Studies on a trace cell lytic activity associated with alpha-lactalbumin

alpha-Lactalbumin (alpha-LA) has been examined with a new and sensitive method for determination of lysozyme activity. Samples of bovine, human, equine, and rat alpha-LA exhibited cell lytic activity, from 2 X 10(-6) to 45 X 10(-6) of the specific activity of hen eggwhite lysozyme. The activity was chromatographically inseparable from bovine and human alpha-LA. Bovine serum albumin and purified beta-lactoglobulin were inactive. The pH profiles and reaction kinetics of bovine and human alpha-LA showed differences from those of the corresponding milk lysozymes, indicating that their lytic activities were not likely to have resulted from trace lysozyme content. Thus, it appears that a weak cell lytic activity is inherent to alpha-LA.     

降钙素原的原核表达及单克隆抗体制备

目的:高效表达与纯化可溶性重组人PCT蛋白,制备高灵敏度和高特异性的抗人PCT医用诊断单克隆抗体。方法:大肠杆菌表达重组人PCT蛋白后,利用饱和硫酸铵沉淀和亲和层析方法纯化PCT蛋白后,经质谱、Western blot和间接ELISA法进行性质鉴定和分析重组蛋白的表达与免疫反应性;重组蛋白免疫小鼠,经细胞融合及筛选制备抗PCT单克隆抗体(m Ab)。结果:在大肠杆菌中高效表达了人PCT蛋白;重组人PCT蛋白具有良好的免疫反应性与免疫原性;经筛选获得7株抗PCT单克隆抗体细胞株,经ELISA鉴定,筛选抗体可与PCT抗原有良好的特异性反应。结论:利用重组人PCT蛋白免疫制备了抗人PCT单克隆抗体,为进一步研发PCT快速诊断试剂提供了原料。     

抗HLJ1单克隆抗体的制备及抗原检测方法的建立

为制备抗人肝脏DnaJ-like蛋白(Human liver DnaJ-like protein, HLJ1)的单克隆抗体, 并建立免疫组化和双抗体夹心ELISA检测HLJ1的方法。采用淋巴细胞杂交瘤技术, 获得两株能稳定分泌抗HLJ1单克隆抗体的杂交瘤细胞株A4C7和 C4C8。经鉴定, 两株单抗的亚类均为IgG1, 并且效价高、特异性好。以单抗A4C7和C4C8作为一抗, 对人胎肝组织石蜡切片进行免疫组化染色, 结果表明, 两株单抗均为阳性染色, 且HLJ1主要定位于胎肝细胞的胞浆。选取A4C7进行HRP酶标记, 并以HRP- A4C7作为酶标抗体, 以C4C8作为包被抗体, 建立双抗体夹心ELISA方法, 并进行棋盘滴定确定抗体的最佳工作浓度。该检测方法的线性范围是15~750 ng/mL, 灵敏度下限达15 ng/mL, 特异性良好。所建立的免疫组化和双抗体夹心ELISA 法可用于快速、灵敏地检测组织及血清中的HLJ1蛋白, 为HLJ1的肿瘤相关性研究提供了有力的工具。     

Immunoenzyme determination of human granulocyte-macrophage colony-stimulating factor using monoclonal antibodies

A set of seven hybridomas producing monoclonal antibodies (MAbs) to the human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was obtained. The properties of the monoclonal antibodies were characterized, and pairs of MAbs specific to different non-overlapping epitopes of GM-CSF were identified. A sensitive and simple method of two-site ELISA for GM-CSF was developed on the basis of two MAbs. According to this method, one MAb is absorbed onto a microtiter plate and another is labeled with biotin and used for the detection of GM-CSF bound to the first MAb. MAb labeled with biotin, in its turn, was visualized with the streptavidin-horseradish peroxidase conjugate. The sensitivity of this test was no less than 0.5 ng/ml, and a linear dose-response relationship was observed within a concentration interval from 0.5 to 32 ng/ml. No cross-reactivity was found with human tumor necrosis factor-alpha, granulocyte colony-stimulating factor, interleukin-2, or interleukin-3 in this test system.     

