Detection of high CD44 expression in oral cancers using the novel monoclonal antibody,C44Mab-5

CD44 is a transmembrane glycoprotein that regulates a variety of genes related to cell-adhesion, migration, proliferation, differentiation, and survival. A large number of alternative splicing isoforms of CD44, containing various combinations of alternative exons, have been reported. CD44 standard (CD44s), which lacks variant exons, is widely expressed on the surface of most tissues and all hematopoietic cells. In contrast, CD44 variant isoforms show tissue-specific expression patterns and have been extensively studied as both prognostic markers and therapeutic targets in cancer and other diseases. In this study, we immunized mice with CHO-K1 cell lines overexpressing CD44v3-10 to obtain novel anti-CD44 mAbs. One of the clones, C₄₄Mab-5 (IgG₁, kappa), recognized both CD44s and CD44v3-10. C₄₄Mab-5 also reacted with oral cancer cells such as Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 using flow cytometry. Moreover, immunohistochemical analysis revealed that C₄₄Mab-5 detected 166/182 (91.2%) of oral cancers. These results suggest that the C₄₄Mab-5 antibody may be useful for investigating the expression and function of CD44 in various cancers.     

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody.

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody. International Journal for Parasitology 22: 527-531. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting Opisthorchis viverrini antigen in faecal extracts of four groups of individuals. These were 24 patients with O. viverrini infection only (group 1), 31 patients with O. viverrini and other parasitic infections (group 2), 141 patients with other parasitic infections (group 3) and 21 normal, parasite-free individuals (group 4). The first antibody used in the ELISA was polyclonal immunoglobulin G prepared from the serum of a rabbit previously immunized with crude extract of O. viverrini. The second antibody was monoclonal antibody specific to an antigen located in the worm tegument and muscular tissue. Sensitivity of the assay was 31% while specificity was 100%. Considerations for improving the sensitivity are discussed.     

Detection of bluetongue virus group-specific antigen using monoclonal antibody based sandwich ELISA

A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for use in the sandwich ELISA. The MAb, designated as 5B5 was specific to VP7 and belongs to IgG2a subclass and was selected for the development of the sELISA in this study. The MAb had a titer of 1:25 with BTV and 1:2 with the rVP7 protein. The sELISA is based on capturing of BTV antigen with VP7 specific MAb followed by detection using BTV polyclonal antiserum raised in rabbits. The assay was evaluated with six cell culture adapted serotypes of BTV that have been isolated from India, 1, 2, 15, 17, 18 and 23. The assay could detect BTV antigen as early as day 8 in blood. It was also successfully applied for the detection of BTV group specific antigen in clinical samples of blood, washed RBCs, buffy coat and plasma. A total of 102 field samples from animals, suspected of being infected with BTV, were tested and 29.42% were positive. The blood samples were also amplified in cell culture which improved the sensitivity of the assay. Results confirmed that the sELISA is rapid and specific.     

Detection of invasive Candida albicans infection using a specific 99mTc-labeled monoclonal antibody for the C. albicans germ tube

Accurate diagnosis is critical for effective treatment of the invasive infection by Candida albicans. Here, we investigated whether a ^99m technetium (Tc)-labeled Fab’ fragment of the monoclonal antibody specific for the C. albicans germ tube could specifically identify an invasive C. albicans infection. The germ tube of C. albicans was used as an immunogen to obtain monoclonal antibodies and the Fab’ fragment of MAb03.2 C1–C2 with highest affinity and specificity was labeled with ^99mTc. In vitro binding assays showed that the labeled Fab’ preferentially bound to the germ tubes of C. albicans (4.23 ± 0.17 × 10² Bq per 1 × 10⁷ cells). These values were significantly higher than those for blastospores of C. albicans, blastospores of heat-killed C. albicans, Aspergillus fumigatus, Staphylococcus aureus, and Escherichia coli (P < 0.05). By using in vivo biodistribution and planar imaging with single photon emission computed tomography, we demonstrated a significant specific accumulation of radioactivity in C. albicans-infected tissues. In summary, ^99mTc-MAb03.2 C1–C2 Fab’ is able to specifically accumulate in C. albicans-infected tissues, but not in tissue infected with A. fumigatus or bacteria or in a sterile inflammation. This study provides a new and specific radiopharmaceutical for the diagnosis of invasive C. albicans infections.     

