IgG2a Kappa Research Articles

Development of high-affinity monoclonal antibody using CD44 overexpressed cells as a candidate for targeted immunotherapy and diagnosis of acute myeloid leukemia.

CD44s antigens have been suggested as an efficient biomarker for cancer stem cells. Current study aimed to develop a hybridoma that producing a high affinity murine anti-human CD44 monoclonal antibody for early diagnostic laboratory tests of some cancer. To make hybridoma against CD44, mice were immunized with MDA-MB-468 cells. Resulted hybridomas using three culture media were screened by indirect ELISA, then cloned by limiting dilution, and isotype was determined after obtaining ascitic fluid and antibody purification. We obtained a stable secreting clone, capable of secreting a high-affinity monoclonal antibody against CD44 protein, IgG2a kappa, with the affinity of 5.4 × 10-8 M without cross-reactivity. We could establish a hybridoma in a native form. This stable and high-affinity anti-CD44 mAb has a potential for diagnostic procedures and laboratory research. Thus, it could be exploited as a suitable tool for target-specific diagnosis and even treatment in several cancers.

Purification and characterization of monoclonal IgG antibodies recognizing Ebola virus glycoprotein.

PurposeThe goal of this study was to purify and characterize Ebola virus glycoprotein (GP)-specific IgG antibodies from hybridoma clones.Materials and MethodsFor hybridoma production, mice were injected by intramuscular-electroporation with GP DNA vaccines, and boosted with GP vaccines. The spleen cells were used for producing GP-specific hybridoma. Enzyme-linked immunosorbent assay, Western blot assay, flow cytometry, and virus-neutralizing assay were used to test the ability of monoclonal IgG antibodies to recognize GP and neutralize Ebola virus.ResultsTwelve hybridomas, the cell supernatants of which displayed GP-binding activity by enzyme-linked immunosorbent assay and the presence of both IgG heavy and light chains by Western blot assay, were chosen as a possible IgG producer. Among these, five clones (C36-1, D11-3, D12-1, D34-2, and E140-2) were identified to secrete monoclonal IgG antibodies. When the monoclonal IgG antibodies from the 5 clones were tested for their antigen specificity, they recognized GP in an antigen-specific and IgG dose-dependent manner. They remained reactive to GP at the lowest tested concentrations (1.953–7.8 ng/mL). In particular, IgG antibodies from clones D11-3, D12-1, and E140-2 recognized the native forms of GP expressed on the cell surface. These antibodies were identified as IgG1, IgG2a, or IgG2b kappa types and appeared to recognize the native forms of GP, but not the denatured forms of GP, as determined by Western blot assay. Despite their GP-binding activity, none of the IgG antibodies neutralized Ebola virus infection in vitro, suggesting that these antibodies are unable to neutralize Ebola virus infection.ConclusionThis study shows that the purified IgG antibodies from 5 clones (C36-1, D11-3, D12-1, D34-2, and E140-2) possess GP-binding activity but not Ebola virus-neutralizing activity.

Detection of Abrin Holotoxin Using Novel Monoclonal Antibodies.

Abrin, a member of the ribosome-inactivating protein family, is produced by the Abrus precatorius plant. Having the potential to pose a severe threat to both human and animal health, abrin is classified as a Select Agent by the U.S. Department of Health and Human Services. However, an immunoassay that is specific for intact abrin holotoxin has not yet been reported. In this study, seven new monoclonal antibodies (mAbs), designated as Abrin-1 through Abrin-7 have been developed. Isotyping analyses indicate these mAbs have IgG1, IgG2a, or IgG2b heavy-chains and kappa light-chains. Western blot analyses identified two abrin A-chain specific mAbs, Abrin-1 and Abrin-2, and four B-chain specific mAbs (Abrin-3, -5, -6, and -7). A sandwich enzyme-linked immunosorbent assay (ELISA), capable of detecting a mixture of abrin isoforms and agglutinins was developed using B-chain specific Abrin-3 for capture and A-chain specific Abrin-2 as detector. The ELISA is highly sensitive and detects 1 ng/mL of the abrin holotoxin in phosphate-buffered saline, nonfat milk, and whole milk, significantly below concentrations that would pose a health concern for consumers. This ELISA also detects native abrin in plant extracts with a very low background signal. The new abrin mAbs and ELISA should be useful for detecting this potent toxin in the milk supply chain and other complex matrices.