Protein radiohalogenation: observations on the design of N-succinimidyl ester acylation agents

In previous studies we have demonstrated that antibodies radioiodinated with N-succinimidyl 3-iodobenzoate (SIB) are less susceptible to loss of radioiodine in vivo than antibodies iodinated directly by electrophilic substitution on their tyrosine residues with Iodogen. Since the Bolton-Hunter reagent, N-succinimidyl 3-(4-hydroxy-3-iodophenyl)propionate, is identical with SIB except that it contains a hydroxyl group on the aromatic ring and a two-methylene spacer, a comparison of their coupling chemistry and in vivo behavior was performed to better understand the structural requirements for a useful iodinated acylation agent. Protein concentration and pH had a significant effect on the coupling efficiency of both SIB and the Bolton-Hunter reagent; however, protein-labeling yields with SIB were generally higher by a factor of 2. Paired-label biodistribution studies in mice demonstrated that thyroid uptake (a monitor of dehalogenation) of antibody labeled by the Bolton-Hunter method was twice that of antibody labeled with SIB but only 7% of that observed for antibody labeled with Iodogen. These results suggest that even minor differences in iodination site can profoundly alter the retention of label on a protein in vivo.     

A new enzyme-linked immunosorbent assay with two monoclonal antibodies to specific epitopes measures human lecithin-cholesterol acyltransferase

We established five monoclonal antibodies that reacted with human LCAT and recognized different epitopes on LCAT. These are mouse anti-human LCAT monoclonal antibodies designated 36487, 36454, 36442, 36405, and 36486, which react with the peptides corresponding to human LCAT amino acid residues R159-E179, M258-S273, S274-S294, D352-S376, and N415-E440, respectively. We also successfully used two of these antibodies to develop an ELISA, which uses a solid phase monoclonal antibody, 36486, that reacts with the C-terminus of LCAT, and a detection monoclonal antibody, 36487, that reacts with an epitope located in the center of the LCAT primary structure. We observed a significant positive correlation between the values of LCAT protein determined with ELISA and LCAT activity determined with liposome substrate (r = 0.871, P < 0.001) or the endogenous self-substrate method (r = 0.864, P < 0.001), and we obtained inter- and intra-assay coefficients of variation less than 6.1%, minimum detection limit of 0.1 microg/ml. Highly specific monoclonal antibodies will be useful in the study of the molecular pathology of LCAT. Therefore, this precise and sensitive LCAT assay will help clarify the role of this enzyme in the metabolism of HDLs, and can be used for diagnostic purposes in investigating liver function. We obtained five monoclonal antibodies that recognized different epitopes on LCAT and developed a sandwich-type ELISA. Highly specific monoclonal antibodies provide a sensitive and specific analytical system for measurements of LCAT protein.     

Properties and application to immunoassay of monoclonal antibodies to neuron-specific γγ enolase

Two monoclonal antibodies to human and bovine neuron-specific γγ enolase have been produced in the isolated hybrid cell lines, which were obtained by fusion between γγ-immunized mouse spleen cells and mouse myeloma cells (P3-NS-1/1-Ag4-1), followed by a screening procedure with an enzyme immunoassay. The monoclonal antibody to human γγ enolase (E1-G3) and that to bovine γγ enolase (B1-D6) consisted of γ2a/κ and γl/κ immunoglobulin chains, respectively. Both antibodies could bind with the respective antigen with a molar ratio of about 1:1, and were found to be specific for the γ subunit of enolase, showing reactivities with human γγ and αγ, rat γγ and αγ, and bovine γγ enolases. However, the antibodies did not cross-react with the α or β subunit of human and rat enolase isozymes. Both antibodies could partially inhibit the activity of γγ and αγ enolases. E1-G3 antibody inhibited γγ and αγ enolase activity by 70 and 30%, respectively, and B1-D6 antibody, by 90 and 40%, respectively. Both antibodies had no effect on the activity of αα and ββ enolases of human and rat origins. The applicability of E1-G3 and B1-D6 antibodies to the sandwich-type enzyme immunoassay for neuron-specific enolase (enolase γ subunit) was examined, and it was found that the assay system using E1-G3 and B1-D6 as the labeled antibodies were sufficiently sensitive for the assay of serum neuron-specific enolase concentrations.     

