Glutaraldehyde (GA)-hapten adducts, but without a carrier protein, for use in a specificity study on an antibody against a GA-conjugated hapten compound: histamine monoclonal antibody (AHA-2) as a model

In our recent study on monoclonal antibodies (mAbs AHA-1-5) against glutaraldehyde (GA)-conjugated histamine (HA), we identified one mAb (AHA-2) which can detect neuronal HA in the rat brain with an immunocytochemistry method (ICC) [Fujiwara et al. (1999) J. Biochem. 126, 503-509]. In the present study the specificity of AHA-2 mAb for use for ICC has been examined by means of competitive experiments involving HA and analogs, all of which had been allowed to react with GA followed by sodium borohydride, but not allowed to couple with the carrier protein. It was demonstrated that the antibody distinguished alterations in the chemical structure of the molecule, showing decreased immunoreactivity with all the GA-adducts of (R)-(-)-alpha-methylhistamine, 1- and 3-methylhistamine, L-histidine, and 1- and 3-methyl-L-histidine. On the other hand, AHA-1 mAb only reacted with GA-adducts of 3-MeHA (3-MeHA-GA) and HA (HA-GA), to almost the same degree, in relatively high concentration ranges. AHA-3, 4, and 5 mAbs reacted about 10-times more strongly with 1-MeHA-GA than with HA-GA, but reacted very little or not at all with the other analogs. These results may suggest that AHA-2 mAb recognized both the non-substituted imidazole and alpha-methine groups of a HA molecule in addition to the conjugation site of GA including the part(s) reduced with NaBH(4), and especially the imidazole group more strictly than the other mAbs. This may partly explain why AHA-2, among the five AHA mAbs, can detect neuronal HA with an ICC method. The present ELISA method for GA-hapten adducts should be applicable to other antibodies against GA-conjugated biologically active amines or amino acids, thus allowing the study of antibody specificity for ICC more easily and accurately than was previously possible with hapten-protein conjugates as antigens.     

Monoclonal antibodies against glutaraldehyde-conjugated histamine: application to immunocytochemistry

 We have developed mouse monoclonal antibodies (AHA-1–5, all IgG₁ sub-isotype mAbs) against histamine (HA) conjugated to bovine serum albumin using glutaraldehyde-NaBH₄. Among these, AHA-1 mAb was found to be the most useful for HA immunocytochemistry (ICC) in terms of specificity and sensitivity without non-specific immunobinding. AHA-1 was demonstrated to be specific to HA with an enzyme-linked immunosorbent assay (ELISA) binding test, simulating the ICC of tissue sections, and not reactive to any of the other amino acids and peptides with N-terminal histidine tested. By use of this antibody, indirect immunoperoxidase staining was observed in rat stomach fixed with glutaraldehyde (GA) in combination with NaBH₄ reduction. In contrast, no immunoreactivity was seen in tissue fixed only with GA. Absorption controls indicated that the immunostaining could be completely inhibited by GA-conjugated HA, which was consistent with the results of an ELISA inhibition test. No cross-reactivity occurred with other GA-conjugated amino acids. ICC staining was dense in the cytoplasm of gastric enterochromaffin-like cells and very weak in mast cells. A new finding was that staining was noticed in some cell bands of the intermediate layer between the stratum lucidum and the stratum corneum of the stratified squamous epithelium of the gastric cardia, esophagus, tongue, and skin in rats. The results strongly suggest that the monoclonal antibody allowed highly specific detection of HA in animal tis- sues. Accepted: 27 August 1996     