Preparation and Characterization of a Novel Monoclonal Antibody Against the Extracellular Domain of Human Transferrin Receptor.

Human transferrin receptor (hTfR1), a type II homodimeric transmembrane protein, is extensively expressed at low levels in most normal human tissues. However, the expression level of hTfR1 in some tumor tissues and the blood-brain barrier (BBB) is comparatively higher. Therefore, we developed an hTfR1 monoclonal antibody 2C9, which may be utilized to deliver drugs across the BBB through receptor-mediated endocytosis in the future. 2C9 was obtained by hybridoma cells fused by the SP2/0-Ag14 cell line and splenic B cells from hTfR1-immunized BALB/c mice. In this study, we used indirect ELISA, Western blot, and immunofluorescence to explore the characterization and application of MAb 2C9. The results showed that the affinity constant (Kaff) of the MAb 2C9 produced from ascites was 2.85 × 10-8 M and its isotype is IgG2a (Kappa chain). Above all, this report provides a more comprehensive protocol to produce monoclonal antibodies against the extracellular domain of hTfR1.

Relationship between PCNA Proliferating Marker and Angiogenesis

The goal of the study was to evaluate the prognosis significance of intratumor angiogenesis that was compared with PCNA proliferating marker, histology grade, mitotic index, tumor size, and histology type. The study had been realized using different histology types of bitch mammary tumors according to WHO classifications. There were evaluated some potential angiogenesis markers, such as intratumor microvessel density, total microvascular area and perimeter according to microscopic tumor area, and average vessel area and perimeter in different benign and malign mammary tumors. The study was realized in 8 bitch mammary tumors represented by two benign lesions (simple adenoma and fibroadenoma) and 6 carcinomas (grade I, II and III tumors). From all tumors had been harvested fragments represented by 5 mm slices that were fixed in 10% buffered formalin and proceeded by paraffin technique. The slides were stained by usual methods (HE and TM). To realize immunohistochemistry reaction paraffin embedded slices were attached onto silanized glass slides (Dako). PCNA proliferating marker (clone PC10, izotype IgG2a kappa - Dako) was evaluated by counting by 3 evaluators 8-10 high power magnified fields (400x), about 1000 cells (immunomarked nuclei). There was established average PCNA percentage for each tumor type. Intratumor angiogenesis had been established using CD31 marker (clone JC70A, izotype IgG1 kappa - Dako) and evaluated using semiautomatic image analysis method. Both immunohistochemical reactions utilized LSAB technique. To evaluate new methods necessary to establish bitch mammary tumors malignancy, which is important for mammary cancer prognosis, we utilized several malignancy markers. Also, because intratumor angiogenesis quantification provided contradictory results obtained by many authors, this parameter was related and compared with classic and reliable markers (PCNA antigen, mitotic index, histology grade, tumor size, and tumors’ characteristics).

Development of a quantitative antigen-specific cell-based ELISA for the 7G7/B6 monoclonal antibody directed toward IL-2Rα

