Detection with monoclonal antibodies

GalNAc 31 -4(Fuca1 -2Fuca1 -3)GlcNAc carbohydrate epitopes detection with monoclonal antibodies that are characterized by enzymatically synthesized neoglycoproteins. Clycobiology 10, 601-609. 

Purification and characterization of an individual MPC can be simplified by the introduction of various forms of sequence tags and functional protein domains into the subunits of the complex. This is easily accomplished in S. cerevisiae with the use of genetic recombination. These fusions have primarily three different purposes to introduce epitopes that can be specifically detected with monoclonal antibodies, to aid in purification, and to contribute a new function. 

Frankfurt, O. S., Robb, J. A., Sugarbaker, E. V., and Villa, L. (1997) Apoptosis in breast carcinomas detected with monoclonal antibody to single-stranded DNA relation to bcl-2 expression, hormone receptors, and lymph node me-tastases. Clin. Cancer Res. 3(3), 465 471. 

FIGURE 10.4 Distribution of markers in pituitary adenomas. CHR-A, chromogranin A KER (CAMS.2), keratins detected with monoclonal antibody CAMS.2 SYNAP, synaptophysin NSE, neuron-specific enolase KER (AE1/AE3), keratins detected with antibodies AE1 and AE3 HBME-1 CK7, cytokeratin 7 CK19, cytokeratin 19 CFAP, glial fibrillary acid protein. 

Strauchen JA, Dimitriu-Bona A. Malignant fibrous histiocytoma expression of monocyte/macrophage differentiation antigens detected with monoclonal antibodies. Am J Pathol. 1986 124 303-309. 

While lipoproteins are the products of many different genes, the major apolipoproteins share properties distinguishing them from most lipid-free and membrane-associated proteins. For example, apolipoproteins consist of a single polypeptide chain that has relatively little tertiary structure. Most apolipoproteins contain stretches of amphipathic alpha-helix, whose hydrophobic face can be turned to the lipid surface of the particle. The apolipoproteins are flexible, as is reflected in their unusually small free energy of unfolding. As these apolipoproteins expand and contract at the cell surface, different protein domains are exposed that are detectable with monoclonal antibodies. These properties reflect the role of apolipoproteins at the surface of lipoprotein particles whose size changes as they circulate. 

Fenderson BA, O Brien DA, MiUette CF, Eddy EM. Stage-specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies. Dev Biol 1984 103 117-128. 

Figure 15.9 The results of capture ELISA on native RNase A and formalin-treated RNase A. Right panel, native RNase A (curve 1) and unfractionated formalin-treated RNase A (curve 2). Left panel, individual fractions of formalin-treated RNase A monomer (curve 3), dimmer (curve 4), trimer (curve 5), tetramer (curve 6), and a mixture of oligomers with >5 cross-linked proteins (curve 7). The ELISA plate wells were coated with monoclonal antibody against bovine pancreatic RNase A (1 pg/mL) overnight at 4°C and then blocked with bovine serum albumin. The wells were incubated for lh at 37°C in the presence of various concentrations of antigen in lOOpL of PBS. After washing, each plate well received a 1 4000 dilution of horseradish peroxidase conjugated rabbit polyclonal anti-RNase A antibody followed by incubation at ambient temperature for lh. After washing, detection was achieved using a mixture of 2,2,-azino-di-(3-ethylbenzthiazoline-6-sulphonate) and hydrogen peroxide. Absorbance was monitored at 405 nm. See Rait etal.11 for details.

Alston, K., Robinson, R.C., Park, S.S., Gelboin, H.V., and Friedman, F.K. (1991) Interactions among cytochromes P-450 in the endoplasmic reticulum. Detection of chemically cross-linked complexes with monoclonal antibodies./. Biol. Chem. 266, 735-739. 

When primary mouse monoclonal antibodies are of different IgG isotypes/ subclasses, they can be selectively detected with secondary antibodies directed against the corresponding IgG isotype (Fig. 8.2). 

D. J. Bucher, A. Mikhail, S. Popple, P. Graves, G. Meiklejohn, D. S. Hodes, K. Johansson, andP. E. Halonen, Rapid detection of Type A Influenza viruses with monoclonal antibodies to the M protein (Ml) by enzyme-linked immunosorbent assay and time-resolved fluoroimmunoassay, J. Clin. Microbiol. 29, 2484-2488 (1991). 

Krenacs, L., Tiszlavicz, L., Krenacs, T., and Boumsell, F. (1993) Immunohistochemical detection of CDla antigen in formalin-fixed and paraffin-embedded tissue sections with monoclonal antibody OlO. J. Pathol. 171, 99-104. 

The immunocytochemical staining of cytochrome C offers another alternative since, upon exposure to apoptotic stimuli, cytochrome C is rapidly released into the cytosol, an event that may be required for the completion of apoptosis in some systems (L2). The effect of cytosolic cytochrome C is thought to be the activation of caspases. The immunocytochemical staining of cytochrome C localized in mitochondria in healthy cells or diffused in the cell cytoplasm with monoclonal antibody (Promega) after induction of apoptosis, as detected by fluorescence microscopy, can be used for monitoring apoptosis (L2, M7, S8, T6). It is also a simple, rapid, specific mefhod for quanfifafive assessment of apoptotic cells. 