Connexons and cell adhesion: a romantic phase

Recent evidence indicates, that gap junction forming proteins do not only contribute to intercellular communication (Kanno and Saffitz in Cardiovasc Pathol 10:169-177, 2001; Saez et al. in Physiol Rev 83:1359-1400, 2003), ion homeostasis and volume control (Goldberg et al. in J Biol Chem 277:36725-36730, 2002; Saez et al. in Physiol Rev 83:1359-1400, 2003). They also serve biological functions in a mechanical sense, supporting adherent connections between neighbouring cells of epithelial and non-epithelial tissues (Clair et al. in Exp Cell Res 314:1250-1265, 2008; Shaw et al. in Cell 128:547-560, 2007), where they stabilize migratory pathways in the developing central nervous system (Elias et al. in Nature 448:901-907, 2007; Malatesta et al. in Development 127:5253-5263, 2000; Noctor et al. in Nature 409:714-720, 2001; Rakic in Brain Res 33:471-476, 1971; J Comp Neurol 145:61-83 1972; Science 241:170-176, 1988), or mediate polarized movements and directionality of neural crest cells during organogenesis (Kirby and Waldo in Circ Res 77:211-215, 1995; Xu et al. in Development 133:3629-3639, 2006). Since, most data describing adhesive properties of gap junctions delt with connexin 43 (Cx43) (Beardslee et al. in Circ Res 83:629-635, 1998), we will focus our brief review on this isoform.     

Immunoelectron microscopy study of polyamines using a newly prepared monoclonal antibody against spermidine: use of a mixture of glutaraldehyde and paraformaldehyde as a cross-linking agent in the preparation of the antigen

We developed a mouse monoclonal antibody (ASPD-19, IgG3 sub-isotype mAb) against spermidine (Spd) conjugated to bovine serum albumin (BSA) using a mixture of glutaraldehyde (GA) and paraformaldehyde (PFA)-sodium borohydride for applications in immunoelectron microscopic studies. The antibody specificity was evaluated by an ELISA binding test simulating the immunocytochemistry (ICC) of tissue sections. The ASPD-19 mAb is highly specific for Spd and Spm, almost the same degree to each, and can distinguish alterations in the chemical structure of other polyamine (PA) analogs, showing less than 3.2% cross-reaction with N(1)-acetylspermidine, acetylspermine, or N(8)-acetylspermidine. By an indirect immunoperoxidase method using the ASPD-19 mAb, PA-like immunoreactivities were observed in different tissues fixed with Karnovsky fixative (a mixture of GA and PFA) in combination with borohydride reduction. In contrast, immunoreactivity was very low in tissues when the borohydride reduction step was omitted. The PA-like immunoreaction was completely abolished by the adsorption of the ASPD-19 mAb with 100 microg/ml of Spd or Spm, but was inhibited little or none by other PA-related compounds or amino acids. A light microscopic ICC method using ASPD-19 produced immunostaining of PAs in certain cells in rat tissues with high biosynthetic activities (small intestine, pancreas and spinal cord). A pre-embedding immunoelectron microscopic study using rat spinal cord showed PA immunoreactivity located predominantly on free (polysomes) and attached ribosomes of the rough endoplasmic reticulum (Nissl bodies) in the cytoplasm of motor neurons. These results are in complete agreement with the results obtained by our recent ICC method using another mAb (ASPM-29) produced against GA-conjugated Spm.     

Sensitivity in horseradish peroxidase neurohistochemistry: a comparative and quantitative study of nine methods.

Nine currently available methods for HRP neurohistochemistry have been compared with each other on matching tissue sections from four rats and four rhesus monkeys. The nine methods investigated in this report are the diaminobenzidine (DAB) procedures of LaVail JH and LaVail MM (J Comp Neurol 157:303, 1974), of Adams JC (Neuroscience 2:141, 1977) and of Streit P and Reubi JC (Brain Res 126:530, 1977); the benzidine dihydrochloride (BDHC) procedures of Mesulam M-M (J Histochem Cytochem 24:1273, 1976) and of De Olmos J and Heimer L (Neurosci Lett 6:107, 1977); the o-dianisidine (O-D) procedure of De Olmos J (Exp Brain Res 29:541, 1977); the p-phenylenediamine dihydrochloride and pyrocatechol (PPD-PC) procedure of Hanker JS et al., (Histochem J 9:789, 1977) and the tetramethyl benzidine (TMB) procedures of Mesulam M-M (J Histochem Cytochem 26:106, 1978) and of De Olmos J et al. (J Comp Neurol 181:213, 1978). Quantitative comparisons were based on counts of retrogradely labeled perikarya. The extent of anterograde transport and the size of the injection site were also compared at a more qualitative level. The results indicate that one TMB procedure (Mesulam M-M, J Histochem Cytochem 26:106, 1978) is distinctly superior to each of the other eight procedures in the number of labeled perikarya that it can demonstrate. Furthermore, these differences are statistically significant at better than the 0.05 level of confidence. Differences in sensitivity are most evident when the perikarya contain small quantities of transported HRP. The same TMB method also demonstrates more anterograde transport and a larger injection site than all the other procedures. If less sensitive procedures are employed, afferent or efferent connections that are clearly demonstrated by this TMB procedure are either underestimated or completely overlooked. It is suggested that sensitivity in HRP neurohistochemistry is determined by multiple factors which include the method of fixation, post-fixation storage, the choice of chromogen, the incubation parameters, the type of HRP enzyme that is administered, and the postreaction treatment.     