Studies on cell-clearing activity in alpha-lactalbumin. Effects of calcium removal on activity and conformation.

Effects of calcium removal on the cell-clearing activity of alpha-lactalbumin (alpha-LA) and concomitant changes in conformational structure have been investigated as part of a continuing study of the activity found earlier [McKenzie, H.A. & White, F.H., Jr. (1987) Biochem. Int. 14, 347]. This activity is similar to that of lysozyme, whereby lysis of the bacterial cell wall is catalyzed. However, the specific activity of alpha-LA is on the order of 10(-6) that of lysozyme. Under conditions where activities of apo and native alpha-LA were approximately linear functions of the protein concentration, the maximal ratio of apo to native activity was 5.7:1, determined by comparison of second order velocity constants. The CD spectrum of apo alpha-LA is intermediate between that of the A state and the native protein. By NMR, the conformation of apo alpha-LA is similar to, but distinctly different from, that of the native protein. The apo form did not revert completely to the native state when Ca(II) was resupplied, consistent with a role for this cation in folding. It is suggested that the activity increase may result from a diminished constriction of the "cleft" region in alpha-LA.     

Biodistribution of p-Iodobenzoyl (PIP) labeled antibodies in a murine lymphoma model

A monoclonal antibody against the murine T-cell antigen Thy 1.1 was radioiodinated using N-succin-imidyl p-iodobenzoate (PIP) in an attempt to decrease deiodination of the labeled antibody. The biodistribution of the PIP labeled antibody was compared to Iodogen labeled antibody in Thy 1.1⁺ lymphoma bearing AKR/Cum mice, where the antibody was tumor specific, and AKR/J mice where the antibody reacted with both tumor and normal T-cells. PIP labeling resulted in decreased iodine concentrations in stomach and salivary gland as compared to Iodogen labeling. There was little difference in radioiodine concentrations between the two preparations in tumor, lymphoid tissues or other organs. These results suggest deiodination of intact antibody plays little role in the clearance of radioiodinated anti-Thy 1.1 antibody from tissues.     

Biodegradation of 125I-Labeled monoclonal antibodies after intravenous administration to rats with experimental C6 glioma

Employment of radiolabeled antibodies in biological studies, allows their specific accumulation in organs and tissues to be accurately detected. However, stability of such radiolabeled antibodies depends on both the method of labeling and particular experimental conditions. Therefore, stability of labeled antibodies should be determined in every particular experiment. In this study we have investigated stability of the ¹²⁵I-labeled monoclonal antibodies to gliofibrillary acidic protein (GFAP), endothelial antigen AMVB1, and non-specific mouse IgG at the stage of their synthesis and after their intravenous administration to rats with experimental C6 glioma. Stability of labeled antibodies was determined in blood samples and homogenates of organs by the method of their precipitation with trichloroacetic acid. The ¹²⁵I-radiolabeled antibodies were characterized by high radiochemical putiry, immunochemical activity and stability of the resultant preparations in blood and tissues, and the brain after administration in vivo. Electrophoretic analysis, thin-layer chromatography, and immunohistochemical tests have demonstrated the radiochemical purity, immunochemical competence, and stability of the labeled antibodies in vivo.     

Monoclonal antibody storage conditions, and concentration effects on immunohistochemical specificity

Monoclonal antibodies against a 24,000 dalton intracellular estrogen-regulated protein in human breast cancer cells were used to study storage conditions and the effects of monoclonal antibody concentrations on immunohistochemical antigen localization. Both hybridoma supernatants and ascites fluid obtained from mice injected with hybridoma cells were used as sources of monoclonal antibodies; the monoclonal antibodies in the ascites fluid were concentrated and purified. Both antibody preparations were stored at 4, -20, or -70 degrees C and periodically tested for activity at these storage conditions. There was no difference in activity for the antibodies between storage at -20 and -70 degrees C. However, when highly diluted antibody was stored at 4 degrees C, the activity was lost within 2 weeks if carrier proteins were not added. These monoclonal antibodies were applied to immunohistochemical staining of different mouse and human tissues processed for routine paraffin sections, using the avidin-biotin-peroxidase procedure. A monoclonal antibody of unrelated specificity was used as control. When these antibodies were used at high concentrations, all the different tissues examined were immunostained. With reduction of the antibody concentration, an immunohistochemical dissection of the tissues was seen until specific immunostaining was reached. When even more highly diluted monoclonal antibody was used, heterogeneity in the staining pattern became very high. On the basis of these results, certain immunohistochemical criteria are proposed for the selection of the optimum concentration of monoclonal antibodies for specific antigen detection.     

