[Effect of basic fibroblast growth factor antibody combined with irinotecan on proliferation and apoptosis of small cell lung cancer H223 cells in vitro].

OBJECTIVE: To study the synergistic inhibitory effects of basic fibroblast growth factor (bFGF) monoclonal antibody (bFGF mAb) and irinotecan on the proliferation of small cell lung cancer H223 cells. METHODS: CCK-8 assay and flow cytometry were used to assess the effects of bFGF mAb combined with irinotecan on the proliferation and apoptosis of H223 cells, respectively. Western blotting was performed to analyze the effect of bFGF-mAb combined with irinotecan on AKT and ERK1/2 phosphorylation in the cells. RESULTS: Both bFGF mAb and irinotecan alone inhibited H223 cell proliferation in a dose-dependent manner (P<0.05). The inhibitory rate was significantly higher in H223 cells treated with bFGF mAb + irinotecan (54.30%) than in cell treated with bFGF mAb (18.73%) or irinotecan (21.96%) alone (P<0.05). Both bFGF mAb and irinotecan induced H223 cell apoptosis in a dose-dependent manner (P<0.05), and the combined treatment resulted in a significantly higher early apoptosis rates (6.5%) than treatment with bFGF mAb (2.7%) or irinotecan (4.3%) alone (P<0.05). bFGF mAb and irinotecan, either alone or in combination, significantly inhibited the levels of p-AKT protein and p-ERK1/2 protein without obviously affecting AKT and ERK1/2 protein levels. CONCLUSION: bFGF mAb and irinotecan produce synergistic inhibitory effects on small cell lung cancer H223 cells by suppressing proliferation and promoting apoptosis of the cells, and can effectively block the MAPK/ERK and PI3K/AKT signaling pathways associated with bFGF.

(125)I-labeled anti-bFGF monoclonal antibody inhibits growth of hepatocellular carcinoma.

AIM: To investigate the inhibitory efficacy of (125)I-labeled anti-basic fibroblast growth factor (bFGF) monoclonal antibody (mAb) in hepatocellular carcinoma (HCC). METHODS: bFGF mAb was prepared by using the 1G9B9 hybridoma cell line with hybridization technology and extracted from ascites fluid through a Protein G Sepharose affinity column. After labeling with (125)I through the chloramine-T method, bFGF mAb was further purified by a Sephadex G-25 column. Gamma radiation counter GC-1200 detected radioactivity of (125)I-bFGF mAb. The murine H22 HCC xenograft model was established and randomized to interventions with control (phosphate-buffered saline), (125)I-bFGF mAb, (125)I plus bFGF mAb, bFGF mAb, or (125)I. The ratios of tumor inhibition were then calculated. Expression of bFGF, fibroblast growth factor receptor (FGFR), platelet-derived growth factor, and vascular endothelial growth factor (VEGF) mRNA was determined by quantitative reverse transcriptase real-time polymerase chain reaction. RESULTS: The purified bFGF mAb solution was 8.145 mg/mL with a titer of 1:2560000 and was stored at -20 °C. After coupling, (125)I-bFGF mAb was used at a 1: 1280000 dilution, stored at 4 °C, and its specific radioactivity was 37 MBq/mg. The corresponding tumor weight in the control, (125)I, bFGF mAb, (125)I plus bFGF mAb, and (125)I-bFGF mAb groups was 1.88 ± 0.25, 1.625 ± 0.21, 1.5 ± 0.18, 1.41 ± 0.16, and 0.98 ± 0.11 g, respectively. The tumor inhibition ratio in the (125)I, bFGF mAb, (125)I plus bFGF mAb, and (125)I-bFGF mAb groups was 13.6%, 20.2%, 25.1%, and 47.9%, respectively. Growth of HCC xenografts was inhibited significantly more in the (125)I-bFGF mAb group than in the other groups (P < 0.05). Expression of bFGF and FGFR mRNA in the (125)I-bFGF mAb group was significantly decreased in comparison with other groups (P < 0.05). Groups under interventions revealed increased expression of VEGF mRNA (except for (125)I group) compared with the control group. CONCLUSION: (125)I-bFGF mAb inhibits growth of HCC xenografts. The coupling effect of (125)I-bFGF mAb is more effective than the concomitant use of (125)I and bFGF mAb.

