| Southeast Asian deletion (-SEA) a-thalassaemia is one of the most common single gene inheritance diseases of the human in the world and can lead to Bart’s hydrops fetalis, a serious birth deficiency in homozygotes. Currently,(-SEA) a-thalassaemia is considered a public health problem in southern China, especially in the Guangxi, Guangdong, and Hainan provinces, where the estimated frequencies of heterozygous carriers range from4.5%to14.0%of the population. The effective method to prevent the birth of babies with (--SEA) a-thalassaemia is to perform gene detection for all people of reproductive. However, due to the requirement of specialized skills and equipment as well as the high cost, gene detection can’t be popular in primary hospital, which limits the screening of people for (-SEA) a-thalassaemia deletions.Fortunately, hemoglobin ξ (zeta globin chain), an embryonic globin that is an important biomarker, makes screening for carriers of the (-SEA) a-thalassemia deletion simpler and faster. The zeta globin chain is present in almost all normal fetal cord blood samples but cannot be detected by approximately three months after birth except in (-SEA) a-thalassemia deletion carriers, who express the zeta globin chain in their peripheral blood for their whole lives. The techniques used for the detection of the zeta globin chain include high-performance liquid chromatography (HPLC), immunocytochemistry and sandwich ELISA. Recently, the sandwich ELISA for the zeta globin chain has been shown to be a sensitive, specific and simple method compared with other techniques. We have developed a sandwich ELISA kit that is the first one in China and is used in clinical detection and screening. However, the sandwich ELISA has several shortcomings, such as the requirement of skilled technicians and special equipment/facilities and the relatively long time needed to run the assay (approximately2h); these shortcomings have limited the screening of people for (-SEA) a-thalassemia deletions. Moreover, ELISA is not suitable for conducting individual tests due to the precoated nature of the microplate in the ELISA kit. Therefore, it is necessary to develop a screening system that is simple and fast and can be applied to both high-throughput screening and individual testing. An immunochromatographic assay (ICA) is one type of assay that meets these requirements.ICA is a fast, simple and low-cost technique and the result can be read visually with naked eyes or simple equipment, so it is suitable for both individual testing and on-site screening. Now, ICA has been widely used to qualitation and semi-quantitation detection of disease and forbidden additive. The tracers used in ICA include colloidal gold, phosphorescence microparticle, lactoprene, gelatin, etc. Colloidal goal is the most common tracer used in ICA and it has successfully applied to screen and monitor many substances, such as antibiotics, pathogen and drugs. However, it is difficult to use the colloidal gold-labeled immunochromatographic assay (G-ICA) for the detection of hemoglobin unless the hemoglobin sample is diluted sufficiently to reduce the color because the red color of the hemolysate samples interferes with the readout of the assay. This interference occurs even though colloidal gold particles exist in different colors, such as red, orange and purple. This is one of the main reasons that there are not any ICA for the detection of hemoglobin so far.In order to eliminate interference of red color from hemoglobin on ICA and ensure the sufficient sensitivity at the same time, we chosen fluorescence microsphere instead of colloidal gold as the tracer and develop a fluorescence immunochromatographic assay (FL-ICA) to detect the zeta globin chain in the hemolysates of carriers of the (-SEA) a-thalassemia deletion. This assay can be completed within10min using a simple UV detector and does not suffer from interference from the red background color of the hemolysate. A total of314blood samples were tested by FL-ICA and ELISA. The results of these assays were confirmed by PCR, the standard technique for genetic disease testing. The sensitivity and specificity of this novel FL-ICA were100%and98.0%, respectively; the corresponding values for the ELISA performed simultaneously were100%and99.2%, respectively. In conclusion, a new FL-ICA-a simple, fast, convenient, low-cost method-was developed that may be useful in both high-throughput screening and individual detection of the(-SEA) a-thalassemia deletion in carriers.I. The constitution of fluorescence immunochromatographic assayConstruction of immunochromatographic strip The strip is mainly consist of four parts:the sample pad, the conjugated pad with fluorescently labeled monoclonal antibody (mAb)3H9, the nitrocellulose membrane coated with rabbit-anti-mouse IgG as the control line (C line) and mAb4D11as the test line (T line), and the absorbent pad. Judgement for the result of the immunochromatographic strip Using the best excitation light source to illuminate the fluorescently labeled mAb3H9that is staying