Applications of monoclonal antibodies to human follicle-stimulating hormone in enzyme immunoassays

Monoclonal antibodies against human follicle-stimulating hormone (hFSH) were generated by using an improved hybridoma technique with a semisolid medium in methylcellulose for initial cloning. The generated monoclonal antibodies were characterized with respect to their subunit and epitope specificity as well as cross-reactivity to other glycoprotein hormones. Monoclonal antibodies of high affinity and high specificity to hFSH were finally selected for applications in sandwich enzyme immunoassay. The monoclonal antibody specific to the alpha-subunit of FSH was coated on microtiter wells and served as the first antibody. The other high-affinity monoclonal antibody specific to beta-subunit of FSH was labeled with horseradish peroxidase and served as the second antibody. This immunoassay can be performed within 70 min at room temperature and has a minimum sensitivity of 2 mIU/ml for serum sample.     

Antibody fragments labeled with fluorine-18 and gallium-68: In vivo comparison with indium-111 and iodine-125-labeled fragments

Although monoclonal antibodies have been radiolabeled with many different radionuclides, the application of positron emission tomography (PET) to the imaging of radiolabeled antibodies has been limited to the investigation of a small number of long-lived radionuclides. In this study, we labeled F(ab′)₂ fragments of a mouse monoclonal antibody (BB5-G1) specific for a human parathyroid surface antigen with the positron emitting radionuclides, gallium-68 and fluorine-18. The biodistribution of the fragments was evaluated in a nude mice model and the results were compared to those obtained with fragments labeled with iodine-125 and indium-111 using conventional labeling techniques. All labeled fragments bound to human parathyroid tissue implanted in nude mice, with parathyroid-to-muscle ratios reaching as high as 10:1, 4 h after administration. A major difference was observed in the uptake and clearance of the various labeled fragments through the kidney. The halogen activity cleared, but the metal radioactivity was retained in the kidney. The results indicate that the fluorine-18 or gallium-68 labeled fragment may be useful for parathyroid imaging with positron emission tomography.     

The hyaluronate receptor is a member of the CD44 (H-CAM) family of cell surface glycoproteins [published erratum appears in J Cell Biol 1991 Feb;112(3):following 513]

The present study was undertaken to determine the relationship between the hyaluronate receptor and CD44 (H-CAM), cell-surface glycoproteins of similar molecular weights that have been implicated in cell adhesion. In initial experiments, a panel of monoclonal antibodies directed against CD44 were tested for their ability to cross react with the hyaluronate receptor. These antibodies immunoprecipitated [3H]hyaluronate binding activity from detergent extracts of both mouse and human cells, indicating that the hyaluronate receptor is identical to CD44. In addition, one of these antibodies (KM-201 to mouse CD44) directly blocked the binding of labeled hyaluronate to the receptor and inhibited hyaluronate dependent aggregation of SV-3T3 cells. CD44 has also been implicated in lymphocyte binding to high endothelial venules during lymphocyte homing. Interestingly, the monoclonal antibody Hermes- 3, which blocks lymphocyte binding to the high endothelial venules of mucosal lymphoid tissue, had no effect on the binding of labeled hyaluronate. Furthermore, the binding of lymphocytes to high endothelial cells of lymph nodes and mucosal lymphoid tissue was not significantly affected by treatment with agents that block the binding of hyaluronate (hyaluronidase, excess hyaluronate and specific antibodies). Thus, CD44 appears to have at least two distinct functional domains, one for binding hyaluronate and another involved in interactions with mucosal high endothelial venules.     