Colloidal gold nanoparticle probe-based immunochromatographic assay for the rapid detection of chromium ions in water and serum samples.

An immunochromatographic assay (ICA) using gold nanoparticles coated with monoclonal antibody (McAb) for the detection of chromium ions (Cr) in water and serum samples was developed, optimized and validated. Gold nanoparticles coated with affinity-purified monoclonal antibodies against isothiocyanobenzyl-EDTA (iEDTA)-chelated Cr(3+) were used as the detecting reagent in this completive immunoassay-based one-step test strip. The ICA was investigated to measure chromium speciation (Cr(3+) and Cr(6+) ions) in water samples. Chromium standard samples of 0-80 ng mL(-1) in water were determined by the test strips. The results showed that the visual lowest detection limit (LDL) of the test strip was 50.0 ng mL(-1). A portable colorimetric lateral flow reader was used for the quantification of Cr. The results indicated that the linear range of the ICA with colorimetric detection was 5-80 ng mL(-1). The ICA was also validated for the detection of chromium ions in serum samples. The test trips showed high stability in that they could be stored at 37°C for at least 12 weeks without significant loss of activity. The test strip also showed good selectivity for Cr detection with negligible interference from other heavy metals. Because of its low cost and short testing time (within 5 min), the test strip is especially suitable for on-site large-scale screening of Cr-polluted water samples, biomonitoring of Cr exposure, and many other field applications.

[Mass spectrometry identification and immune cross-reactivity of a minor shrimp allergen-sarcoplasmic calcium binding protein from Litopenaeus vannamei].

AIM: To identify sarcoplasmic calcium-binding protein (SCP) as a minor shrimp allergen by mass spectrometry, and to analyze the immune cross-reactivity among crustacean SCPs. METHODS: The M(r); 21 000 allergen from Litopenaeus vannamei was identified by MALDI-TOF/TOF-MS. BLAST and ClustalW were used to compare amino acid sequence identity of the allergen among crustaceans. The puritifed M(r); 21 000 allergen was injected subcutaneously in mice to produce the specific polyclonal antibodies to analyze immune cross-reactivity of the allergen with proteins from 8 other species of crustaceans by Western blotting. RESULTS: The M(r); 21 000 shrimp allergen was identified as SCP. Sequence comparison revealed that SCP had 81%-100% amino acid identity among crustaceans. Western blotting showed that the proteins with M(r); about 21 000, corresponding to SCP from Metapenaeus ensis, Penaeus monodon, Oratosquilla oratoria, Macrobrachium rosenbergii, Procambarus clarkii, Portunus pelagicus, Charybdis feriatus, Eriocheir sinensis were recognized by polyclonal antibodies against SCP of Litopenaeus vannamei. CONCLUSION: SCP is a minor shrimp allergen, and SCPs have a high sequence homology and strong immune cross-reactivity among crustaceans, which can be used as detective, diagnostic and safe immunotherapeutic agents for subjects with shrimp allergy.

[Preparation and application of monoclonal antibodies against Heavy Metal Chromium].

AIM: To produce the monoclonal antibodies (mAb) against Heavy Metal Chromium, and develop a competitive inhibition enzyme-linked immunosorbent assay (ciELISA) for the detection of Chromium. METHODS: Chromium ions was chelated with bifunctional chelating agent Isothio-cyanobenzyl- EDTA firstly and then conjugated with bovin serum alburmin(BSA) and ovalbmin(OVA) respectively to complete antigens . Four female BALB/c mice were immunized with Cr-iEDTA-BSA, the Hybridoma lines secreting monoclonal antibodies against Chromium ions were established by the hybridoma technology. The titer and the specificity of the antibodies were characterized in the way of indirect enzym-elinked immunosorbent assay (iELISA) and ciELISA. RESULTS: After immunization and cell fusion, three hybridomaes which can stably secrete mAbs against chromium were obtained. The antibody titer was up to 1×10(5);, only had 9.62% cross-reactivity to Fe(3+); and little to other ions. Linear detection of ciELISA for Chromium covered a range from 0.783 µg/L-50 µg/L with the limit at the level of 1 µg/L. An excellent correlation of results obtained by ciELISA and ICP-AES when the concentration more than 1 µg/L. CONCLUSION: The monoclonal antibody against Chromium with high sensitivity and specificity has been generated and a competitive inhibition enzyme-linked immunosorbent assay for the detection of Chromium ions has been established successfully, which was met the the national standard of water quality nonitoring.

