was identified using mass-spectrometric techniques and N-terminal sequencing. These data were used to identify the protein through the SWISSPROT protein sequence database and BLAST homology search. These results showed GP-1D8 had some similarity to human albumin precursor. Co-immunoprecipitation assays of lysate from MCF-7 cells and mass spectrometric analysis revealed the interaction of GP-1D8 with beta-tubulin. This is the first time human breast carcinoma tissues and MCF-7 cells have been shown to express a 66-kDa glycoprotein similar to human albumin precursor. These results might be important in the detection of novel potential biomarkers and may provide insight into the molecular mechanisms of tumorigenesis.

Efficient purification and preconcentration of erythropoietin in human urine by reusable immunoaffinity column.

In this paper, an efficient method is proposed for purification and preconcentration of erythropoietin (EPO) in human urine samples. The EPO-specific immunoaffinity column (IAC) was generated by covalent immobilization of anti-EPO polyclonal antibodies on Sepharose 4B support. The EPO-binding capacity of the IAC was found to be about 2.0 microg (6.6IU) per 1.5 mL of gel and the activity recoveries of EPO at low concentrations of 7.8, 10 and 120 m IU/mL by the IAC were between 78 and 86%. Substantial cleanup effect was observed when the IAC was applied to human urine samples.

Screening of carbohydrate-specific phage antibodies against recombinant human erythropoietin (rhuEPO) using a phage display antibody library: preliminary study.

This paper is a preliminary report on development of a screening method for carbohydrate-specific phage antibodies against recombinant human erythropoietin (rHuEPO), using a phage display antibody library. rHuEPO was oxidized with sodium periodate or treated with 1,4-dithiothreitol and guanidine hydrochloride for detecting the specificity of obtained phage antibodies. Of 100 phage clones, three initially showed higher carbohydrate-related specificity. One of them (No. 62) bound specifically to the carbohydrate chains of rHuEPO, while the other two (Nos. 63 and 83) might recognize the steric conformation related to both the carbohydrate and the polypeptide chain of rHuEPO. These phage antibodies may serve as useful capture ligands for future development of efficient analytical methods for rHuEPO.

Development of ELISA for metallothionein-II allows determination of heavy metal pollution of fresh water.

Metallothionein (MT), a metal-binding protein induced primarily by heavy metals in vertebrates, is considered a biomarker for environmental heavy-metal contamination. To investigate heavy metal pollution in the freshwater environment, MT-I and MT-II were purified from livers of crucian carp (Carassius carassius) by gel exclusion chromatography and ion exchange chromatography. To detect the purified MT-II, a specific monoclonal antibody (mAb) against crucian carp MT-II was produced from the hybridoma strains by cell-cell fusion. By using Enzyme-Linked Immunosorbent Assay (ELISA) with this mAb, the purified crucian carp MT-II was detected with a high specificity and sensitivity. There was a good correlation between the amount of MT-II in carp livers and the concentration of heavy metals in water. ELISA was then used to evaluated the degree of heavy metal pollution in two freshwater systems. The results indicate that the MT-II content in carp liver tissue can be used as an indicator of environmental heavy-metal pollution.

Synthesis and preliminary biological evaluation of the 99mTc labeled nitrobenzoimidazole and nitrotriazole as tumor hypoxia markers.

1-(3-1,2,4-Nitrotriazole-1-yl)-propanhydroxyiminoamide and 1-(6-nitrobenzoimidazole-1-yl)-propanhydroxyiminoamide were synthesized and radiolabeled with (99m)Tc. The (99m)Tc labeled complexes continuously accumulated in hypoxic murine sarcoma S180 cells in vitro but not in aerobic cells. Biodistribution results in mice bearing S180 tumor indicated that the tracers could localize in tumor and eliminate from it slowly. These results suggested that the (99m)Tc labeled nitrobenzoimidazole and nitrotriazole might be the novel tumor hypoxia markers.

Isolation and characterization of an anti-recombinant erythropoietin single-chain antibody fragment using a phage display antibody library.

The production of a large amount of specific antibodies against erythropoietin (EPO) is necessary for both clinical treatment and doping control. However, the weak immunogenicity of EPO and the side effects of excessive injection make the conventional immunological protocol rather inefficient and time-consuming. In this study, a single-chain antibody fragment of variable region (scFv) against recombinant human erythropoietin (rHuEPO) was produced after three rounds of panning a phage display antibody library. The selected scFv-B2 was expressed in soluble form in Escherichia coli DH5alpha F' and purified by His-bond nickel affinity chromatography with a yield of about 1-2 mg of antibody in 1 L of the culture supernatant. The molecular weight of the scFv was estimated to be 29 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the affinity constant was found to be 1.0 x 10(8) L mol(-1) based on a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). The potential ability of the scFvs for immunopurification of rHuEPO from related sample was demonstrated by using a double-antibody sandwich ELISA. The reported method is a very powerful tool to produce specific antibodies for rHuEPO detection demands.

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.

Codon optimization of MTS1 and its expression in Escherichia coli.

MTS1, which encodes a protein named p16, is an important gene involved in tumorigenesis. To increase the expression of p16 in Escherichia coli, MTS1 was synthesized de novo by recursive PCR, with codons optimized towards E. coli. Studies indicate that N-terminal amino acids of p16 had negative impact on its expression in E. coli. The function of p16DeltaN8 is not affected by the absence of N-terminal eight amino acids, compared with p16. p16DeltaN8 was expressed in E. coli, which reached 22% of total cell proteins. Purified p16DeltaN8 (purity was 98%) was delivered into A875 (melanoma), MCF7 (breast cancer), and HeLa (cervical cancer) cells by lipofectin. Results show purified p16DeltaN8 remarkably inhibited the growth of A875 and MCF7 cells, whereas it had little effect on HeLa cells.

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.

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.