Immunogold labeling of keratin filaments in normal human epidermal cells with two anti-keratin monoclonal antibodies

We report on application of the highly sensitive and specific immunogold labeling method for ultrastructural investigation of keratin intermediate filament antigens in human epidermal cell suspensions. Triton X-100 pretreated cells proved accessible to the colloidal gold conjugate, thus enabling keratin filament bundles to be labeled. Anti-keratin KL1 and KL2 monoclonal antibodies were raised in mice after immunization with either human stratum corneum-isolated keratins or keratins extracted from human epidermal cells suspensions, respectively. Immunoelectron microscopy confirmed immunofluorescence and immunoperoxidase results of epidermal keratinocyte staining, and revealed two different antibody reactivity patterns: KL2 reacted with keratin filaments in keratinocytes of all epidermal layers, whereas antigen to KL1 was detected only on keratin of the suprabasal layers, not on the basal keratinocyte tonofilaments. The monoclonal antibody-recognized epitopes were specific for the keratin filaments. Vimentin-rich cells (melanocytes) were not stained in the same epidermal cell suspensions. Additionally, two distinct ultrastructural patterns of keratin filament epitope labeling were observed. KL1 and KL2 monoclonal antibodies react with two different antigenic determinants, depending on the stage of keratinocyte differentiation, and may therefore be used for immunohistochemical studies of various keratin-containing cells in normal and pathologic conditions.     

An immunochemical assay model system for the sensitive detection of pyruvate dehydrogenase complex (PDHc) and its decarboxylating subunit pyruvate dehydrogenase (E1).

An immunochemical enzyme immunoassay model system was developed and compared for maximum sensitivity with a radioimmunoassay method and the classic enzyme activity method for the detection of pyruvate dehydrogenase complex (PDHc) and its decarboxylating subunit, pyruvate dehydrogenase (E1), isolated from Escherichia coli. Cross-linked large molecular weight antibody-enzyme conjugate systems are compared with heterobifunctional singular antibody conjugates substituted with high levels of horseradish peroxidase. Both polyclonal and monoclonal antibodies generated to the Escherichia coli PDHc and E1 antigens were used to develop a double-antibody sandwich microtiter plate enzyme-linked immunosorbent assay. It is demonstrated that a double sandwich immunochemical assay system can be quantitative for PDHc, can detect PDHc in crude cell lysates and has levels of sensitivity of 2.0.10(-16) mol for the detection of PDHc. This assay model system provides specific antibody selection criteria and coupling methods needed to select specific antisera that cross-react with human PDHc. This rapid and sensitive immunochemical assay method clearly demonstrates that sensitive mass assay systems can be developed for the detection of PDHc. Different from Western blot, this methodology could be used to generate mass assays which could be applied to the rapid detection of mammalian antigens (employing the corresponding antibodies) implicated in a number of pyruvate dehydrogenase deficiencies associated with human disorders.     

Stability of the molten globule state of a domain-exchanged chimeric protein between human and bovine alpha-lactalbumins

A domain-exchanged chimeric alpha-lactalbumin (alpha-LA), which consisted of the alpha-domain of human alpha-LA and the beta-domain of bovine alpha-LA, was constructed. Like native alpha-LA, the chimeric protein was in a molten globule state in the absence of Ca(2+) at neutral pH and low salt concentration. The stability of the molten globule state of the constructed chimeric protein was identical to that of the recombinant human protein and was higher than that of the recombinant bovine protein. The stability of the molten globule state of alpha-LA is defined by the stability of the alpha-domain.     

