Protein A-Sepharose Affinity Chromatography Research Articles

Mass-Production and Characterization of Anti-CD20 Monoclonal Antibody in Peritoneum of Balb/c Mice.

Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of monoclonal antibody against CD20 in order to diagnostic application in leukemia and lymphomas disorders. Hybridoma cells that produce monoclonal antibody against human CD20 were administered into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After twelve days, approximately 7 ml ascetic fluid was harvested from the peritoneum of each mouse. Evaluation of mAb titration was assessed by ELISA method. In the present study, we describe a protocol for large scale production of MAbs. We prepared monoclonal antibodies (mAbs) with high specificity and sensitivity against human CD20 by hybridoma method and characterized them by ELISA. The subclass of antibody was IgG2a and its light chain was kappa. Ascetic fluid was purified by Protein-A Sepharose affinity chromatography and the purified monoclonal antibody was conjugated with FITC and Immunofluorescence was done for confirming the specific binding. The conjugated monoclonal antibody could have application in diagnosis B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells.

Purification of monoclonal antibody against PGRP and its activity for small cell lung cancer in vitro

Objective To explore the effect of purification on monoclonal antibody (MAb) against PGRP by Protein A-Sepharose affinity chromatography, and to provide some based data for the purification of other antibody using the same method. Methods The ascites which include MAb was purified by Protein A-Sepharose affinity chromatography. The purity and activity of MAb was tested by SDS-PAGE and ELISA. The biological function was identified by flow cytometer and immunohistochemistry. Results The average concentration of protein in ascites before purification is 23.62 mg/ml. Before and after purification, the total protein is 148.79 mg and 146.67 mg, respectively. The recovery coefficient of protein is 98.58%. The concentration of MAb in ascites is 5.21 mg/ml averagely. The MAb purity is more than 95 %. The immunoactivity of purified antibody is higher than that of unpurified antibody. Conclusion The purity of MAb against PGRP purified by Protein A-Sepharose affinity chromatography is very high. The immunoactivity of purified antibody is higher than that of unpurified antibody. So the ProteinA-Sepharose affinity chromatography is a rapid, convenient and reliable method for the purification of MAb Against PGRP. Key words: Chromatography, affinity; Antibodies, monoclonal; Purify

Significant Improvements in the Performance of an Established Affinity Chromatography Procedure Employed to Purify a Monoclonal Antibody in 100 Purification Cycles

Protein A-Sepharose affinity chromatography is a very successful method for the purification of immunoglobulins for pharmaceutical use. However, the chromatography efficiency and lifetime of this method have to be always adjusted to specific chromatography conditions (biological source, buffers, flow rates, antibody properties, temperature, protein concentration, cleaning protocol, etc). This study sought to demonstrate improvements in the performance of an established affinity chromatography procedure employed to purify the CB.Hep-1 monoclonal antibody (mAb) used in the purification of the active pharmaceutical ingredient of a Hepatitis B vaccine. In conclusion, the relative poor mAb recovery observed in 150 mM PBS; pH 8.0/100 mM citric acid; pH 3.0 buffer system conditions was attributed to the inefficacy of the elution buffer to disrupt completely interactions between the matrix and mAb. In this regard, retention of the CB.Hep-1 mAb into the matrix was helped by the ligand coupled to the matrix and not by unspecific interactions. The 1.5M glycine-NaOH/3M NaCl; pH 9.0/200 mM glycine-HCl; pH 2.5 buffer system significantly improved the affinity chromatography recovery without affecting mAb purity, molecular homogeneity, ligand leakage and mouse DNA content in 100 purification cycles. Thus, application of 1.5M glycine-NaOH/3M NaCl; pH 9.0/200 mM glycine-HCl; pH 2.5 as buffer system allowed the reduction of the CB.Hep-1 mAb and Hepatitis B vaccine costs, respectively.

The Tip of the Coiled-coil Rod Determines the Filament Formation of Smooth Muscle and Nonmuscle Myosin

Myosin II self-assembles to form thick filaments that are attributed to its long coiled-coil tail domain. The present study has determined a region critical for filament formation of vertebrate smooth muscle and nonmuscle myosin II. A monoclonal antibody recognizing the 28 residues from the C-terminal end of the coiled-coil domain of smooth muscle myosin II completely inhibited filament formation, whereas other antibodies recognizing other parts of the coiled-coil did not. To determine the importance of this region in the filament assembly in vivo, green fluorescent protein (GFP)-tagged smooth muscle myosin was expressed in COS-7 cells, and the filamentous localization of the GFP signal was monitored by fluorescence microscopy. Wild type GFP-tagged smooth muscle myosin colocalized with F-actin during interphase and was also recruited into the contractile ring during cytokinesis. Myosin with the nonhelical tail piece deleted showed similar behavior, whereas deletion of the 28 residues at the C-terminal end of the coiled-coil domain abolished this localization. Deletion of the corresponding region of GFP-tagged nonmuscle myosin IIA also abolished this localization. We conclude that the C-terminal end of the coiled-coil domain, but not the nonhelical tail piece, of myosin II is critical for myosin filament formation both in vitro and in vivo.