Interleukin-2 receptor α (IL-2Rα, CD25) has been identified as a valuable target for immunotherapy. The 7G7/B6 monoclonal antibody, a mouse IgG2a kappa, recognizes an epitope of the IL-2Rα peptide, other than that identified by anti-Tac. This antibody is currently being explored for potential therapeutic and diagnostic applications. Here, we show a cell-based enzyme-linked immunosorbent assay (CbELISA) method for quantitative measurement of the binding activity of the 7G7/B6 antibody to the Kit-225-iG3 cell line expressing IL-2Rα antigen on the cell surface. The cell- and antigen-specificity of the assay was established using specific cell lines and irrelevant control antibodies. Satisfactory binding curves were demonstrated with Kit-225-iG3 cells grown between 3 and 25 passages in culture and at seed densities of 2×10 5–4×10 6 cells/well. The assay shows reproducible dose–response curves in the concentration range of 10–1000 ng/ml. The assay validation data presented here indicate that this CbELISA assay is quantitative, reproducible, robust, precise, and can be used to test the biological activity, lot to lot comparison, and stability of 7G7/B6 monoclonal antibody.

Generation of a high-producing clone of a humanized anti-B-cell lymphoma monoclonal antibody (hLL2)

LL2 is a murine immunoglobulin (Ig)G2a-kappa anti-B-cell monoclonal antibody with proven targeting and therapeutic efficacy in the management of non-Hodgkin's lymphoma (NHL). The authors had previously generated a humanized LL2 (hLL2) that demonstrated binding properties identical to those of LL2. Nevertheless, the productivity of the cell line was insufficient for large-scale production of the antibody for clinical studies. Therefore, the authors chose an amplifiable system for the generation of hLL2.The hLL2 sequences were ligated into the expression vector pdHL2, which has a dhfr amplifiable gene, and were incorporated into the SP2/0 cells by electroporation. A methotrexate (MTX) resistant clone producing hLL2 was identified. Stepwise increases in MTX concentrations, from 0.1 to 5 microM, and subcloning of the cells by limiting dilution were performed.By amplifying the dhfr and hLL2 genes with stepwise increases in the MTX concentration, the antibody production was enhanced from its original 1.4 to 70 +/- 5 mg per liter of culture media. Subsequent subcloning further improved the productivity. Immunoreactivity of the antibody was conserved, as proven by enzyme-linked immunosorbent assay and cell-binding assays. By isoelectrofocusing, the isoelectric point (pI) of the antibody was measured at approximately 9.6. The productivity of the clone was not affected by culture conditions or storage of the cells in liquid nitrogen.By means of gene amplification, the authors have generated a high-producing hLL2-IgG clone suitable for production of the quantity of antibody necessary for clinical diagnostic and therapeutic trials of NHL patients.

Generation of a high-producing clone of a humanized anti-B-cell lymphoma monoclonal antibody (hLL2).

LL2 is a murine immunoglobulin (Ig)G2a-kappa anti-B-cell monoclonal antibody with proven targeting and therapeutic efficacy in the management of non-Hodgkin's lymphoma (NHL). The authors had previously generated a humanized LL2 (hLL2) that demonstrated binding properties identical to those of LL2. Nevertheless, the productivity of the cell line was insufficient for large-scale production of the antibody for clinical studies. Therefore, the authors chose an amplifiable system for the generation of hLL2. The hLL2 sequences were ligated into the expression vector pdHL2, which has a dhfr amplifiable gene, and were incorporated into the SP2/0 cells by electroporation. A methotrexate (MTX) resistant clone producing hLL2 was identified. Stepwise increases in MTX concentrations, from 0.1 to 5 microM, and subcloning of the cells by limiting dilution were performed. By amplifying the dhfr and hLL2 genes with stepwise increases in the MTX concentration, the antibody production was enhanced from its original 1.4 to 70 +/- 5 mg per liter of culture media. Subsequent subcloning further improved the productivity. Immunoreactivity of the antibody was conserved, as proven by enzyme-linked immunosorbent assay and cell-binding assays. By isoelectrofocusing, the isoelectric point (pI) of the antibody was measured at approximately 9.6. The productivity of the clone was not affected by culture conditions or storage of the cells in liquid nitrogen. By means of gene amplification, the authors have generated a high-producing hLL2-IgG clone suitable for production of the quantity of antibody necessary for clinical diagnostic and therapeutic trials of NHL patients.