Zinc binding in bovine alpha-lactalbumin: sequence homology may not be a predictor of subtle functional features

alpha-Lactalbumin (alpha-LA), a calcium-binding protein, also possesses zinc-binding sites comprising a single strong site and several weaker secondary sites. The only site found by X-ray crystallography (Ren et. al., J. Biol. Chem. 1993;268:19292) was Glu 49 of human alpha-LA, but zinc binding had never been measured in solution for human alpha-LA. This residue was genetically substituted by Ala in bovine alpha-LA and the metal-binding properties of the resulting desMetE49A protein were compared with those for native alpha-LA by fluorescence methods. Surprisingly, desMetE49A alpha-LA and the native bovine protein had similar affinities for both Zn(2+) and Ca(2+). Genetic substitution of other possible candidates for Zn(2+) chelating residues, which included Glu 25, did not alter the affinity of bovine alpha-LA to Zn2+; however, substitution of Glu 1 by Met resulted in the disappearance of strong Zn(2+) binding. A proposed site involves Glu 1, Glu 7, Asp 11, and Asp 37, which would participate in strong Zn(2+) binding based on their propinquity to Glu 1. Human alpha-LA, which has a Lys at position 1 rather than Glu, binds zinc with a reduced affinity compared with native bovine alpha-LA, suggesting that the site identified from the X-ray structure did not correspond to strong zinc binding in solution.     

An alternate form of Ku80 is required for DNA end-binding activity in mammalian mitochondria

Mammalian mitochondrial DNA end-binding activity is nearly indistinguishable from that of nuclear Ku. This observation led to the hypothesis that mitochondrial DNA end-binding activity is in part dependent upon Ku80 gene expression. To test this hypothesis, we assayed for Ku activity in mitochondrial extracts prepared from the xrs-5 hamster cell line that lacks Ku80 mRNA expression. Mitochondrial protein extracts prepared from this cell line lacked the DNA end-binding activity found in similar extracts prepared from wild-type cells. Azacytidine-reverted xrs-5 cells that acquired nuclear DNA end-binding activity also acquired mitochondrial DNA end-binding activity. Western blot analysis of human mitochondrial protein extracts using a monoclonal antibody specific for an N-terminal epitope of Ku80 identified a protein with an apparent molecular weight of 68 kDa. This mitochondrial protein was not detected by a monoclonal antibody specific for an epitope at the C-terminal end of Ku80. Consistently, while both the N- and C-terminal Ku80 monoclonal antibodies supershifted the nuclear DNA end-binding complex on an electrophoretic mobility shift assay, only the N-terminal monoclonal antibody supershifted the mitochondrial DNA end-binding complex. To confirm that the 68 kDa Ku protein was not a consequence of nuclear protein contamination of mitochondrial preparations, highly purified intact nuclei and mitochondria were treated with proteinase K which traverses the pores of intact nuclei but gains limited access into intact mitochondria. Ku80 in purified intact nuclei was sensitive to treatment with this protease, while the 68 kDa Ku protein characteristic of purified intact mitochondria was resistant. Further, immunocytochemical analysis revealed the co-localization of the N-terminal specific Ku80 monoclonal antibody with a mitochondrial-targeted green fluorescence protein. Mitochondrial localization of the C-terminal Ku80 monoclonal antibody was not observed. These data are consistent with the hypothesis that a C-terminally truncated form of Ku80 is localized in mammalian mitochondria where it functions in a DNA end-binding activity.     

Effect of conditions of monoclonal antibody adsorption on antigen-binding activity

The dependence of the antigen-binding activity of immobilized antibodies on pH of a saturating buffer has been investigated. We analyzed 28 monoclonal antibodies (MCAs) produced by various hybridomas to three virus antigens, i.e., the nuclear p23 protein of hepatitis C virus (C core protein p23), p24 protein of HIV 1, and the surface antigen of hepatitis B virus (HBsAg). Antibodies were adsorbed on the surfaces of immune plates in acidic (pH 2.8), neutral (pH 7.5), and alkaline (pH 9.5) buffers. The binding of labeled antigens, i.e., biotinylated or conjugated with horseradish peroxidase, with immobilized antigens was tested. It was shown that 10 out of 28 analyzed MCAs (36%) considerably better preserved their antigen-binding activity if their passive adsorption was carried out on the surface of polystyrene plates in an acidic buffer (pH 2.8). This approach allowed constructing a highly sensitive sandwich method for HBsAg assay with a minimal reliably determined antigen concentration of 0.013–0.017 ng/ml. The described approach may be recommended for the optimization of sandwich methods and solid-phase competitive methods.