Study on the scale-up of human IgG3 purification using protein A affinity chromatography.

The purification of human IgG3 subclass out of IgG (Immunoglobulin-G) was studied using protein A-Sepharose affinity chromatography. The effect of operational parameters such as flow rate, ionic strength, pH and size of sample was investigated, and the process was scaled-up 10-fold. The use of 0.5 m NaCl in the loading buffer had a dramatic effect in the purity of IgG3 recovered in the flowthrough fraction (values in the order of 97% were consistently obtained). This was attributed to a more effective binding of IgG subclasses 1, 2 and 4 to protein A (well known classical mechanism based in Fc fragment) and in some extent to a decrease in the binding of subclass 3 to protein A by the alternative mechanism based in the Fab fragment. The increase in residence time also increased in a relevant way the purity of IgG3. This is attributed to an increased effectiveness of the mechanisms mentioned above. The recovery yields in the IgG3 rich fraction were in the range 21-32% and are possibly a consequence of binding to protein A by the alternative mechanism and also due to deactivation during processing.

Secondary structure of a fibrillin-1 eight-cysteine motif.

University of Manchester, Wellcome Center For Cell/Matrix Research, 2.205 Stopford, Manchester, MI3 9PT *University of Stirling, Scotland Eight-cysteine motifs occur within the fibrillin family of extracellular proteins which includes fibrillin-I and fibrillin-2, and the latent transforming growth factor-p binding proteins (LTBPs) 1-3 [1,2]. The importance of these motifs is highlighted by identification of mutations in eight-cysteine motifs resulting in Marfan syndrome [3], and the fact that one such module in LTBP-I binds TGF-P latency associated peptide via a disulphide linkage [4]. The signature of the eight-cysteine motifs is the distribution of cysteine residues, three of which are contiguous. While biochemical analysis suggests that all cysteine residues are involved in intramolecular disulphide bonds [5], the structure of the eight-cysteine motifs of fibrillin have not been defined. This report compares computer assisted modelling based on the primary sequence with the secondary structure determined by circular dichroism on recombinant fibrillin-I eight-cysteine motifs. Fibrillin1 cDNA encoding three domains, an eight-cysteine motif flanked by two epidermal growth factor (EGF) motifs, was generated by RT-PCR. The sequence was expressed in COS-1 cells using the mammalian expression vector signal plg tail (Invitrogen), into which an enterokinase site was introduced. The hsion protein contained an Fc tail which allowed purification by protein A-Sepharose affinity chromatography. Enterokinase cleavage allowed release of the recombinant protein. Protein purity was assessed by SDS-PAGE on 15% gels and by mass spectrum determination by MALDI-TOF. Circular dichroism was conducted using a JASCO J-600 with scan rates at 10 nm min -’ with a time constant of 2 seconds. Data were collected from 280-195 run. Fitting ofthe data was conducted using CONTM procedure which fits a-helix and Psheet quantities to spectral data when compared to a structural database.

