Antibodies In Ascites Fluid Research Articles

Scale-up production and characterization of anti-human cardiac troponin I monoclonal antibody in ascitic fluid of balb/c mice

ABSTRACTThe Human Cardiac Troponin I (hcTnI) is a 210 amino acid protein, 23 kDa in molecular weight. This biomarker is commonly used to diagnose myocardial infarction, micro injury, and acute coronary syndrome (ACS) in patients referring to emergency departments. The American Heart Association (AHA) and European Society of Cardiology (ESC) proposed troponin I as the gold biomarker for early detection of heart attack, especially in myocardial infarction (MI). Therefore, developing monoclonal antibodies against this biomarker could help in for early detection of heart attack. Hybridoma technology is a well-known technique introduced to produce monoclonal antibodies in specialized cells. The aim of this study was to produce large scale of monoclonal antibody against human cardiac troponin I using Hybridoma technology in order to design a diagnostic kit. The monoclonal antibody was produced using conventional Hybridoma technology in ascitic fluid of mouse and characterized for its ability to detect Human Cardiac Troponin I in a rapid test system. The results indicate the successful detection of Troponin I using the obtained monoclonal antibody. According to the achieved results it seems that ascites production of monoclonal antibody is very versatile, inexpensive, and economically useful for monoclonal antibody production.

Anti Adeno-Associated Virus 2 (AAV) Antibody Profile in Ovarian Cancer Ascitic Fluid: Implications for AAV Intraperitoneal Gene Therapy

Objective: Recombinant adeno-associated virus (rAAV) type 2 is a common vector used in gene therapy. However, the presence of anti-AAV2 neutralizing antibodies or other neutralizing factors can significantly limit effective transduction. Intraperitoneal gene therapy could enable local delivery of the target gene directly to ovarian cancer cells. Until now, there have been no reports on the presence of anti-AAV antibodies in ascitic fluid, which might limit the effectiveness of rAAV as a candidate vector for intraperitoneal gene therapy. Thus, the characterization of the preexisting neutralization antibodies in ascitic fluid will provide insight into successful intraperitoneal gene therapy. Methods: The study was conducted on 23 ascitic fluid samples obtained from women with stage 3 and 4 ovarian cancer. The samples were collected to determine the presence of anti-AAV antibodies with ELISA test and the presence of neutralizing antibodies with neutralizing assay. Results: Our results indicate that anti-rAAV antibodies are present in 70%, whereas neutralizing factors/antibodies are present in 78% of analyzed ascitic fluid samples. This correlation provides evidence for the presence of additional, different from antibodies, currently unknown factors in ascites, which are able to inhibit AAV2 infection in the absence of anti-AAV antibodies. Conclusion: The presence of neutralizing antibodies against rAAV or other neutralizing factors in ascitic fluid should be taken into account during intraperitoneal gene therapy, because they might limit effective intraperitoneal gene therapy with rAAV as a vector.

Large scale generation and characterization of anti-human IgA monoclonal antibody in ascitic fluid of BALB/c mice.

Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori.

Interferon γ improves the vaccination potential of oncolytic parvovirus H-1PV for the treatment of peritoneal carcinomatosis in pancreatic cancer