Monoclonal antibodies targeting basic fibroblast growth factor inhibit the growth of B16 melanoma in vivo and in vitro.

Up-regulated basic fibroblast growth factor (bFGF or FGF-2) plays an important role in the development and metastasis of melanoma; therefore, neutralizing antibodies to bFGF may suppress melanoma growth. In this study, we have developed three monoclonal antibodies against bFGF (anti-bFGF mAbs), which display remarkable anti-tumor and anti-angiogenic effects in vitro and in vivo. Anti-bFGF mAbs significantly inhibit the proliferation and induce apoptosis of B16 cells, and show inhibitory effects on the migration of B16F10 cells and the tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Treatment of B16 melanoma spheroids with anti-bFGF mAbs in vivo results in significant reduction in tumor size and prolonged survival time of animals. Moreover, TUNEL (terminal transferase dUTP nick end labeling) assay and CD31 staining confirmed the increase of apoptosis and decrease of intratumoral microvessel density in tumor sections from animals treated with anti-bFGF mAbs. Our data indicate that anti-bFGF mAbs are potential therapeutic candidates for melanoma therapy by effectively suppressing the melanoma growth through inhibition of angiogenesis and induction of apoptosis in the tumor.

[A Paralytic shellfish poisoning GTX2,3 mimic peptide isolated from phage display: characterization and potential applications].

AIM: To screen peptide mimics of PSP (Paralytic shellfish poisoning) GTX2,3 from a random 12-mer phage display peptide library and to identify and characterize the specificity and accuracy of the peptide mimotope of GTX2,3. METHODS: The monoclonal antibody against GTX2,3 (mAb E(9);F(10);) was used as a target to screen the 12-mer phage display peptide library and the specificity of phage clones were identified by sandwich ELISA and blocking assay. The peptide sequences of positive phage clones were determined and analyzed by DNA sequencing. The affinity and specificity of synthetic peptide were identified by a competitive ELISA. RESULTS: The 20 clones were identified to be specific reactivity with the mAb E(9);F(10);. Amino acid sequence analysis revealed seven different types of mimotope sequence, most of which contained a common motif DXLXPP(X presents random acid amino), X was random amino acid. The Phage No.2(phage 2), the clone with mimotope sequence WPSLDXLXPPSY showed the strongest binding, and inhibited the reactivity of the mAb with GTX2,3. Another ELISA result showed that synthetic peptide-1 (SP-1) which contain mimotope amino acid sequence was able to inhibit the mAb- GTX2,3 interaction. CONCLUSION: The mimotope peptide of GTX2,3 is obtained by using the phage display technology. The results also showed that SP-1 bind to mAb E(9);F(10); at the same site as GTX2,3.

[Identification, isolation, purification of allergens in shrimp and allergic analysis for the main allergens].

AIM: To identify the allergens in shrimp, and to isolate, purify and analyze the main allergen components. METHODS: The total shrimp proteins were extracted by PBS, the allergens were identified with 11 shrimp allergic patients' serum IgE by Western blot. Three main shrimp allergens 21,000, 36,000 and 80,000 were purified by ammonium sulfate precipitation, Sephadex G-50 chromatography and DEAE-exchange chromatography. Western blot and indirect ELISA were used to confirm and analyze allergenicity of the three main allergens. RESULTS: Western blot demonstrated that there were nine allergen components in shrimp proteins, and IgE binding to 21,000, 36,000 and 80,000 shrimp allergen by 5, 7, 5 (36.4%, 63.6%, 45.5%) of 11 shrimp allergic patients' sera. Results of indirect ELISA showed that the binding absorbency of allergic patients' sera IgE with the three main purified allergens were all higher than that with shrimp protein extraction. CONCLUSION: There are at least 9 allergens in shrimp; the 21,000, 36,000 and 80,000 proteins are the main allergen components; and the 36 000 protein is the primary main allergen, with the highest allergenicity and sensitization rate. In further investigation, we will use monoclonal antibody technique to verify weather the 21,000, 36,000, 80,000 allergens have common epitope, which will lay a foundation for shrimp allergy clinical diagnosis and shrimp allergen vaccine design.

Colloidal gold probe-based immunochromatographic assay for the rapid detection of lead ions in water samples.