Conformation of the hypervariable region L3 without the key proline residue.

The refined structure of the Fab fragment of the monoclonal antibody CRIS-I (IgG2a kappa) against the leukocyte differentiation antigen CD5, determined at 1.9 A resolution with an agreement R-factor of 18.3%, reveals a variant of the canonical conformations proposed for the light chain complementarity determining region L3 (CDR-L3). This is the first Fab structure available with a kappa light chain in which the CDR-L3 lacks the key proline residue in either position 94 or 95. The conformation found could be significant for about 10% of the murine IgG molecules with kappa light chains without proline in their CDR-L3 sequences.

Crystallization of intact monoclonal antibodies.

Attempts were made to crystallize four monoclonal antibodies, one IgG2a kappa and three IgG1 kappa. Using a PEG 3350 screen combined with detergents, and developed from our experiments with an IgG2a kappa antibody specific for canine lymphoma cells, crystals have now been obtained of two of these four immunoglobulins, an antiphenytoin and an antiphenobarbital antibody. A complex between the antiphenobarbital antibody and its drug antigen crystallized as well. The antibody for phenytoin has, to this point, produced only clustered microcrystals, marginally suitable for X-ray analysis. Single crystals of the IgG1 kappa antibody against phenobarbital, however, were characterized by X-ray diffraction to be primitive monoclinic, with unit cell dimensions a = 67 A, b = 193 A, c = 74 A, and beta = 110 degrees. These crystals have an entire IgG1 kappa molecule as the asymmetric unit and they diffract to at least 3.2 A resolution.

Evidence for the extended helical nature of polysaccharide epitopes. The 2.8 A resolution structure and thermodynamics of ligand binding of an antigen binding fragment specific for alpha-(2-->8)-polysialic acid.

The antigen binding fragment from an IgG2a kappa murine monoclonal antibody with specificity for alpha-(2-->8)-linked sialic acid polymers has been prepared and crystallized in the absence of hapten. Crystals were grown by vapor diffusion equilibrium with 16-18% polyethylene glycol 4000 solutions. The structure was solved by molecular replacement methods and refined to a conventional R factor of 0.164 for data to 2.8 A. The binding site is observed to display a shape and distribution of charges that is complementary to that of the predicted conformation of the oligosaccharide epitope. A thermodynamic description of ligand binding has been compiled for oligosaccharides ranging in length from 9 to 41 residues, and the data for the largest ligand has been used in a novel way to estimate the size of the antigen binding site. A model of antigen binding is presented that satisfies this thermodynamic data, as well as a previously reported requirement of conformational specificity of the oligosaccharide. X-ray crystallographic and thermodynamic evidence are consistent with a binding site that accommodates at least eight sialic acid residues.

Dose-related comparison of antibody-dependent cellular cytotoxicity with chimeric and native murine monoclonal antibody 17-1A

Chimeric 17-1A antibody (IgG1 kappa) was constructed by linking variable region genes of murine monoclonal antibody 17-1A with genes for human kappa light chain and gamma 1 heavy-chain constant regions. This study was undertaken to compare in vitro antibody-dependent cellular cytotoxicity (ADCC) between the chimeric 17-1A (IgG1 kappa) and native murine 17-1A antibody (IgG2a kappa) with human peripheral blood mononuclear cells (PBMNC) against 7 human tumor (1 colon, 6 pancreas) cell lines. ADCC activity was measured by chromium-release assay. When freshly-isolated PBMNC from healthy donors were used for effector cells, significantly higher ADCC activity of chimeric antibody compared to murine antibody at optimal antibody dose (10 micrograms/mL) and lower doses (to 0.6 micrograms/mL) was observed against tumor cells with relatively high 17-1A expression. This high ADCC activity of the chimeric antibody persisted even when freshly-isolated monocyte-depleted PBMNC was used. When interleukin-2 activated PBMNC were used, comparable increases in ADCC were observed with both chimeric and murine antibody. These results suggest that chimeric 17-1A antibody is a more effective mediator of in vitro ADCC activity with human freshly-isolated PBMNC than the native murine antibody and this may be a better choice for clinical cancer trials evaluating possible immunotherapy with monoclonal antibody.