Oncolytic viruses with their capacity to specifically replicate in and kill tumor cells emerged as a novel class of cancer therapeutics. Rat oncolytic parvovirus (H-1PV) was used to treat different types of cancer in preclinical settings and was lately successfully combined with standard gemcitabine chemotherapy in treating pancreatic ductal adenocarcinoma (PDAC) in rats. Our previous work showed that the immune system and particularly the release of interferon-gamma (IFNγ) seem to mediate the anticancer effect of H-1PV in that model. Therefore, we reasoned that the therapeutic properties of H-1PV can be boosted with IFNγ for the treatment of late incurable stages of PDAC like peritoneal carcinomatosis. Rats bearing established orthotopic pancreatic carcinomas with peritoneal metastases were treated with a single intratumoral (i.t.) or intraperitoneal (i.p.) injection of 5x108 plaque forming units of H-1PV with or without concomitant IFNγ application. Intratumoral injection proved to be more effective than the intraperitoneal route in controlling the growth of both the primary pancreatic tumors and peritoneal carcinomatosis, accompanied by migration of virus from primary to metastatic deposits. Concomitant i.p. treatment of H-1PV with recIFNγ resulted in improved therapeutic effect yielding an extended animal survival, compared with i.p. treatment with H-1PV alone. IFNγ application enhanced the H-1PV-induced peritoneal macrophage and splenocyte responses against tumor cells while causing a significant reduction in the titers of H1-PV-neutralising antibodies in ascitic fluid. Thus, IFNγ co-application together with H-1PV might be considered as a novel therapeutic option to improve the survival of PDAC patients with peritoneal carcinomatosis.

Cloning and Prokaryotic Expression of Rat RVLG and Preparation of Mouse Anti-RVLG Polyclonal Antibody

Abstract In order to identify rat ovarian germ cells, the authors expressed and purified rat RVLG protein in E. coli cells and prepared a mouse anti-rat RVLG polyclonal antibody. The rat RVLG cDNA was obtained from rat testicle tissue by RT-PCR and was cloned into the vector pMD19-T. Sequence analysis proves that the cloned RVLG cDNA fragment was 60 bp longer than that released in the GenBank (NM_001077647), resulting from an alternative splicing of the RVLG pre-mRNA. The RVLG cDNA was doubly digested with the restriction endonucleases BamH I and EcoR I, and then was extracted from gel and inserted into the prokaryotic expression vector pGEX-4T-1. The recombinant expression plasmid pGEX- RVLG was verified for successful construction and then was transformed into E. coli BL21(DE3) for induction to express the GST-RVLG fusion protein by IPTG. The GST-RVLG fusion protein was expressed in E. coli BL21 (DE3) at a high level, which accounts for more than 10% of the total bacterial cellular protein. The purified RVLG protein was used as an antigen to immunize KM mouse for the production of polyclonal antibody in ascitic fluid followed by celiacly injecting the mouse with S180 cells. The mouse anti-rat RVLG antibody was analyzed by ELISA, Western blotting, and Immunohistochemistry for its specificity and titer. The antibody could recognize RVLG protein specifically and its titer is about 1:20 000. These results confirm that the mouse anti-rat RVLG polyclonal antibody with high affinity and specificity has been prepared successfully, and lays a foundation for the authors' ongoing research on the specific expression of RVLG in rat ovary.

Antigen Binding of a Mouse Monoclonal IgG1 Is Inactivated by Heating but Not by Freeze/Thaw Cycling

A mouse monoclonal antibody in ascitic fluid or in purified form was subjected to heating or repeated freeze/thaw cycles. Heating the antibody at 80°C for 10 min completely destroyed its ability to bind to antigen. Unpurified antibody in ascitic fluid or purified antibody in buffer was frozen to -196°C in liquid nitrogen and then thawed at room temperature. Purified antibody was also slowly frozen at -4°C followed by thawing at room temperature. Regardless of the manner of freeze/thaw treatment or whether the antibody was purified or not, immunologic competence for its antigen was not lost. The results call into question the belief that freezing and thawing in all cases are necessarily deleterious to antibody structure and function.