The universality of immuno-PCR for ultrasensitive antigen detection

Immuno-PCR is a relatively new method for ultrasensitive antigen detection that utilises polymerase chain reaction (PCR) from a secondary antibody-target DNA conjugate to detect the specific binding of a primary antibody to its target antigen [l] . There have been several reports of the use of immuno-PCR, but the universality of a single protocol for low-level detection of a variety of antigens has not been confirmed. We are seeking to measure the very low levels of tissue DNA damage (adducts) that are expected to be formed following incidental human exposure to potentially carcinogenic alkylating agents. Detection and quantitation of such low-level damage is technically difficult. This is the case for the powerfully promutagenic DNA base lesion 06-methylguanine, which may be produced in human DNA following exposure to methylating agents such as the environmentally ubiquitous nitrosamines. To assess the feasibility of using immuno-PCR for detection of very low levels of a DNA adduct and the universality of the method for detecting a variety of different antigens, we established an immuno-PCR protocol using purified rabbit polyclonal antibodies recognising four biochemically diverse antigens: pyruvate dehydrogenase complex (PDC; oligomeric; Mr 8.5 x lo6), the DNA repair enzyme 06-methylguanine-DNA methyltransferase (MGMT; monomeric; Mr 2 1,700), an oligopeptide from the N-terminal end of human MGMT (20-mer; Mr 2,3 10) and a DNA adduct (06-methylguanosine) conjugated to BSA (06-MeG-BSA; Mr 67,600). This protocol is a modification of the method of Zhou et a1 [2], the basic principle of which relies upon detection of antigen-bound antibody by the sequential use of (i) a second antibody-streptavidin conjugate directed against the primary antibody, (ii) a biotinylated target DNA (which will bind to streptavidin with high affinity) and (iii) PCR for the detection of the immobilised target DNA. Our modifications include (i) the biotinylated target DNA for PCR, which was produced using a fragment of the gene for ahydroxynitrile lyase from the Cassava plant. (A BamH 1 restriction enzyme site was added to this fragment for subsequent digestion and fill-in labelling of the 5'-end of the cut DNA using biotinylated-14-dATP and the Klenow fragment of DNA polymerase), (ii) using 96-well polycarbonate plates for thermocycling and (iii) using NeutrAvidin (Pierce) instead of streptavidin. PCR products were analysed by agarose gel electrophoresis, with ethidium bromide staining of amplified DNA. Antigen detection limit was defined by the point of disappearance of the ethidium bromide signal. A standard peroxidase-based sandwich enzyme linked immunosorbent assay (ELISA) was performed with antigen coated on 96 well plates to serve as a comparison for the immunoPCR. Serum IgG was purified by protein A-Sepharose affinity chrom-atography for use in both immuno-PCR and ELISA.

Identification of Myelin Basic Proteins in Circulating Immune Complexes Associated with Lepromatous Leprosy

Circulating immune complexes (CIC) were first measured in lepromatous patients (LL) by the 125I-C 1q binding assay and the polyethylene glycol (PEG) precipitation test. High levels were found by both methods (95 and 90% of positives, respectively). LL-CIC were investigated for the presence of neural antigens. CIC were precipitated in 3.5% PEG, filtered through protein A-Sepharose affinity chromatography, eluted with glycine-HCI, pH 2.8, and washed with PBS; fractions after CIC dissociation were studied by SDS-PAGE and Western blotting. The LL-CIC PEG precipitates and the glycine-HCI eluates were positive in 76 and 71% respectively against anti-myelin basic proteins (MBP) monoclonal antibody, showing a single band at 15-25 kDa similar to the one obtained incubating MBP with anti-MBP. No reaction was detected with CIC-PBS fractions; strips were incubated with other anti-neural antibodies such as anti-glial fibrillary acidic proteins, anti-S-100, and anti-neuarofilaments, without any reactivity. Our results demonstrate that LL-CIC contain MBP as an antigen; its significance could be related to the pathogenesis of leprosy since the liberation of MBP after Mycobacterium leprae nerve damage may elicit anti-MBP autoantibodies to myelin breakdown, which reacts with peripheral nerve MBP inducing CIC formation. This mechanism may be important in demyelination and destruction of nerve in leprosy.

Identification of an acute-phase reactant in murine infections with Trypanosoma brucei.

A 42-kDa protein appeared at a much higher concentration in plasma from Trypanosoma brucei-resistant (C57BL/6) mice after infection than in plasma from trypanosome-susceptible (C3H/He) mice. This protein was purified by sequential steps of gel filtration, protein A-Sepharose affinity chromatography, isoelectric focusing, and ammonium sulfate precipitation. The purified protein was identified as a subunit of the acute-phase reactant haptoglobin. Causes of elevated plasma haptoglobin and its implications for resistance to trypanosomiasis are discussed.

Trypanosoma cruzi: Involvement of IgG isotypes in the parasitemia control of mice immunized with parasite exoantigens of isoelectric point 4.5

In a previous work we demonstrated that Trypanosoma cruzi exoantigens of pI 4.5 (Ea 4.5), whose most important epitopes are glucidic, are able to induce a partially protective immune response in mice. To ascertain the involvement of antibody isotypes in this protection, we immunized mice with Ea 4.5 plus Bordetella pertussis as adjuvant. The analysis of immune response by skin test revealed the occurrence of specific immediate type hypersensitivity on Day 15 after the last immunization. By ELISA and using Ea 4.5 as antigen, specific IgG1 antibody was detected. When formaldehyde-fixed epimastigotes were used as antigen, binding of IgG1 and IgG2 was observed. Trypomastigotes incubated for 1 hr at 33 °C with the immune sera and then injected in normal syngeneic mice produced a significantly lower parasitemia than trypomastigotes incubated with the control sera. This capacity of anti-Ea 4.5 sera was resistant to 56 °C for 2 hr and was diminished after the absorption of immune sera with the carbohydrate moiety of Ea 4.5. The assay with the immune IgG1 and IgG2, separated through protein A-Sepharose affinity chromatography, showed that IgG1 retains most of this capacity. Purified immune IgG1 revealed two antigenic bands of molecular weight between 50 and 55 kDa in SDS-PAGE of Ea 4.5.