Four ricin chain A-based immunotoxins directed against the common chronic lymphocytic leukemia antigen: in vitro characterization

The common B-chronic lymphocytic leukemia (B-CLL) antigen (cCLLa) appears to be ideal for targeted immunotherapy in that it is the most prevalent and disease-restricted marker in B-CLL. To assess this potential, we developed four immunotoxins (ITs) of anti-cCLLa monoclonal antibody CLL2m (an IgG2a kappa), using ricin chain A (RTA) or its deglycosylated derivative (dgA), each conjugated to either the whole IgG molecule or its Fab' fragment. Each IT was tested in vitro for specificity and cytotoxic activity (assessed by protein synthesis inhibition [PSI] and by cell kill [CK] in the clonogenic assay) against B-CLL cells. RTA-based anti-CD5 ITs and enriched normal B and T lymphocytes were used as controls. Each IT exhibited antigen-specific, dose-dependent activity. Thus, whereas B-CLL cells exhibited dose- dependent PSI and CK (whether the B-CLL clone was CD5+ or CD5-), normal B (cCLLa-/CD5-) and T lymphocytes (cCLLa-/CD5+) remained unaffected. IT potency was independent of toxin glycosylation, but was slightly influenced by antibody valence; divalent ITs were twice as potent as monovalent ITs (IC50, 2.3 v 7.1 x 10(-11) mol/L; CK, 2.6- v 2.0-log reached with 524 v 1,072 IT molecules bound/cell, respectively). In the presence of ammonium chloride or Verapamil, IT-induced CK was enhanced 10- to 80-fold. These data suggest that the cCLLa is a promising target for IT-based immunotherapy of B-CLL in vivo and ex vivo.

Induction, via immunization with a monoclonal antiidiotypic antibody, of viral envelope specific Ab3 antisera that neutralize retrovirus infectivity.

We have developed five mouse monoclonal antiidiotypic antibodies to the 35/56 (Ab1) rat monoclonal that neutralizes retroviral infectivity by binding to the gp70f epitope of murine leukemia retrovirus. The anti-Id nature of these five Ab2s was evidenced by their inability to react with a panel of six other rat IgG2a kappa monoclonals isotype-matched to the 35/56 anti-gp70f mAb1, including two to the distinct epitopes "g" and "h" of gp70, or to normal rat IgG2a. On the basis of several competition assays four mAb2 were clearly either directed to the paratope of anti-gp70f mAb1 (.1C7, .1B, and .E) or not (.A, representing a noninternal image Ab2 alpha anti-Id). The P3E8 mAb2 was difficult to classify. Based on relative efficiency in these assays, .1C7 was chosen for further study, and upon injection was able to induce Ab3 responses in C57BL/6, BALB/c, and CBA mice. The fact that the Ab3 activity was detected in a competitive ELISA in which the hyperimmune antisera blocked the binding of Ab1 to Ab2, plus the ability to raise Ab3 neutralizing antibodies in three different mouse strains were consistent with .1C7 as an internal image Ab2 beta anti-Id. These results thus indicate the potential for internal-image monoclonal antiidiotypic antibody-based vaccines for retroviral diseases.

Characterization of a glial associated antigen GA-1 by monoclonal antibody.

A glial antigen (GA-1) was identified by monoclonal antibodies (MAb) raised against C6 rat glioma cells. MAb-7D3 (IgG2a kappa) revealed GA-1 as a single protein band with a Rf value of 0.09 by the use of basic-PAGE Western blot. SDS-PAGE Western blot and radioimmunoprecipitation (RIP) further resolved GA-1 into two subunits with a molecular weight of 200 and 78 kDa respectively. Subcellular localization by immunocytochemical staining revealed its cytosolic presence with a punctate pattern perinuclearly. Significant expression of GA-1 may be detected in 4 glioma or glial cell lines derived from rat brain. However, no expression may be detected in the rest of the 18 mammalian cell lines or primary neural cell cultures examined. All of the above data thereby suggest that GA-1 may be glial specific whereas the epitope of GA-1 defined by MAb-7D3 is species (rat) specific.