Monoclonal antibodies to an extracellular component of Pyricularia grisea

Monoclonal antibodies were produced from fusions of myeloma cells with splenocytes from BALB/c mice immunized with an extract of a liquid culture fluid of an isolate of Pyricularia grisea race IB—49. The hybridomas were screened with the immunogen, and three were selected and propagated in mice for antibodies in ascites fluid. The monoclonal antibodies 3C3 and 4E10, isotype IgG3, and 10G9, isotype IgA, were characterized by indirect ELISA, Western immunoblotting, and immunoelectron microscopy. All three monoclonal antibodies reacted similarly with the antigens in various serological techniques used and did not cross-react with any unrelated fungal isolates representing 11 genera but reacted positively with all 20 races or isolates of P. grisea. The monoclonal antibodies bound to a high Mr protein component (113 × 103) in extracts of liquid culture fluids and not to conidial antigens. The epitopes recognized by the monoclonal antibodies were densely distributed in the cell walls and lomasomes of hyphae and much less so in the cytoplasm of conidia. It is suggested that the antigenic component was synthesized in the cytoplasm of fungal cells, accumulated in the lomasomes, and secreted through cell walls as an extracellular molecule. The monoclonal antibodies could detect homologous antigen at about 60 ng fungal protein/mL and a fivefold dilution of the extracts of infected rice tissue by ELISA. Key words: fungus, monoclonal antibodies, ELISA, immunoassays.

Production of polyclonal antibodies in ascitic fluid of mice: time and dose relationships.

Further studies of the humoral immune response have been undertaken using ovine immunoglobulin G as the immunogen and BALB/C mice as the recipients. Mice immunized by the intraperitoneal route weekly for five weeks with an emulsion of the immunogen in Freund's complete adjuvant and given an intraperitoneal injection of pristane on day 14 produce relatively large volumes of ascitic fluid. A simple fluoroimmunoassay is used to determine specific antibody titres. The mice do not lose condition, remain active and eat normally throughout, although all show extensive granuloma formation within the peritoneal cavity. Tapping is performed before ascitic fluid production is excessive and each study is complete within 49 days, with a maximum of 5 taps. Only a single immunization site is required, since no higher antibody titres are obtained using combined intraperitoneal plus intramuscular plus subcutaneous immunization routes. A dose response study has shown that relatively large amounts (100 micrograms) of ovine immunoglobulin G must be injected to evoke a maximum response. Studies are in progress of various adjuvants and potential modulators of the immune response and of ways in which the immunogenicity of macromolecules can be changed. This approach can also be used to obtain antibodies for diagnostic purposes more rapidly than in rabbits and sheep.

Production of polyclonal antibodies in ascitic fluid of mice: technique and applications.

Balb/c mice immunised intraperitoneally with insulin developed significantly higher antibody concentrations in their serum than mice immunised subcutaneously. The antibody response was dose related, 50 ug amounts being more effective than either 20 ug or 5 ug amounts. In contrast, the antibody titres in the ascitic fluid of mice immunised with growth hormone were significantly higher after 5 ug when compared to 25 and 100 ug amounts. Repeated intraperitoneal injections of an emulsion of sheep IgG or human growth hormone in complete Freund's adjuvant, together with a single intraperitoneal injection of Pristane, induced ascites formation in most mice within 5 weeks. Up to 90 mL could be obtained from a single mouse by abdominal tapping, and the antibodies derived from the ascitic fluid proved suitable for application in a radioimmunoassay.

Efficient conjugation of DTPA to an IgM monoclonal antibody in ascites fluid

We have developed a simple, rapid, and reproducible method for conjugating the bifunctional metal chelator diethylenetriaminepentaacetic acid (DTPA) to an IgM monoclonal antibody (MoAb) without first isolating the MoAb from the ascites fluid. Treatment of the protein mixture in the ascites fluid with cyclic DTPA anhydride (cDTPAA) followed by HPLC purification on a size exclusion column allowed isolation of the DTPA-IgM conjugate which could then be labeled with 111 In ⩾80% yield. Over the range of total protein concentrations used (11–44 mg/mL), the number of DTPA molecules per molecule of IgM was approximately one-half the molar ratio of cDTPAA to total protein. We have used this method to prepare an 111In labeled anti-Thy 1.2 IgM, a MoAb with specificity for a murine cell-surface antigen found on normal and malignant T cells and neuroectodertmal tissues. Analysis of the DTPA-IgM conjugate prior to and after 111In labeling using indirect immunofluorescence flow cytometry and a target-cell binding assay showed that the antigen specificity of this anti-Thy 1.2 MoAb is not substantially altered by the presence of up to 8 DTPA molecules per IgM molecule.