One-step immunochromatographic assay (ICA) has been developed using colloidal gold-labeled monoclonal antibody probe for the rapid detection of lead ions in water samples. The ICA was based on the theory of competitive reactivity, and the results can be easily judged based on the presence or absence of a red colored test line with visual detection. Under optimal conditions, this method shows high detecting sensitivity with a LOD (limit of detection) of 50 ng/ml. Stability test indicates that the immunochromatographic strips are stable for 8 weeks at room temperature. During practical application, nanometer TiO2 is used to enrich the lead ions in water samples. The ICA is successfully applied in the measurement of lead ion concentrations in local water samples, and the results are highly consistent with that of ICP-MS. Detecting lead ions with ICA can be done within 4 min and is very useful for the rapid onsite testing.

Selection and characterization of a human neutralizing antibody to human fibroblast growth factor-2.

Compelling evidences suggest that fibroblast growth factor-2 (FGF-2) plays important roles in tumor growth, angiogenesis and metastasis. Molecules blocking the FGF-2 signaling have been proposed as anticancer agents. Through screening of a human scFv phage display library, we have isolated several human single-chain Fv fragments (scFvs) that bind to human FGF-2. After expression and purification in bacteria, one scFv, named 1A2, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor. This 1A2 scFv was then cloned into the pIgG1 vector and expressed in 293T cells. The purified hIgG1-1A2 antibody showed a high binding affinity of 8x10(-9)M to rhFGF-2. In a set of vitro assays, it inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-1A2 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells. For the first time, we generated a human anti-FGF-2 antibody with proven in vitro anti-tumor activity. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on FGF-2 signaling for growth and survival.

A competitive indirect enzyme-linked immunoassay for lead ion measurement using mAbs against the lead-DTPA complex.

Immunoassays for quantitative measurement of environmental heavy metals offer several advantages over other traditional methods. To develop an immunoassay for lead, Balb/c mice were immunized with a lead-chelate-protein conjugate to allow maximum exposure of the metal to the immune system. Three stable hybridoma cell lines were obtained through spleen cells fusion with Sp2/0 cells. One cell line, 2A11D11, produced mAbs with preferential selectivity and sensitivity for Pb-DTPA than DTPA, exhibiting an affinity constant of 3.34 + or - 0.24 x 10(9) M(-1). Cross reactivity (CR) with other metals were below 1%, except for Fe(III) with a CR less than 5%. This quantitative indirect ELISA for the lead ion was used to detect environmental lead content in local water sources; importantly, the results from the immunoassay were in excellent agreement with those from ICP-MS. Development of immunoassays for metal ions may thus facilitate the detection and regulation of environmental pollution.

[Cloning of the variable region genes from hybridoma against bFGF and expression of single chain antibody fragments in E.coli HB2151].

AIM: To construct and express the single chain antibody (scFv) in E.coli HB2151 by cloning the variable region genes from hybridoma against bFGF. METHODS: Total RNA was extracted from hybridoma cell line B2F3 secreting mAbs against bFGF and the cDNA was amplified by retropolymerase chain reaction (RT-PCR). V(L) and V(H) were fused by a short peptide linker containing 15 amino acids (Gly(4)Ser)(3) using splice-overlap extension PCR to construct the scFv gene. The sequences of the scFv were analyzed by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd and Ig Blast data base in GenBank. The scFv gene was inserted into pCANTAB-5E vector and expressed in E.coli HB2151. RESULTS: The V(H) gene contained 375 base pairs and encoded 125 amine acid residues. The V(L) gene contained 399 base pairs and encoded 133 amine acid residues. There were four FRs, three CDRs and two characteristic cysteine residues in the V(H) gene and the V(L) gene, respectively. The scFv gene contained 789 base pairs and encoded 263 amine acid residues with the structure of V(H)-linker-V(L). Restriction endonuclease digestion and DNA sequencing proved that the expression vector of pCANTAB-5E-scFv was constructed correctly. SDS-PAGE and ELISA analysis showed that scFv was successfully expressed in E.coli HB2151 and the expression protein had specific antigen binding activity. CONCLUSION: The variable region genes of anti-bFGF mAbs have been cloned successfully and single chain antibody fragments have been constructed and expressed, which will be a great help to the study of humanized antibodies against bFGF.