Restriction fragment length polymorphism (RFLP) analysis provides evidence for a high degree of homology of mitochondrial DNAs from rat hepatomasVersus normal rat livers

Where differences have been reported between tumor and normal mitochondrial DNA (mtDNA), they have generally involved limited modifications of the genome (Taira et al., Nucleic Acids Res. 11:1635, 1983; Shay and Werbin, Mutat. Res. 186:149, 1987). However, Corral et al. (Nucleic Acids Res. 16:10935, 1988; 17:5191, 1989) observed recombination between cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 6 (ND6), two genes normally on opposite sides of the circular mitochondrial genome. In rat hepatoma mtDNA COI and ND6 were reported to be separated by only 230 base pairs (Corral et al., 1988, 1989). We have performed RFLP analysis on mtDNA from normal rat livers and rat hepatomas, using COI and ND6 probes. Additional experiments compared end-labeled DNA fragments produced by EcoRI and HindIII digestion of mtDNA. These studies failed to provide any evidence for genetic recombination in rat hepatoma mtDNA, even in the same cell line used by Corral et al. Rather, they support the conclusion that mtDNA from tumor and normal tissues exhibits a low degree of heterogeneity.     

Heterogeneous expression of glutamine synthetase mRNA in rat liver parenchyma revealed by in situ hybridization and Northern blot analysis of RNA from periportal and perivenous hepatocytes

Using radiolabeled specific cDNA glutamine synthetase mRNA could be detected by in situ hybridization exclusively within those few perivenous hepatocytes which stained immunocytochemically for glutamine synthetase. This localization of glutamine synthetase mRNA was recently reported by Moorman et al. [(1988) J. Histochem. Cytochem. 36, 751-755]. Biotinylated cDNA was not suitable for mRNA detection because of a very high background staining under the conditions of in situ hybridization. Dot blot and Northern blot analysis of RNA isolated from periportal and perivenous subfractions of hepatocytes also demonstrated the exclusive perivenous localization of two hybridizable glutamine synthetase mRNAs of length 2.8 and 1.6 kilobases. These results indicate that the unique heterogeneity of glutamine synthetase in rat liver parenchyma is controlled at the pretranslational level.     

Construction and characterization of single-chain antibodies against human insulin-like growth factor-I receptor from hybridomas producing 1H7 or 3B7 monoclonal antibody

Recombinant antibody consisting of the single-chain variable fragment (scFv) of 1H7 monoclonal antibody against insulin-like growth factor-I receptor (IGF-IR) and human IgG(1) Fc domain, scFv-Fc, has been found to exhibit inhibitory effects on breast cancer growth in vitro and in vivo [Li et al. (2000) Cancer Immunol. Immunother. 49, 243; Sachdev et al. (2003) Cancer Res. 63, 627]. Various types of scFvs from hybridomas producing 1H7 or 3B7 mAb were constructed using conventional phage display technology to further characterize the specificity and affinity of anti-IGF-IR mAbs. Binding studies performed using either phage antibodies or soluble scFv proteins to IGF-IR or insulin receptor (IR) and IGF-IR pre-incubated with mAbs suggested that (i) 1H7 and 3B7 bind to IGF-IR but do not bind to its structurally related IR, (ii) either the VL-VH or VH-VL sequence order does not apparently affect specificity for IGF-IR and (iii) 1H7 and 3B7 bind the independent epitopes, located in or near the N-terminal (440-514) and C-terminal (62-184) domains of the alpha subunit, respectively. This study not only revealed new information on binding regions for two anti-IGF-IR mAbs, but also provided the scFv genes as tools for further manipulation of the affinity or development of new IGF-IR-targeted cancer therapeutics.     