Use of high-performance liquid chromatography for the purification of antibodies and antibody conjugates and the study of antibody-antigen interactions

The purification and characterization of polyclonal and monoclonal antibodies and antibody-enzyme conjugates by high-performance liquid chromatography (HPLC) using a Protein Pak 300 SW column and a Protein Pak DEAE anion-exchange column is described. The following polyclonal antibodies were examined: (i) donkey anti-mouse immunoglobulin G (IgG), (ii) mouse IgG, (iii) sheep anti-human IgG and (IV) goat anti-human factor VIII. HPLC was used to analyse the purity of horseradish peroxidase conjugates of rabbit anti-mouse IgG and donkey anti-mouse IgG. In the case of donkey anti-mouse IgG, each stage of the production of the purified antibody and antibody-enzyme conjugate was monitored by HPLC. HPLC was used to examine monoclonal mouse anti-human T cell and mouse anti-human apoliproprotein B antibodies. The presence of antibody in ascites fluid from mice bearing Landschütz ascites tumour cells was also detected. The antigen-antibody reaction between human serum albumin and anti-human serum albumin was demonstrated using HPLC and this procedure should offer a novel method for studying antigen-antibody interaction.

34] The effect of pristane on ascites tumor formation and monoclonal antibody production

Preinjection of mice 10 days before intraperitoneal injections of hybridomas gives optimum development of ascites tumors with respect to the rate at which tumors form, percentage of mice developing tumors, volume of ascites fluid produced, and concentration of monoclonal antibody in ascites fluid. The dose of pristane injected has little or no effect on ascites formation down to 0.1 ml of pristane.

A simple general method of exactly comparing different binding activities and antigenic properties of antibodies by obtaining duplicate copies from a single isoelectric focusing gel

We report a simple general method of exactly comparing different binding activities and/or antigenic properties of antibodies in ascites fluid after fractionation on a single isoelectric focusing gel. Two copies of the gel are made on nitrocellulose: 1 copy is overlaid with the radioiodinated form of one overlay protein and the other copy is overlaid with the radioiodinated form of another. By superimposing X-ray films obtained after radioautography, any 2 binding activities or antigenic properties of antibodies can be compared exactly and in detail. The procedure is simple, highly sensitive, and independent of the molecular weight of the overlay protein. This is accomplished without requiring isolation of the antibodies, denaturing agents, precipitation, chemical fixation or other modification reactions.

Monoclonal antibodies to the chemotherapeutic agent methotrexate: Production, properties and comparison with polyclonal antibodies

Hybridomas that secrete monoclonal antibodies to the chemotherapeutic agent methotrexate (MTX) were produced by fusion of FO myeloma cells with splenocytes obtained from BALB/c mice immunized with L-MTX covalently bound to keyhole limpet hemocyanin (KLH). When the MTX/KLH molar ratio was 430 1 or 530 1 (high MTX series), 238 412 (58%) or 157 405 (39%), respectfully, of antibody producing hybridomas secreted only antibodies to MTX. In contrast, only 15 412 (40%) or 54 405 (13%) hybridomas secreted only antibodies to KLH. When the molar ratio was 50 1 (low MTX series), only 14 343 (4%) of the wells obtained anti-MTX and 93 343 (27%) contained antibodies to KLH. The predominance of anti-MTX over anti-KLH secretors in the 2 high MTX series, and the reverse predominance of anti-KLH secretors over anti-MTX hybridomas in the low MTX series was highly significant ( P < 0.001). The anti-MTX antibodies were mainly IgG1 with fewer IgG2a and IgG2b subclasses. All were kappa chain isotypes. No IgM or IgA antibodies were detected. Titers and binding constants of monoclonal anti-MTX antibodies in ascites fluid obtained from mice injected with selected cell lines were higher than those of conventional rabbit, mouse, or goat antibodies. Based on results of competitive inhibition radioimmunoassays, the ascites monoclonal antibodies were at least as specific as the conventional antibodies for MTX compared to structurally related compounds. IgG2a and IgG2b antibodies had high titers in complement mediated hemolysis of sheep erythrocytes that contained MTX on the cell surface (E-MTX) and also bound 125I-labeled protein A when complexed to MTX on these cells. IgG1 antibodies were relatively weak in the hemolysis assay and failed to bind 125I-labeled protein A directly. Hemolytic activity and 125I-labeled protein A binding were markedly enhanced when E-MTX cells were treated sequentially with IgG1 anti-MTX then rabbit IgG antibodies to mouse IgG1.