Purification of autoantibodies to myelin basic protein by antigen specific affinity chromatography from cerebrospinal fluid IgG of multiple sclerosis patients Immunoreactivity studies with human myelin basic protein

Immunoglobulin G (IgG) was purified by single-step protein A-Sepharose (Pharmacia TM) affinity chromatography from the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients and controls. Autoantibodies to myelin basic protein (anti-MBP) were isolated from the purified IgG fraction by two-step antigen specific affinity chromatography. Anti-MBP in the context of whole CSF or in purified from reacts equally to MBP prepared from non-MS or MS brain tissue. Kinetic studies of anti-MBP titers demonstrate that when anti-MBP is reacted with increasing amounts of non-MS or MS MBP, the autoantibody is immunoabsorbed by either antigen in vitro. Immunoabsorbtion of anti-MBP by MBP or its synthetic peptides may also possible in vivo as a potential therapeutic tool.

Immunoglobulin heavy chain associated protein in multiple sclerosis cerebrospinal fluid

Immunoglobulins G, A, and M (IgG, IgA and IgM) were isolated from multiple sclerosis (MS) cerebrospinal fluid (CSF) and sera by Protein A-Sepharose (PAS) affinity chromatography or using solid-phase immunoabsorbents. An isoelectric point heterogenous protein with apparent mol. wt of 74,000–80,000 was seen consistently when CSF Igs were immunoaffinity purified but was never seen when PAS was used for Ig purification. Control experiments exclude the binding of this protein non-specifically either to Sepharose or to Igs or to other CSF proteins. Analysis of purified Igs under non-reducing conditions leads to some reduction in staining pattern indicating that a portion of the protein may be disulfide linked to itself or to other CSF proteins. Immunoblot analysis of CSF Igs separated on 2-DE gels is consistent with a portion of the total CSF Ig protein existing as “free” heavy (H)-chains. PAS requires dimeric Fc fragment of Ig for binding; the differences in 2-DE gels of PAS and immunoaffinity purified Igs may be due to interaction of the 74,000–80,000 protein with “free” H-chain, which nevertheless, still leaves this complex available for binding by anti-H chain antisera. It has been previously suggested that the cytotoxicity of “free” H-chains is abrogated in the absence of the complementary L-chains by H-chain binding proteins; the protein reported here has features similar to these proteins reported previously and may be involved in some way in regulating Ig production in the MS central nervous system.

Large-scale purification of a murine antimelanoma monoclonal antibody

The 9.2.27 monoclonal antibody (murine IgG 2a), which recognizes a human melanoma-associated 250 kDa glycoprotein/proteoglycan, was produced in batch-suspension cultures and purified by staphylococcal protein A-Sepharose affinity chromatography. Initially, antibody production was performed in sub-lots of eight fermenters (280 1 per run). The cells were removed by centrifugation and the antibody from supernatant was concentrated 10–20-fold using ultrafiltration. The antibody was adsorbed onto a 500-ml protein A-Sepharose column at pH 8.4, followed by extensive washing with phosphate-buffered saline, pH 8.4, to remove endotoxins and other protein contaminants. The antibody was desorbed at pH 4.5. A 60–140-fold purification of antibody with a 100–1000-fold reduction of endotoxins was achieved. 2–4 g of highly purified antibody were obtained from each column run. Pooled fractions were concentrated by ultrafiltration, formulated and sterile filled as final product. Analysis of the final product included but was not limited to sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focusing, high-performance liquid chromatography, as well as evaluations of specific activity, endotoxin level, protein A concentration, bovine immunoglobulin G level, DNA concentration, mycoplasma contamination, sterility and reverse transcriptase activity.

Purification of murine monoclonal antibodies by affinity chromatography using protein A-Sepharose

A simple method is described for the isolation of murine monoclonal immunoglobulin G subclasses using protein A-Sepharose affinity chromatography.