Conformational and sequential epitopes on the human granulocyte-colony stimulating factor molecule (hG-CSF) and their role in binding to human placenta receptors.

Monoclonal antibodies (mAbs) named 8C2 and 6E3, directed against the recombinant human granulocyte colony-stimulating factor (hG-CSF), were used as probes to study the cytokine orientation on its binding to receptors from human placenta. Competition enzyme linked immunoabsorbent assays (ELISA) revealed that mAb 8C2 would be directed to a linear epitope, whereas mAb 6E3 would delimit a more assembled epitope. Gel-filtration high performance liquid chromatography (HPLC) of the immune complexes formed by incubating [(125)I]hG-CSF with each mAb showed that epitope 8C2, but not 6E3, was altered after cytokine iodination. In addition, mAb 6E3 completely inhibited [(125)I]hG-CSF binding to human placental microsomes. Although [(125)I]mAb 6E3 was unable to bind to preformed hG-CSF-receptor complexes, [(125)I]mAb 8C2 did recognize hG-CSF previously bound to receptors, suggesting that epitope 8C2 would remain accessible in the hG-CSF-receptor complex. To identify the cytokine region defined by mAbs, hG-CSF was digested with different proteolytic enzymes: Arg-C, Glu-C, trypsin and alpha chymotrypsin. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation. Results showed that mAb 6E3 would be directed to a conformation-dependent epitope located close to the hG-CSF binding domain and included into the sequence 1-122/123, whereas mAb 8C2 recognized the region 41-58, which represents a linear epitope left exposed after cytokine binding to receptors from human placenta.     

Comparative studies of the antigens recognized by sperm-immobilizing monoclonal antibodies.

Characteristic properties of the antigens recognized by sperm-immobilizing monoclonal antibodies (SI-mAbs) from different sources were compared by ELISA competitive inhibition assay, Western blot analysis, chromatographic analysis, and enzymatic digestion studies. Among 9 SI-mAbs, human mAb H6-3C4 and three mouse mAbs--2C6, 2B6, and 2E5--also possessed strong sperm-agglutinating activity. Binding of human mAb H6-3C4 to sperm was strongly inhibited by the three mouse mAbs (2C6, 2B6, and 2E5), but not by the rat or the other four mouse mAbs. SDS-PAGE revealed that mAb H6-3C4 and three mouse mAbs recognized the same antigen molecules of 15-25 kDa present in both sperm extracts and seminal plasma. Chemical treatments with trifluoromethanesulfonic acid and sodium metaperiodate destroyed the antigen determinants recognized by the above four mAbs, as detected by both ELISA and antibody absorption tests. Western blot analysis revealed that the antigens were susceptible to treatments with papain, proteinase K, and N-glycanase, but resistant to trypsin, V8 protease, and thermolysin. These results indicate that one of the major antigens recognized by mAbs with sperm-immobilizing action may be a sperm membrane-associated glycoprotein of 15-25 kDa and the epitope may involve N-linked oligosaccharides.     

Monoclonal antibodies to different epitopes on a cell-surface enzyme, human placental alkaline phosphatase, effect different patterns of labeling with protein A-colloidal gold

Two monoclonal antibodies (mAbs) to different epitopes on human placental alkaline phosphatase (PLAP), both of the immunoglobulin G2a heavy-chain class and having similar affinities for PLAP, were compared for their ability to label the enzyme on the HeLa cell surface. In one type of experiment employing [125I]-labeled mAbs, the results demonstrated quantitative differences in binding of the mAbs to the cells. At saturating levels, the number of molecules of mAb E5 bound to the cells was almost eight times the number of mAb B10 molecules bound. In another type of experiment, mAbs were indirectly visualized on the cell surface using protein A tagged with colloidal gold particles in transmission electron microscopy. Only one of the antibodies (E5) displayed a clustered distribution of PLAP that previously had been observed with rabbit polyclonal antibodies and goat anti-rabbit IgG-labeled gold (J Histochem Cytochem 33:1227, 1985). The other antibody (B10) showed less frequent and more scattered labeling; three to four times more gold particles were visualized in each cluster on cells bound by mAb E5 compared to cells bound by B10. These results are consistent with the idea that not all epitopes on a membrane-bound antigen may be equally accessible for antibody binding. Even identical epitopes on different PLAP molecules are not equally hindered by other membrane components, since at least some of the PLAP molecules are labeled by the more sterically hindered mAb B10. Quantification of the number of gold particles employing the more abundantly bound mAb E5 provides an average estimate of seven to eight molecules of PLAP in each cluster. Because of inefficiencies in labeling, however, this value is probably lower than the real number.     

Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display

Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat. Thus, understanding mechanisms of antibody-mediated viral inhibition and neutralization escape is critical. Here, a robust yeast display system for fine epitope mapping of viral surface hemagglutinin (HA)-specific antibodies is demonstrated. The full-length H5 subtype HA (HA0) was expressed on the yeast surface in a correctly folded conformation, determined by binding of a panel of extensively characterized neutralizing human monoclonal antibodies (mAbs). These mAbs target conformationally-dependent epitopes of influenza A HA, which are highly conserved across H5 clades and group 1 serotypes. By separately displaying HA1 and HA2 subunits on yeast, domain mapping of two anti-H5 mAbs, NR2728 and H5-2A, localized their epitopes to HA1. These anti-H5 mAb epitopes were further fine mapped by using a library of yeast-displayed HA1 mutants and selecting for loss of binding without prior knowledge of potential contact residues. By overlaying key mutant residues that impacted binding onto a crystal structure of HA, the NR2728 mAb was found to interact with a fully surface-exposed contiguous patch of residues at the receptor binding site (RBS), giving insight into the mechanism underlying its potent inhibition of virus binding. The non-neutralizing H5-2A mAb was similarly mapped to a highly conserved H5 strain-specific but poorly accessible location on a loop at the trimer HA interface. These data further augment our toolchest for studying HA antigenicity, epitope diversity and accessibility in response to natural and experimental influenza infection and vaccines.     

Passive immunization against oral AIDS virus transmission: An approach to prevent mother‐to‐infant HIV‐1 transmission?

To develop immunoprophylaxis regimens against mother-to-child human immunodeficiency virus type 1 (HIV-1) transmission, we established a simian-human immunodeficiency virus (SHIV) model in neonatal macaques that mimics intrapartum mucosal virus exposure (T.W. Baba, J. Koch, E.S. Mittler et al: AIDS Res Hum Retroviruses 10:351-357, 1994). We protected four neonates from oral SHIV-vpu+ challenge by ante- and postpartum treatment with a synergistic triple combination of immunoglobulin (Ig) G1 human anti-HIV-1 neutralizing monoclonal antibodies (mAbs) (T.W. Baba, V. Liska, R. Hofmann-Lehmann et al: Nature Med 6:200-206, 2000), which recognize the CD4-binding site of Env, a glycosylation-dependent gp120, or a linear gp41 epitope. Two neonates that received only postpartum mAbs were also protected from oral SHIV-vpu+ challenge, indicating that postpartum treatment alone is sufficient. Next, we evaluated a similar mAb combination against SHIV89.6P, which encodes env of primary HIV89.6. One of four mAb-treated neonates was protected from infection and two maintained normal CD4+ T-cell counts. We conclude that the epitopes recognized by the three mAbs are important determinants for achieving protection. Combination immunoprophylaxis with synergistic mAbs seems promising to prevent maternal HIV-1 transmission in humans.     

A monoclonal antibody against the glutaraldehyde-conjugated polyamine, putrescine: application to immunocytochemistry