The effect of pre-injection of mice with pristane on ascites tumour formation and monoclonal antibody production

Experiments were performed to determine whether for mice pre-injected with pristane (2,6,10,14-tetramethylpentadecane) there is an optimum interval before injection of hybridomas in order to maximize ascites fluid formation and yield of monoclonal antibodies. With injections of 0.5 ml of pristane followed by injections of 5 × 10 5 hybridoma cells making monoclonal antibodies against mammalian carbamyl phosphate synthetase, a pre-injection time of 10 days was optimum with respect to (a) the time taken for the ascites tumours to appear; (b) the percentage of mice developing ascites tumours (all mice developed tumours over a span of 2 days); and (c) the concentration of monoclonal antibodies in ascites fluid: 11 mg/ml compared with 7 mg/ml for the next highest group.

The separation of three antibody populations from anti-poly(A)·poly(U) antibodies elicited in mice or rabbits and antigenic features of poly(A)·poly(U)

Anti-poIy(A)·poly(U) antibodies in ascitic fluid of DDY mice immunized with poly(A)·poly (U)-methylated bovine serum albumin complexes were fractionated into three major antibody populations, Ab-1, Ab-2 and Ab-3, by precipitating with poly(I)·poly(C), poly(A)·poly(U), and poly(A)-2 poly(U), respectively. Antibody population one, Ab-1, reacted with various double-stranded RNAs [poly(I)-poly(C), poly(A)·poly(U), and rice dwarf virus ribonucleic acid (RDV-RNA)] and poly(A)-2 poly(U). Ab-2 reacted with poly(A)-poly(U) and poly(A)·2 poly(U), but not with poly(I)·poly(C) and RDV-RNA, while Ab-3 reacted only with poly(A)·2 poly(U). Although both Ab-1 and Ab-2 reacted with poly(A)·poly(U), the two populations were distinguishable by their different reactivities against chemically modified antigens and oligonucleotides. In contrast to Ab-1, acetylation at the furanose 2'-position of poly(U) resulted in a dramatic decrease in the complement fixation reactivity of Ab-2. Also, Ab-2 was capable of binding with complexes of hexa- to heptaadenylates and poly(U), whereas Ab-1 required oligoadenylates of longer chain lengths (9–10 chain length) for binding. Therefore, it appears that poly(A) poly(U) possesses unique antigenic determinants which are recognizable only by Ab-2, in addition to those determinants which are common to a variety of double-stranded RNAs.

Characterization of monoclonal antibody specific for human alpha interferon.

A mouse hybridoma cell line has been isolated, which secretes a monoclonal antibody specific of human leukocyte (alpha) interferon. The antibody secreted by the hybridoma belongs to the IgG1 class. It neutralizes biological activities (cellular and antiviral) of alpha interferon. Mass production of the antibody in ascitic fluid has been obtained. A convenient method of purification of the IgG from the ascitic fluid on DEAE-Trisacryl M is described.