[Expression of human anti-HBsAg single chain Fab gene in Pichia pastoris].

AIM: To investigate the relation of the affinity activity to the structure of recombinant Fab. METHODS: The human anti-HBsAg single chain Fab gene was obtained by overlaping PCR from the H and L chains of Fab, and was introduced into the expression system of Pichia pastoris. The expression vector of single chain Fab was transformed into GS115 cells by the method of LiCl transformation. The transformants were obtained and cultured in shake flask. The culture supernatant of the recombinant yeast was deposited in (NH(4))(2)SO(4) and purified by affinity chromatography. The affinity of culture supernatant of recombinant yeast and purified single chain Fab was analyzed by ELISA. RESULTS: The analysis of SDS-PAGE and Western blot showed that the single chain Fab gene was expressed successfully in Pichia pastoris, and the expression quantity was about 5-10 mg/L in shake flask. The recombinant Fab was purified by affinity chromatography and the purity reached 97.8%. The ELISA analysis showed that the recombinant single chain Fab had good HBsAg binding properties. CONCLUSION: The single chain Fab gene could be expressed successfully in Pichia pastoris and the recombinant single chain Fab can efficiently bind with HBsAg.

[Purification of recombinant humanized anti-HBsAg Fab antibody by affinity chromatography].

AIM: To establish a steady purification method for producing recombinant humanized anti-HBsAg Fab from yeast fermentation supernatant. METHODS: Humanized anti-HBs scFv was used to immunize BALB/c mice to obtain anti-scFv monoclonal antibody (mAb) which was used to purify recombinant humanized anti-HBsAg Fab from yeast fermentation supernatant by affinity chromatography. RESULTS: The purity of purified recombinant anti-HBsAg Fab was above 95% and its recovery rate was about 75%-85%. CONCLUSION: An efficient affinity chromatography suitable for purification of recombinant humanized anti-HBsAg Fab in large-scale was established.

[Construction of eukaryotic vector and expression of chimeric antibody against rh-bFGF].

AIM: To express a human-mouse chimeric antibody against rh-bFGF antigen in eukaryotic cells. METHODS: The VL and VH genes were amplified and cloned from the hybridoma 1F11 secreting anti-rh-bFGF mouse monoclonal antibody (mAb) and the CL and CH genes were amplified and cloned from the plasmid pMDHC and pMDLC. They were inserted into eukaryotic expression vectors after sequencing. The recombinant plasmids were then transformed into CHO-dhfr- cells for expression. The humanization and antigen specificity of expressed products were identified by indirect ELISA. The relative molecular mass (Mr) of expressed products was determined by SDS-PAGE. RESULTS: The cloned antibody genes were identified to be functional by sequencing. The chimeric antibody could be detected in the culture supernatant of transformed CHO cells. The humanization and specificity of the chimeric antibody to rh-bFGF were confirmed by ELISA. CONCLUSION: The mouse-human chimeric antibody against rh-bFGF has been expressed successfully in eukaryotic cells, which lays the foundation for its further clinical research.

[Production of recombinant humanized anti-HBsAg Fab antibody by fermentation].

In order to produce recombinant human anti-HBsAg Fab antibody in Pichia pastoris, the recombinant yeast was fermented using fed-batch system in a 30 L bioreactor. The fermentation temperature was 30 degrees C, the pH was 5.0 approximately 5.3, and the DO was 20% approximately 30%. The recombinant Fab antibody was purified from crude culture supernatant by ion exchange and analyzed by SDS-PAGE and western blot and ELISA. When the absorbance (OD600) of broth reach 300 at the end of fed-batch phase, the induced phase was initiated. The results showed that recombinant human anti-HBsAg Fab antibody was high-level expressed in recombinant Pichia pastoris using a fed-batch fermentation system. Both chains of the Fab were successfully expressed upon methanol induction. After 192 h of induction, the expression level of recombinant Fab (soluble) reached 412 mg/L. The recombinant Fab antibody was purified effectively by ion-exchange chromatography from the fermentation supernatant to a purity of 95%. And the affinity activities of the purified recombinant Fab antibdy and fermentation supernatant were detected, and both of them showed high affinity activities. The results demonstrated that recombinant human anti-HBsAg Fab antibody could be high level produced by fed-batch fermentations in Pichia pastoris. Which can be efficiently used in industrial production.