Studies on macromolecular activators of phagocytosis from platelets (MAPPs) using anti MAPP murine monoclonal antibodies

1-MAPP(290 kD) and s-MAPP(150 kD) are glycoproteins which have been shown to be involved in released products from platelets (PRPr) and to activate monocyte and neutrophil phagocytosis via the Fc receptors. Three IgG 2a (kappa) murine monoclonal antibodies against 1-MAPP and nine IgG1 (kappa) antibodies against s-MAPP have been raised. By affinity chromatography using these monoclonal antibodies, active MAPPs were obtained from the supernatant of outdated platelet concentrates in the presence of 60% saturated ammonium sulphate and from PRPr-rich medium, and substantial amounts of non-functioning immunoreactive MAPPs were obtained from the precipitate. Fab fragments of one of the two anti 1-MAPP and of two of the four anti s-MAPP monoclonal antibodies inhibited the function of their corresponding MAPPs, but none of them could inhibit the other MAPPs. 1-MAPP and s-MAPP in the platelets were visualized by indirect fluorescent antibody technique using Fab fragments of the antibodies.

T560: an (H-2b x H-2a) F1 hybrid, phosphorylcholine (PC)-binding, murine B cell lymphoma that bears receptors for IgA and IgG, presents antigen and secretes IL-4.

We describe T560, a tissue culture-adapted B lymphoma derived from the gut-associated lymphoid tissue (GALT) of a (B10 x B10.H-2a H-4b)F1 hybrid mouse. This lymphoma is interesting and useful not only because it bears an unusual IgA receptor, fully described elsewhere, but also because it is potentially capable of presenting antigen to T cells restricted by the MHC of either parent. Here we document that T560 cells are IgG2a kappa +, Ia+, B220+, J11d.2+, CD3-, CD4-, CD5-, Mac 1-, Mac 2-, non-specific esterase-. They bind bromelain-treated mouse RBC (BrMRBC) in a PC chloride-inhibitable manner but do not bind SRBC, ox RBC (ORBC) or TNP-ORBC. Two lines, T560.1 and T560.2, and several clones are available. T560.1 and its clones contain low numbers of IgA rosette-forming cells (RFC), intermediate numbers of IgG2a RFC and moderately high numbers of IgG2b RFC; T560.2 and its clones contain moderately high numbers of IgA RFC and low numbers of both IgG2a and IgG2b RFC. Both lines stimulate both B10 and B10.A cells in mixed lymphocyte reactions (MLR) and present keyhole limpet hemocyanin (KLH) to KLH-reactive T cells. T560.2 populations are, however, more efficient possibly because they have somewhat higher proportions of brightly fluorescent Ia+ cells and secrete larger quantities of lymphokine than T560.1 cells. They present PC-conjugated KLH (PC-KLH) approximately 20 times more efficiently than unconjugated KLH, suggesting that their PC binding receptors function in antigen uptake. They constitutively produce IL-1, IL-4 and IL-6, but not IL-2, IL-5 or TGF beta. Neither their IgA nor their IgG receptor expression is affected by IL-4 or by IFNs-alpha, -beta, or -gamma. In their ability to bind BrMRBC and secrete IL-4, they resemble the CH12 lymphoma but differ from it in that they are of F1 hybrid origin, are CD5-, bear IgG2a rather than IgM, do not bind sheep erythrocytes and have a receptor for IgA not present on CH12.

Sensitivity of receptors for IgA on T560, a murine B lymphoma, to phorbol myristate acetate and to phosphatidylinositol-specific phospholipase C.