A method for producing anti-DNA antibodies in ascitic fluid of mice

Immunological methods for the production of antibodies to DNA in ascitic fluid of DDY, A/He, and C57B1/6 mice were investigated. Relatively large volumes of ascitic fluid containing antibodies for DNA in high concentration were obtainable after seven injections of denatured ssDNA-MBSA complexes emulsified with CFA into peritoneal cavities of DDY mice. The quantity of anti-ascitic fluid from 4 to 5 DDY mice was equivalent to that of antiserum from one rabbit. DDY mice accumulation of ascites is much greater than in other strains, and the ascitic fluid contains antibodies of higher titer regardless of sex. The antibody titers in ascitic fluid are not affected by alteration of the ascites volume, and are proportional to those of the corresponding sera. Antibodies to DNA in ascitic fluid of these mice occurred exclusively in IgG as in DNA antisera.

A simple rapid method for production of viral antibody in mice.

A technic for production of antibody in mouse peritoneal fluid was described by Munoz(1) utilizing intraperitoneal inoculations of egg albumin or bovine serum albumin mixed in Freund's adjuvant. Ascites production was induced, however, in only 1/2 the animals tested. Both Herrmann and Kasel and their associates (2,3) used ascites fluid from immunized mice as a source of viral antibodies. Considerable quantities were induced by intraperitoneal injection of Sarcoma 180 cells by the first group and bacterial-adjuvant mixture(4) by the latter. A modification of these methods utilizing Ehrlich tumor cells has proved consistently reliable, required less time or fewer injections and is here described in detail. Each group of 8 mice, 4 test and 4 control animals from the same litter, was kept together from birth to 21 days of age when the experiment was begun. Undiluted viral infected fluids removed from monkey renal cell cultures maintained on BAF medium(5) with 5% horse serum served as antigens. Each mouse was injected intramuscularly in the hind-quarter with 0.25 ml of viral antigen mixed with a rapid to and fro movement in the same syringe with 0.25 ml of Freund's complete adjuvant (Difco Laboratories). A second dose was given in the same manner one week later. One week after the second inoculation each animal received 0.25 ml of the viral antigen without adjuvant intramuscularly and 0.1 ml was administered slowly by the intravenous route. At the same time each mouse was given intraperitoneally 0.1 ml of undiluted ascitic fluid from mice previously inoculated by the same route with Ehrlich's ascites tumor cells. Approximately 1–5 × 107 cells were injected and 7 days later the ascitic fluid was harvested aseptically with needle and syringe. Table I summarizes the results with several different viruses. Following immunization it is evident that neutralizing antibodies developed to virtually the same titer in both the serum and ascitic fluid. This has been observed by others for both neutralizing and HI antibodies(2,3,6). It will be recalled that by immunoelectrophoretic technics mouse peritoneal fluid has been found to be similar in composition to mouse serum and that egg albumin antibodies in ascitic fluid have been found in the gamma globulin fraction(7). It should be noted that the pooled ascitic fluids from one group of control animals (Group 6) contained a very low level of neutralizing activity. No explanation for this finding is apparent. The failure to induce formation of adenovirus antibodies probably was due to the low concentration of the viral antigen.

Studies of antibodies in ascitic fluid of individual mice.

Employing mice with ascites, individual patterns of change in hemolysin and ovalbumin titer of ascitic fluid were studied for 4 inbred strains of mice. Mice received both primary and secondary immunizations of sheep erythrocytes. There was evidence of antibody titers 2 days after immunization. Characteristically, after primary immunization, rapid rises in titer occurred which attained their peaks within 7 days and were minimal after 15-20 days. Following secondary immunization, peak titers usually were higher, attained later and tended to diminish more gradually than following primary immunization. Some strain differences in the pattern of antibody titer changes were manifest. Ovalbumin titers measured by the PCA reactions were evident from the 12 th day, increased gradually to a peak, generally 32-42 days after immunization, then diminished more gradually thereafter.