Detection of a neoantigen on human C3bi and C3d by monoclonal antibody

A neoantigen was detected on human C3bi and C3d by using the monoclonal antibody (MoAb) 130. The antibody bound to EC3bi and EC3d cells but not to EC3b. Although highly purified C3bi or C3d strongly inhibited the binding of the antibody to EC3d, highly purified C3c had no such effect. Native C3, C3b, or C3(H2O) inhibited this binding only weakly. The neoantigen was also detected in serum after activation with zymosan or heat-aggregated IgG, and it was found bound to the aggregated IgG and zymosan particles. Plasma samples from patients with immunologic disorders were tested for this neoantigen, and 25 out of 43 samples tested were found to have levels of neoantigen corresponding to 2 to 11.5% complement activation, whereas 13 out of 14 normal donor plasmas contained amounts of neoantigen indicating much less than 1% complement activation.     

Visualization of cellular focal contacts using a monoclonal antibody to 80 kD serum protein adsorbed on the substratum

We have obtained a monoclonal antibody to 80 kD protein of calf serum; this protein easily and uniformly adsorbs on glass from serum-containing media. Indirect immunofluorescence staining of chick and mouse embryo fibroblasts cultured in the presence of calf serum, fixed with formaldehyde and permeabilized with Triton X-100, revealed black non-fluorescent strips and dots under the ventral cell surface, whereas all other parts of the substratum under and between cells were highly fluorescent. The distribution of non-fluorescent regions coincided with the distributed of focal contacts of cells with the substratum, revealed by interference reflection microscopy, as well as with the distribution of vinculin-containing plaques. The dark regions were also associated with the ends of microfilament bundles revealed by immunofluorescence with an anti-actin antibody. Thus, non-fluorescent regions seen after anti-80 kD staining are parts of the substratum under the focal contacts. Visualization of focal contacts with anti-80 kD provides very contrasting and high resolution pictures. Evidence is presented that 80 kD protein is adsorbed to glass in the areas of focal contacts, but the antibodies used for staining cannot penetrate these contacts.     

Flow cytometric analysis of mouse sperm using monoclonal anti-sperm antibody A-1

We have previously prepared an anti-mouse sperm monoclonal antibody (A-1) which inhibited sperm penetration into the egg zona pellucida. By indirect immunofluorescence (IIF), the A-1 antibody was shown to recognize an antigen localized in the acrosomal area of sperm. This antibody bound negligibly to fresh sperm, while binding to methanol-fixed sperm was almost complete. After methanol fixation, no sperm that penetrated into the zona were immunoreactive for this antibody. In the present study we examined the localization and fate of A-1 antigen during the acrosome reaction by IIF and flow cytometry (FCM). Cauda epididymal sperm were treated with either calcium ionophore A23187 or zona solution, immunostained indirectly, and subjected to FCM. Treatment with A23187 reduced the percentage of immunoreactive sperm to 59% from the 80% obtained in the untreated sperm. The treatment also reduced the average fluorescence intensity per fluorescence-positive spermatocyte to 65 channels, while this intensity was 89 channels in the untreated sperm. A similar result was obtained from treatment with zona solution. The proportion of sperm that was immunoreactive with A-1 antibody was reduced to 55% by incubation in zona-containing media from the 80% obtained in zona-free media. On the other hand, neither A23187 nor the zona solution affected the immunoreactivity or the fluorescence intensity of caput epididymal sperm, while the A-1 antigen was present in both the immature sperm from the caput epididymis of adult mice and in the mature sperm from the cauda epididymis of the same mice. These findings suggest that the intramembrane antigen recognized by the A-1 monoclonal antibody is released from sperm as a result of the acrosome reaction. © 1994 Wiley-Liss, Inc.     