on the lines in nitrocellulose membrane, we can judge the result based on the appearance of the C line and T line. The test is considered as failure if the C line is not visible. If the C line is visible, then the appearance of two lines indicates a positive result, whereas the appearance of only the C line indicates a valid negative result.II. Establishment of fluorescence immunochromatographic assay to screen the (-SEA) a-thalassemia carrier.Based on the sandwich ELISA system established in our laboratory, which has been approved by State Food and Drug Adimistration of China (SFDA document) in2011, anti-zeta globin chain monoclonal antibody4D11was still used as capture antibody and fluorescence labled mAb3H9was used as detecting antibody for development of FL-ICA. There are3experimental conditions should be optimized for FL-ICA:①the blood sample should move from the sample pad to the absorbent pad smoothly and uniformly;②the ideal release of fluorescently labeled3H9antibody on the conjugated pad;③the coloration of control line and testing line on NC membrane can be shown clearly and correelyt. The final parameters for FL-ICA were ascertained as follows:the fluorescent microspheres with a diameter of100nm was chosen as tracer and labeled to25ug mAb3H9per milligram fluorescence microspheres. The fluorescent microsphere-labeled mAb3H9was passively immobilized on glass-fiber membrane with6ul/cm; the capture antibody mAb4D11drew onto nitrocellulose membrane was dispensed to3mg/ml, which was benefit for avoiding false positive and false negative; the whole blood specimens were lysed completely by ddH2O and then diluted with dilution buffer containing5%skimmed milk powder. Ⅲ. Application of immunochromatographic assay in detection of the zeta chainThe immunochromatographic strip, assembled with the optimized sample pad, conjugated pad, nitrocellulose membrane and absorbent pad, was used to detect the hemolysate samples from a normal individual and a (-SEA) deletion carrier. The appearance of only a single band, in the control region (C line) in samples for a normal individual was judged to be negative, whereas the two bands, one in the control region (C line) and other in the test region(T line) in samples from (-SEA) deletion carrier and Bart’s hydrops fetalis were judged to be positive. Also, the established fluoresecenc immunochromatographic strip with favourable sensitivity show a positive result even the hemolysate was diluted to1250folds. To evaluate the specificity and accuracy of the FL-ICA in screening for (-SEA) deletion carriers,314hemolysate samples prepared from peripheral blood were tested in parallel with both FL-ICA and sandwich ELISA. Using PCR as the standard diagnostic tool, the sensitivity was100%for both assay and the specificity is98.0%for the FL-ICA compared with99.2%for the ELISA. The results indicated that the novel FL-IC with a favourable sensitivity and specificity could match to the requirement of clinical test. PART2ABSTRACTImmunoglobulin (Ig) as very important molecules in immune system have been considered as special product of B lymphocytes. In2003, however, Xiaoyan Qiu et al proposed and proved that Ig also can be derived and expressed by human cancer cells of epithelial origin. The Ig derived from non-B cells, such as human epithelial cancer cells and some normal epithelial cells are named as non B-Ig. Now, the presence of non-B cell has been gradually accepted and confirmed by more and more researchers who also proved that cancer-derived Ig is not only expressed by many kinds of cancer cells, but also plays an important role on the malignization, migration as well as proliferation of cancer cells.In2008, using an mAb RP215as a therapeutic antibody candidate for cancer, Lee et al found that CA215, on the heavy chains of IgG derived from various types of cancer cells, has a high homology to the heavy chains of human IgG, except a carbohydrate-associated epitope located in the variable region (V region) of Ig heavy chains (H chain). The CA215was proved to express in different degrees in many kinds of cancers such as ovary, cervix, endometrium, breast, stomach and colon cancer. Targeting to CA215, RP215monoclonal antibody can not only inhibit the proliferation and induce apoptosis of cultured cancer cells in vitro, but also inhibit the grown of tumor in the nude mouse experiments. Therefore, CA215, as a pan cancer biomarker, is considered as an ideal target for clinical monitor and treatment. Recently, Dr. Lee has developed the mAb RP215into a humanized antibody, which may approach to be a therapeutic antibody. Also, another effective way of targeting CA215for immunotherapy is therapeutic tumor vaccines. However, CA215C cannot directly become a vaccine candidate because it is a carbohydrate-associated epitope(s) that is a thymus independent antigen(TI-Ag) with weak immunogenicity and difficult to induce both specific cellular immune response and effective secondary antibody response. Our objective is to screen the peptide mimics to CA215C, the special epitope(s) locating