We developed a mouse monoclonal antibody (mAb; APUT-32, IgG1 subisotype mAb) against putrescine (Put) conjugated to bovine serum albumin using a glutaraldehyde (GA)-sodium borohydride procedure, for applications in immunocytochemistry (ICC). The antibody specificity was evaluated by an ELISA binding test, simulating the ICC of tissue sections. APUT-32 mAb was highly specific to Put, and distinguished alterations in the chemical structure of other polyamine (PA) analogs, showing 3.8% crossreaction with cadaverine, 3.3% with spermidine (Spd), and 2.3% with 1,3-diaminopropane. Comparable results in immunoreactivity of APUT-32 mAb were obtained with the ELISA inhibition test. By the indirect immunoperoxidase method using the APUT-32 mAb, Put-like immunoreactivities were observed in the cytoplasm of HeLa and MCF-7 cell lines fixed with GA in combination with NaBH4 reduction, but almost no immunoreaction was seen in the cytoplasm of the human melanoma BD cell line. On the other hand, the same method but using a previously prepared ASPM-29 mAb, specific for spermine (Spm) and Spd, produced intense immunostaining in the cytoplasm of all the three cell types. The Put-like immunoreaction was completely abolished by absorption of the APUT-32 mAb with 10 microg/ml Put-human serum albumin conjugate prepared using GA and NaBH4. HPLC analysis was also performed for the levels of each of the PAs in the three types of cell, showing that the levels of Put detected were much lower than those of Spm and Spd, and were strikingly different in the three cell lines among which the human melanoma BD cell line contained the lowest levels of Put. These results strongly suggest that APUT-32 mAb reacts specifically with Put in the tumor cells and therefore has the potential as a new tool for elucidating the biological roles of Put in cells and tissues.     

Identification of amino acids of influenza virus HA responsible for resistance to a fusion inhibitor, Stachyflin

We have recently described a novel hemagglutinin (HA) conformational change inhibitor of human influenza virus, Stachyflin (Yoshimoto et al, Arch. Virol., 144, 1-14, 1999). Stachyflin-resistant variants of human influenza A/WSN/33 (H1N1) virus were isolated in vitro and the nucleotide sequences of their HA genes were determined. The relation of amino acid substitutions and Stachyflin resistance was analyzed with in vitro membrane fusion between HA-expressing cells and octadecylrhodamine (R18)-labelled chick erythrocytes (RBC). The amino acid substitutions, lysine to arginine at position 51 or lysine to glutamic acid at position 121 of the HA2 subunit of the HA protein was enough to confer a Stachyflin-resistant phenotype of HA protein. The molecular mechanism of anti-HA conformational change activity of Stachyflin is discussed.     

Photofootprinting of drug-binding sites on DNA using diazo- and azido-9-aminoacridine derivatives

It is demonstrated that DNA photofootprinting analysis of the intercalating depsipeptide echinomycin, and the minor groove-binders distamicyn, 4',6-diamidino-2-phenylindole (DAPI) and Hoechst 33258 can be performed using 9-[6-(2-diazocyclopentadienylcarbonyloxy)hexylamino]acridine (DHA) [Nielsen et al. (1988) Nucleic Acids Res. 16, 3877-3888] or 2-methoxy-6-azido-9-aminoacridine (MAA) [Jeppesen et al. (1988) Nucleic Acids Res. 16, 5755-5770]. Both the extent of the drug-binding sites and their relative strength can be determined with either reagent. DNA has the advantage of giving virtually sequence-uniform DNA photocleavage. On the other hand, structural changes in the DNA are detected by MAA. Using the 232-base-pair EcoRI-PvuII pUC19 restriction fragment, it is found that cleavage protection by distamycin, DAPI and Hoechst 33258 all require an (A.T)4 sequence, whereas protection by echinomycin was confined to a G + C-rich 8-base-pair region.     

Characterization of the anti-DnaJ monoclonal antibodies and their use to compare immunological properties of DnaJ and its human homologue HDJ-1

Escherichia coli DnaJ (Hsp40) is suspected to participate in rheumatoid arthritis (RA) pathogenesis in humans by an autoimmune process. In this work a set of 6 anti-DnaJ monoclonal antibodies (mAbs) was raised and localization of the epitopes recognized by the mAbs was investigated. Western blotting and enzyme-linked immunosorbent assay (ELISA) experiments showed that the mAbs efficiently bound only native antigen. Using DnaJ mutant proteins with deletions of specified domains and ELISA, we found that AC11 mAb reacted with the best conserved in evolution N-terminal J domain, whereas BB3, EE11, CC5, CC8, and DC7 bound to the C-terminal part after residue 200. Mapping performed with the use of a random peptide library displayed by filamentous phage indicated that (1) AC11 mAb bound to a region between residues 33-48, including D-34 which belongs to the HPD triad, present in all DnaJ homologues, (2) BB3 recognized residues localized in the 204-224 region, (3) EE11 recognized the 291-309 region, (4) CC5--the region 326-359, and (5) CC8--the 346-366 region. All these mAbs, as well as the polyclonal antibodies against the N- or C-terminal domain, bound efficiently to HDJ-1, human Hsp40. These results show the presence of a significant immunological similarity between bacterial DnaJ and human HDJ-1, which is not restricted to the evolutionarily conserved parts of the proteins, and suggest that HDJ-1 could be a possible target of immune response triggered by DnaJ.     