Detection of a glycosphingolipid antigen in bladder cancer cells with monoclonal antibody MRG-1

Summary The monoclonal antibody MRG-1 has been evaluated for the immunohistochemical detection of the type 3 chain of blood group A in human normal bladder epithelium and bladder tumours. Light microscope examination of paraffin sections demonstrated that this antigen was present in normal epithelium and superficial bladder tumour in patients with blood group A or AB, but was absent in the invasive type of bladder tumour. In normal epithelium, the plasma membrane was positive for this antigen, and the cytoplasm was diffusely stained. In superficial transitional cell carcinoma, the plasma membrane was negative, whereas the cytoplasm was intensely stained in the perinuclear region. This pattern was different from that observed for type 1 and 2 group A antigen, which was recognized mainly at the plasma membrane. However, in superficial transitional cell carcinoma, the staining was also seen on the plasma membrane. The pattern of the localization of this antigen in this carcinoma was influenced by the treatment of organic solvents. Electron microscopical observations confirmed that this antigen was localized on the plasma membrane and also in the Golgi apparatus of the superficial tumour.These results proved that the type 3 chain of blood group A is present in human bladder epithelium and low grade tumours in correspondence with the blood type, but disappears in tumours with high malignant potential. However, its expression is independent of the expressions of the other subtypes which have been studied. Furthermore, the changes in the staining pattern caused by pretreatment with organic solvents suggested possible differences in the microenvironment of the glycolipids containing this type of sugar chain.     

Detection of P-glycoprotein on endothelial and endocrine cells of the human pancreatic islets by C 494 monoclonal antibody

Summary P-glycoprotein, an integral membrane protein acting as an energy-dependent efflux pump, has been detected immunocytochemically in the human pancreatic islets using C 494 monoclonal antibody. Intense P-glycoprotein immunoreactivity was found in both endothelial cells of islet blood capillaries and in endocrine cells. Strong expression of P-glycoprotein has been found in the capillary blood vessels at blood-tissue barrier sites and in numerous kinds of cells with secretory/excretory function. Therefore the present findings suggest that P-glycoprotein may play a role in controlling the composition of the extracellular fluids and the intracellular milieu of endocrine islet cells and possibly in regulating their secretory activity.     

Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Monoclonal antibody F7-26 was obtained by fusion of mouse myeloma cells with spleen cells isolated from a mouse immunized with DNA treated by nitrogen mustard (HN2). Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to the drug dose. Two-parameter analysis for the antibody binding and DNA content showed no binding of antibody to replicating DNA in control cells. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Binding of monoclonal antibody specific for the determinants expressed on ssDNA to the cells treated with alkylating agents may be attributed to local DNA denaturation. Potentiation of L-phenylalanine mustard cytotoxicity by buthionine sulfoximine or hyperthermia was accompanied by increased antibody binding to cellular DNA. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.     

A monoclonal antibody to C1q which appears to interact with C1r2C1s2-binding site

A monoclonal antibody (SB-4) to human C1q was prepared. The equilibrium constant of the antibody for C1q was found to be greater than 10(10) M-1. It has been shown that the antibody binds to the A-B chain dimer, probably via the B chain of C1q. Pepsin digestion of C1q at pH 4.5, which fragments the globular regions but leaves the collagenous region intact, allowed the demonstration that the antigenic site is located in the collagenous region of the molecule. The effect of the antibody on haemolytic activity has shown that it is capable of inhibiting the formation of EAC1 cells from EAC1q cells plus C1r and C1s but is incapable of inhibiting the C1 activity of performed EAC1 cells. This indicates that the binding of the antibody to the collagenous portion of the B chain of C1q probably prevents interaction between C1q and the C1r2-C1s2 complex.     

Sculpting therapeutic monoclonal antibody N-glycans using endoglycosidases

Immunoglobulin G (IgG) monoclonal antibodies are a prominent and expanding class of therapeutics used for the treatment of diverse human disorders. The chemical composition of the N-glycan on the fragment crystallizable (Fc) region determines the effector functions through interaction with the Fc gamma receptors and complement proteins. The chemoenzymatic synthesis using endo-β-N-acetylglucosaminidases (ENGases) emerged as a strategy to obtain antibodies with customized glycoforms that modulate their therapeutic activity. We discuss the molecular mechanism by which ENGases recognize different N-glycans and protein substrates, especially those that are specific for IgG antibodies, in order to rationalize the glycoengineering of immunotherapeutic antibodies, which increase the impact on the treatment of myriad diseases.     

Cell cycle analysis using the monoclonal antibody Ki-67

We determined by flow cytometry the proportion of cells in cycle with the monoclonal antibody Ki-67 and also in S-phase after incorporation of bromodeoxyuridine (BrdUrd) with monoclonal antibody to BrdUrd. The monoclonal antibody Ki-67 was useful to detect a nuclear antigen present only in proliferating cells but not expressed in resting (Go) cells. Cell preparation to measure BrdUrd amount incorporated into cellular DNA was difficult but this anti-BrdUrd antibody was useful for measuring the rate of DNA synthesis and for the analysis of precious cell kinetics. These antibodies may provide useful information of cell kinetics.     