in CA215, from a random phage display peptide library using an anti-CA215C monoclonal antibody RP215. The peptide mimics for CA215C would convert the carbohydrate-associated epitope(s) into peptide epitope, changing the TI-Ag into thymus dependent antigen (TD-Ag). Furthermore, peptide-carrier conjugate or multiple antigenic peptides (MAP) will be synthesized aiming at induce effective secondary immune response and protective immunity in vivo.In our work, dozens of CA215C mimotope clones were screened from12-mers linear phage display peptide library by using mAb RP215. Finally, three mimotope sequences were selected for synthesis of peptide mimics. The antisera from Balb/c mice immunized with peptide mimics conjugated with BSA could bind with CA215but not human IgG The results suggested that the synthetic peptides may be mimetic to epitopes of CA215C.I. Screening of mimotopes to CA215C from phage displayed peptide librariesScreening of mimotope clones specifically binding with RP215The mAb RP215, kindly provided by G. Lee(University of British Columbia, UBC), was identified to recognize the carbohydrate-associated epitope of CA215derived from cancer cells. The CA215C mimotopes were screened from12-mer linear phage display peptide library by using mAb RP215. After3round of selection,50phage clones were selected to be identified the antigenicity by sandwich ELISA coating with mAb RP215as capture antibody and other appropriate object as negative control to exclude non-specific reaction. Thirty three of50phage clones were defined as positive clones binding to anti-CA215C mAb specifically.Analysis of Nucleotide and amino sequence from positive phage clones Thirty of33positive phage clones were chosen to perform DNA sequencing. The amino acid sequences deduced from DNA sequences were analyzed and showed five conservative sequences. The phage clone No.1, No.2, No.5, No.6, No.11and No.31possess the consensus sequence Ⅰ:E-LWR; clones No.4, No.12, No.25and No.36share the conserved sequence Ⅱ:E-HWR; clone No.18, No.21and No.39phage clones possessed the conserved sequence Ⅲ:E-LW; clones No.3and No.40had the consensus sequence IV:EDLW and clone No.7and No.42owned the conserved sequence Ⅴ:E-W. There is a sequence E-(-)WR appeared in conserved sequence I, Ⅲ and Ⅲ, while sequence E-(-)LW shared in conserved sequence I, IV, and Ⅴ.Ⅱ. The synthesis and antigenicity identification of peptide mimics to CA215CSelection of the dominant sequences In order to select the dominant sequences from the seventeen peptide sequences belonged to five conserved sequences, mAb RP215was coated with a serial of concentration to test the binding to phages with representative sequence of conserved sequences. Finally, the peptide sequence of clone No.2from conserved sequence I, No.42from conserved sequence V and No.13from irregular sequence were selected to be synthetized, based on their better affinity with mAb RP215, and termed as R2, R42and R13.The identification for antigenicity of peptide mimics The antigenicity of peptide mimics was identified by sandwich ELISA and competitive ELISA. Among the3linear peptide mimics with biotin on the N-terminal as tag, both R2and R42could specifically bind to mAb RP215against CA215C, while R13could not. The results of competitive ELISA showed that both R2and R42could accordingly inhibit the binding of phage clones No.2and No.42to mAb RP215in a dose-dependent manner with an inhibition above70%. The data suggest that R13does not mimic to epitopes of CA215C, while both R2and R42, simulating the conserved epitopes on corresponding R2and R42phage clones binding to mAb RP215, may mimic to the epitopes of CA215C.Ⅲ. The evaluation for immunogenicity of peptide mimicsAntigen specificity of peptide mimics conjugated with different carriers Two peptide mimics, R2and R42, were separately conjugated with both mouse IgG (peptide-mIgG) and BSA (peptide-BSA), and then the antigenicity of conjugate was identified. The results showed that both peptide-mIgG and peptide-BSA could bind to mAb RP215, but. peptide-mIgG conjugate could also bind with goat-anti-human IgG, while peptide-BSA conjugate could not. It suggests that peptide-mIgG conjugate may form a cross-reaction epitope with human IgG, while peptide-BSA conjugate may be not.Evaluation of immunogenicity of peptide-BSA conjugate Two peptide mimics were conjugated to BSA as experimental vaccine, and Balb/c mice were immunized with peptide-BSA conjugate emulsion using Freud’s adjuvant. The antisera from mice were proved to be capable of binding to CA215, but not to human IgG Competitive ELISA showed that antisera could inhibit the binding of CA215to mAb RP215in a dose-dependent manner. The results suggested that antibodies against CA215were produced in all of mice immunized with the two kinds of peptide-BSA conjugate, R2-BSA and R42-BSA. In conclusion, we obtained the two synthetic peptide mimics to conserved epitode of CA215C, R2and R42, may be expected to be vaccine candidate for tumor immunotherapy. |
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