Enzyme-linked immunosorbent assay of changes in serum levels of growth hormone (cGH) in common carps (Cyprinus carpio)

The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1-14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1-6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1-7, 9 and 10) were used in fluorescent staining and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions.     

N-succinimidyl 3-[(131)I]iodo-4-phosphonomethylbenzoate ([(131)I]SIPMB), a negatively charged substituent-bearing acylation agent for the radioiodination of peptides and mAbs

An important criterion in design of acylation agents for the radioiodination of internalizing monoclonal antibodies (mAbs) is to maximize the retention of radioiodine in the tumor following mAb intracellular processing. We have previously shown that labeling methods that generate positively charged catabolites have enhanced tumor retention. Herein we have extended this strategy to investigate the potential utility of labeling internalizing mAbs with an acylation agent that yielded labeled catabolites that would be negatively charged at lysosomal pH. The negatively charged acylation agent, N-succinimidyl 3-[(131)I]iodo-4-phosphonomethylbenzoate ([(131)I]SIPMB), was prepared from its tin precursor, N-succinimidyl 4-di-tert-butylphosphonomethyl-3-trimethylstannylbenzoate (tBu-SPMTB), in 40% radiochemical yield. The free acid, 3-[(131)I]iodo-4-phosphonomethylbenzoic acid ([(131)I]IPMBA), was also prepared from the corresponding precursor, 4-di-tert-butylphosphonomethyl-3-trimethylstannylbenzoic acid (tBu-PMTBA), in 80% radiochemical yield. The rapidly internalizing mAb L8A4 was conjugated to [(131)I]SIPMB in 25-40% yield with preservation of its immunoreactivity. Internalization and processing in the U87DeltaEGFR glioma cell line was studied in a paired label format with L8A4 labeled with (125)I using the Iodogen method. Retention of initially bound radioactivity in these cells at 24 h from [(131)I]SIPMB-labeled mAb was approximately 6-fold higher than that for directly labeled mAb. Catabolite analysis demonstrated that this difference reflected an order of magnitude higher retention of low molecular weight species in these cells. The [(131)I]SIPMB-L8A4 conjugate was intact over the first 2 h; thereafter, lysine-[(131)I]SIPMB was the predominant catabolite. In contrast, L8A4 labeled using Iodogen rapidly gave rise to mono-[(125)I]iodotyrosine within 2 h, which then cleared rapidly from the cells. These results suggest that SIPMB could be a potent candidate for labeling internalizing mAbs and warrant further study.     

Expression of recombinant anti‐colorectal cancer large single‐chain monoclonal antibody in insect cells

Colorectal cancer is the third most commonly diagnosed cancer in the world. Monoclonal antibody (mAb) CO17‐1A recognizes the tumor‐associated antigen GA733‐2, a cell surface glycoprotein highly expressed in colorectal carcinoma cells which is applicable for preventing and curing colorectal cancer. In this study, we tried to produce a new recombinant anti‐colorectal cancer large single chain (lsc) mAb based on mAb CO17‐1A in the baculovirus‐insect cell protein expression system. Two kinds of recombinant lsc mAbs were generated where variable light chain (VL) and heavy chain (HC) of mAb CO17‐1A were fused together by an interchain linker. The only difference between the two mAbs is based on fusion of an ER retention signal (KDEL) at its C‐terminus of HC. Polymerase chain reaction analysis verified the presence of both recombinant genes in the bacmid for generating viral expression vectors in insect cells. Western blot confirmed the expression of lsc mAbs in baculovirus‐infected insect cells. Cell enzyme linked immunosorbent assay (ELISA) showed that the mAbs from cell lysates bound to SW480 and SW620 human colorectal cancer cells. These results indicate that the baculovirus insect expression system can produce anti‐colorectal lsc mAb recognizing human colorectal cancer cells.