Detection of dideoxyosone intermediates of glycation using a monoclonal antibody: characterization of major epitope structures

Glycation or the Maillard reaction in proteins forms advanced glycation end products (AGEs) that contribute to age- and diabetes-associated changes in tissues. Dideoxyosones, which are formed by the long-range carbonyl shift of the Amadori product, are newly discovered intermediates in the process of AGE formation in proteins. They react with o-phenylenediamine (OPD) to produce quinoxalines. We developed a monoclonal antibody against 2-methylquinoxaline-6-carboxylate coupled to keyhole limpet hemocyanin. The antibody reacted strongly with ribose and fructose (+OPD)-modified RNase A and weakly with glucose and ascorbate (+OPD)-modified RNase A. Reaction with substituted quinoxalines indicated that this antibody favored the 2-methyl group on the quinoxaline ring. We used high performance liquid chromatography to isolate and purify three antibody-reactive products from a reaction mixture of N alpha-hippuryl-L-lysine+ribose+OPD. The two most reactive products were identified as diastereoisomers of N1-benzoylglycyl-N6-(2-hydroxy-3-quinoxalin-2-ylpropyl)lysine and the other less reactive product as N1-benzoylglycyl-N6-[2-hydroxy-2-(3-methylquinoxalin-2-yl)ethyl]lysine. Our study confirms that dideoxyosone intermediates form during glycation and offers a new tool for the study of this important pathway in diabetes and aging.     

Characterising the association of latency with α1-antitrypsin polymerisation using a novel monoclonal antibody

α₁-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α₁-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α₁-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α₁-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α₁-antitrypsin augmentation therapy contains latent α₁-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p < 10⁻¹⁴). We conclude that latent α₁-antitrypsin is a by-product of the polymerisation pathway, that the intracellular folding environment is resistant to formation of the latent conformer but that augmentation therapy introduces latent α₁-antitrypsin into the circulation. A suite of monoclonal antibodies and methodologies developed in this study can characterise α₁-antitrypsin folding and conformational transitions, and screen methods to improve augmentation therapy.     

A monoclonal antibody to human immunodeficiency virus type 1 which mediates cellular cytotoxicity and neutralization.

Monoclonal antibodies (MAbs) were raised against human immunodeficiency virus type 1 gp120. One MAb, P4/D10, was found to mediate highly efficient antibody-dependent cellular cytotoxicity and virus neutralization. The reactivity was located to a major neutralizing region (amino acids 304 to 323) on gp120. Five other MAbs with a similar epitopic reactivity did not show any antibody-dependent cellulan cytotoxicity activity but had a virus-neutralizing capacity.     

Features of plasminogen activation in a complex with antiplasminogen monoclonal antibody IV-1C

Antiplasminogen monoclonal antibody IV-1c (IV-1c) with antigenic determinant in V709-G718 site of plasminogen (Pg) protease domain (Druzhina N.N. et al. 1996.) can induce catalytic activity in Pg moiety of the complex. Catalytic activity appeared in Pg-IV-1c complex after approximately 2 h lag-period. Rate of Lys-Pg activation was higher then that of Glu-Pg. Amidolytic activity of Pg-SK equimolar complex was completely inhibited by IV-1c at 2:1 = Pg:IV-1c molar ratio. At constant Glu-Pg concentration increasing of the IV-1c concentration to equimolar of Pg accelerated Pg activation. Subsequent increase of IV-1c concentration inhibited the Pg activation sharply. Increasing of Glu-Pg concentration at constant IV-1c one did not inhibit Glu-Pg activation in Pg-IV-1c complex. The rate dependence of Pg activation from Glu-Pg-IV-1c complex concentration curve had bell-shaped form with maximum at 500 nM. Electrophoretic analysis of components of Glu-Pg-IV-1c complex showed that Lys-Pg and Lys-Pm were not observed at 100 nM complex concentration for 6 h period of reaction. At 680 nM concentration Glu-Pg-IV-1c complex these forms appeared in initial moments of reaction activation after lag-period. Kinetic scheme and peculiarities of Pg activation reaction in Pg-IV-1c complex are discussed.