A GALT-derived B lymphoma, T560, that bears IgAR is described. T560 is IgG2a kappa +, Ia+, B220+, J11d+, Thy-1-, CD3-, CD4-, CD5-, Mac 1-, Mac 2-, nonspecific esterase negative and binds bromelain-treated mouse RBC but not SRBC or ORBC. It presents antigen, secretes IL-1, IL-4 and IL-6 but not IL-2, IL-5 or TGF beta and appears to be related to the Lyt 1+(CD5) lineage of B cells though it lacks Lyt 1. T560 bears IgAR that, on the cell surface, are completely cross-inhibited by low concentrations of IgM and by high concentrations of IgG2a and IgG2b. They do not appear to represent a cell-surface form of galactosyl transferase. They are inducible by high concentrations of IgA, sensitive to trypsin and insensitive to neuraminidase. They are down-regulated by activation of PKC with PMA, but their recovery is not inhibited by cycloheximide, indicating that they are not degraded or shed. They may either lose their affinity for IgA or be internalized without degradation. Seventy percent of IgA receptor activity is lost when T560 is treated with PI-PLC; part of this loss of activity is due to activation of PKC and is inhibited by staurosporine, but approximately 30% of it is not protected by staurosporine indicating that some, or all, of the IgA receptor of T560 is connected to the cell membrane via a GPI linker. The T560 IgA receptor could be related to the poly-Ig or M cell receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on scale-up parameters of an immunoglobulin separation system using protein A affinity chromatography.

Effects of operational and system parameters on process scale-up of murine immunoglobulin (IgG2a kappa) purification using Protein A affinity chromatography are investigated. Parameters studied are those related to sample application, elution, ligand concentration on support, and column size change. Between sample application velocities of 0.004 and 0.030 cm/s (16-108 cm/h), the product concentration profiles in eluate did not show significant differences. With given system parameters, the retention time and bandwidth of the peak could be predicted by moment theory. The mean equilibrium constant during elution showed a strong effect of pH between 2 and 4. Within the range of protein A concentrations studied, 0.15-1.22 mg/mL of gel, the column capacity shows a linear relationship with the concentration of protein A immobilized. Dimensional scale-up of the column in the radial direction increased the total purification capacity linearly as the cross-sectional area increased without increasing pressure drop; however, the product concentration was diluted. Scale-up of the column dimension in the axial direction enables higher concentrations of product in the eluate, although the retention time increases linearly as the gel height increases.

Characterization of a Monoclonal Antibody to Thymidine Glycol Monophosphate

A monoclonal antibody specific for thymine glycol (TG) in irradiated or OsO4-treated DNA was obtained by immunizing with thymidine glycol monophosphate (TMP-glycol) conjugated to bovine serum albumin by a carbodiimide procedure. Screening by dot-immunobinding and enzyme-linked immunosorbant assay (ELISA) procedures gave eight clones that bound OsO4- treated DNA. One of them, 2.6F.6B.6C, an IgG2a kappa, was characterized further. Hapten inhibition studies with OsO4-treated DNA showed that the antibody was specific for TMP-glycol. Among the various inhibitors tested, inhibition was in the order TMP-glycol greater than 5,6-dihydrothymidine phosphate greater than TMP greater than thymidine glycol greater than TG. Inhibition by 5,6-dihydrothymidine, thymidine, thymine, AMP, and CMP was negligible. In OsO4-treated DNA, as few as 0.5 TG per 10,000 bp were detectable by direct ELISA. Inhibition assays could detect as few as 1.5 TG per 10,000 bp. The antibody was equally reactive with native or denatured DNA containing TG. Among the X-irradiated homopolymers dC, dA, dG, and dT, only dT reacted with the antibody. Using an ELISA, the antibody could detect damage in irradiated DNA at the level of 20 Gy. Thus the antibody is of potential use in assays for DNA damage caused by X rays or other agents that damage DNA by free radical interactions.