Immunoglobulin Classes of Anti‐Sendai Virus Antibody Detected by ELISA in Infected Nude Mouse Serum

Sendai virus-infected nude mouse sera obtained on the seventh day after infection or later, in which anti-Sendai virus antibodies were undetectable by hemagglutination-inhibition and neutralization tests, were found to be reactive with the virus antigen by ELISA using horseradish peroxidase-conjugated anti-mouse IgG rabbit IgG. The reactivity was blocked by rabbit anti-Sendai virus antiserum and was not observed against influenza virus which served as a control antigen. Anti-Sendai virus antibody activity of fractions from Sephadex G-200 gel filtration was detected in the IgM fraction when anti-mouse mu chain-specific antiserum was used and in both IgG and IgM fractions when heavy and light chain-specific anti-mouse IgG serum was employed in ELISA. ELISA of the fractions from protein A-Sepharose affinity chromatography of Sendai virus-infected nude mouse sera showed that the eluates at pH 6.0 and pH 3.5 contained IgG1 and IgG2b anti-Sendai virus anti-bodies, respectively, and that the eluate at pH 4.5 contained both IgG2a and IgG3 antibodies.

The use of protein A-sepharose affinity chromatography for separation and detection of specific IgM antibody in acquired rubella infection: A comparison with absorption by staphylococci containing protein A and density gradient ultracentrifugation

Protein A-Sepharose affinity chromatography has been investigated as a means of removing IgG from sera before testing for rubella-specific IgM. The method was compared with whole staphylococci containing protein A and with sucrose density gradient ultracentrifugation. Approximately 98% of IgG was removed by protein A-Sepharose absorption and 95% by whole staphylococcal absorption, but 50–60% of IgM was also removed. One hundred and two sera were tested — 49 had been shown to contain rubella-specific IgM by ultracentrifugation and 53 to be negative. Accepting 2-fold or greater reduction in antibody titre with 2-mercaptoethanol as the criterion for positivity after absorption, 4 of 49 (8%) sera were negative with protein A-Sepharose and 31% whith whole staphylococci. Of the 4 sera negative after protein A-Sepharose treatment three were obtained between 48 and 62 days after infection. It is essential to confirm positive results by demonstrating reduction of IgM antibody remaining after absorption by treatment with 2-mercaptoethanol. It is also essential that all post-absorption antibody be treated with 2-mercaptoethanol to eliminate false positives. Protein A-Sepharose affinity chromatography was more sensitive than absorption with whole staphylococci but less sensitive thn ultracentrifugation. The method has a place in screening convalescent sera collected less than 28 days after infection.

Murine Schistosomiasis mansoni: anti-schistosomula antibodies and the IgG subclasses involved in the complement- and eosinophil-mediated killing of schistosomula in vitro.

Protein A-sepharose affinity chromatography was used to isolate IgG subclasses from the serum of CBA mice chronically infected with Schistosoma mansoni. The subclasses were tested for the presence of two antibodies which are responsible for the death of young schistosomula in vitro; 'lethal antibody' (LA), which kills schistosomula in co-operation with complement and 'eosinophil adherence antibody' (EAA) which causes the death of schistosomula by promoting the adherence of eosinophils to the parasite. LA and EAA were detected only in the IgG fraction of the serum. LA was concentrated in the IgG2a fraction and EAA in the IgG1 fraction. The development of IgG subclasses specific for schistosomula was followed in mice exposed to twenty cercariae by the fluorescent antibody technique. IgG1, IgG2a and IgG2b antibodies were detected 2 weeks after infection and their titres rose steadily to reach high levels by weeks 12 or 14. IgM antibody was not detected until week 6 and IgA until week 10; both were present at lower concentrations than the IgG1 antibodies.

The rapid isolation of ribonuclease-free immunoglobulin G by protein A-sepharose affinity chromatography

A rapid method is described for the simultaneous removal of contaminant ribonuclease activity and isolation of immunoglobulin G from fractionated or whole serum using insolubilized protein A. Protein A, isolated from the Cowan I strain of Staphylococcus aureus, was covalently attached to Sepharose CL-4B resin and used as a specific affinity absorbent for immunoglobulin G. Affinity column-purified immunoglobulin G preparations were examined for the presence of contaminating serum proteins, retention of antibody activity, and retention of antigenic properties. Following chromatography on protein A-Sepharose, immunoglobulin G preparations were devoid of contaminating serum proteins, in particular ribonuclease activity, that are not normally removed using conventional techniques of salt precipitation in combination with ion-exchange chromatography. There was no significant alteration of either antibody activity or antigenic properties of protein A-Sepharose purified immunoglobulin G.