The effect of CVS is T-cell mediated, since no effect was observed in athymic nu/nu mice of BALB/c background which were hereditarily T cell deficient (data not shown). Therefore, we examined the participation of T-cell subsets in the antitumor effect of CVS following their in vivo depletion with monoclonal antibodies in the Meth A and BALB/c system, Fig. (6)-Protocol B. CVS was injected i.t. 3 times from day 2, and tumor i.v. rechallenging was performed on day 9. Anti-CD3 treatment completely inhibited the antitumor effect of CVS (data not shown). Anti-CD4 and anti-CD8 treatment partially inhibited the effect of CVS. Less than 1.0% of the CD3-, CD4-, or CD8-positive cells were detected in the spleen or lymph nodes following in vivo treatment with the corresponding antibodies by flow-cytometric analysis using FACS (Becton Dickinson, USA). 

De Waele, M., De Mey, K., Moeremans, M., De Brabender, M., and Van Camp, B (1983) Immunogold staining for the light microscopic detection of leukocyte cell-surface antigens with monoclonal antibodies. J Histochem. Cytochem 31, 938-944. 

Otsuki, Y., Maxwell, L. E, Magan, S., and Kubo, H. (1990) Immunogold-silver staining method for light and electron microscopic detection of lymphocyte cell-surface antigens with monoclonal antibodies. J Histochem Cytochem. 38, 1215—1221. 

Janssen, P. J., Brinkmann, A. O., Boersma, W. J., and van der Kwast, T. H. 1994. Immunohistochemical detection of the androgen receptor with monoclonal antibody F39.4 in routinely processed, paraffin-embedded human tissues after microwave pretreatment. J. Histochem. Cytochem. 42 1169-1175. 

K4. Kaufmann, O., Hath, B., Spath-Schwalbe, E., Possinger, K., and Dietel, M., Immunohistochem-ical detection of CD10 with monoclonal antibody 56C6 on paraffin sections. Am. J. Clin. Pathol. Ill, 117-122 (1999). 

G14. Golan, M. D., Burger, R., and Loos, M., Conformational changes in Clq after binding to immune complexes Detection of neoantigens with monoclonal antibodies. J. Immunol. 129, 495-497 (1982). 

II.R.5 Enzyme Sandwich Immunoassay with Monoclonal Antibodies for the Detection of HBeAg and anti-HBe... 

Specificity of HSA-1. Every specimen from a large panel of human serum samples from various races and sexes reacted with monoclonal antibody HSA-1. No human serum was tested that did not react, a fact indicating the absence of genetic variation in the antigenic site detected by the HSA-1 antibody. The HSA-1 monoclonal antibody thus fulfills two essential criteria for an immunological probe for the identification of the species of origin species specificity and intraspecies conservation of the antigenic site. 

To improve the specific response against microcystin immimoconjugates, synthetic lipopeptides were used as adjuvants and were found to invoke a greater immune response than the use of classical adjuvants such as Freunds adjuvant. However, an ELISA for the detection of free microcystin was not developed using these antibodies. Cross-reactivities of various microcystin variants and nodn-larin with monoclonal antibodies have been found affecting to the specificity of these antibodies for the recognition of the mentioned toxins. The number of purified microcystin variants that have been tested by ELISA using monoclonal antibodies shows marked differences between methods. 

Hu et al. have prepared an antibody microarray with monoclonal antibodies against anti-AFP, anti-CEA, and RAH. Antibodies were dissolved in a glycerol and a SDS solution at a concentration of 0.10 mg/mL in a final volume of 0.20 pi for one spot in a diameter of 1.5 mm. The previously blocked arrays were incubated against the proteins—a mixture of a-fetoprotein (AFP), CEA, and human IgG. Then an aliquot of the labeled antibodies Sm3+ anti-AFP, Eu3+—anti-CEA, and Au-GAH was added at a concentration of 0.50 pg/ml. The results showed that each labeled antibody displayed clear signals and good reproducibility. Furthermore, they mixed two or more antibodies in one spot, which could also specifically and selectively detect each individual signal (40). 

Fig. 2 Immunoblot analysis of cross-reactivity of monoclonal antibody generated against rat hepatoma thymidylate synthase. T. spiralis muscle larvae crude extract (lane 1 100 pg protein), purified recombinant (lane 2 1 pg protein) or purified muscle larvae endogenous (lane 3 1 pg protein) thymidylate synthase, rat purified regenerating liver endogenous (lane 4 1 pg protein) or rat recombinant (lane 5 1 pg protein) thymidylate synthase and L5178Y crude extract (lane 6 100 pg protein) were separated by SDS polyacrylamide (12%) gel electrophoresis and proteins were transferred into PVDF membrane. Thymidylate synthase was detected by treatment with monoclonal antibody and goat anti-mouse IgG (H + L) - HRP conjugate (BioRad) as a secondary antibody...

L. Roza, K.J.M. van der Wulp, S.J. McFarlane, P.H.M. Lohman, R.A. Baan (1988). Detection of cyclobutane thymine dimers in DNA of human cells with monoclonal antibodies raised against a thymine dimer-containing tetranucleotide. Photochem. Photobiol, 48, 627-633. 

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