Materials

Use 20- to 30-wk-old -30 BALB c mice to generate routine mouse or mouse-rat hybridomas Sprague-Dawley rats are used for rat-rat MAb antibody production. 2. P3X63Ag8.653 (ATTC, Manassas, VA, cat. no. CRL-1580) murine myeloma cell line other mouse, as well as human and rat hybridoma fusion partners, are listed on the ATCC web page 3. Dulbecco's (DMEM)- or Iscove's-modified Eagle's medium, supplemented with 10-20 preselected fetal bovine serum (FBS), 4 mML-glutamine, 1 mM sodium pyruvate,...

Histamine Release From Washed Human Leukocytes

50-mL Polypropylene centrifuge tubes (Corning Inc. Corning, NY cat. no. 25330). 2. 12 X 75-mm Polystyrene test tubes. 4. 1,4-piperazinebis (ethane sulfonic)-buffered saline containing 0.003 human serum albumin, 0.1 D-glucose (PAG) buffer. 5. 1,4-piperazinebis (ethane sulfonic)-buffered saline containing 0.003 human serum albumin, 0.1 D-glucose 10-M CaCl2 and 0.1 mM NaCl2 (PAGCM) buffer.

Eugene Mechetner Summary

Cell fusion protocols that were developed by Kohler and Milstein in the mid-1970s and aimed at producing and characterization of mouse monoclonal antibodies (MAbs) remain the gold standard of hybridoma development. Despite tremendous progress in using MAbs in multiple research, diagnostic, and therapeutic areas, major experimental flaws in designing and carrying out hybridoma experimentation often result in the production of hybridomas exhibiting poor growth parameters and secreting...

Detection of the MDR PGlycoprotein Expression and Function

Acquired and intrinsic multidrug resistance is the major reason for the failure of anticancer chemotherapy. The most important component of clinical multidrug resistance is mediated by P-glycoprotein (Pgp), an ABC transporter encoded by the MDR1 gene and expressed on the membrane of tumor and normal cells. Sensitive and reproducible detection of Pgp expression and function are critical for the development of new MDR1 drugs and clinical protocols aimed at modulating Pgp-mediated multidrug...

Solubilizing Fc Fusion Proteins

The purpose of this chapter is to describe in depth how to genetically engineer, stably transfect, screen, produce, and purify Fc fusion proteins. A general outline of the following procedures is depicted in Fig. 1. Expression of soluble Fc fusion protein is entirely dependent on a mammalian expression system. The protein expression system described here involves the usage of an expression vector (pEE12) that is under the control of a strong viral promoter (6). The viral promoter enables the...

Cell Fusion

Grow 8-azaguanine-resistant P3X63Ag8.653 myeloma cells at a maximum concentration of 0.5 X 106 mL for 3 d refresh medium 24 h before fusion. Harvest and wash the cells immediately before fusion, wash them twice in large volumes of serum-free medium (e.g., 50-mL conical tubes with ice-cold DMEM), and keep on ice until fusing with splenocytes. It is imperative that myeloma cells remain highly viable and completely free of protein in the medium. 2. Place and keep tube with 1.5 mL of 50 PEG...

Calculations

The measurement of multilevel calibrated QuantiBRITE fluorescent beads enables the construction of a standard curve for antigen quantification. Using a Microsoft Excel-spreadsheet referring to CellQuest, provided by BD Biosciences, the sample fluorescence measured can be converted into the term ABC as shown in Fig. 1C for the expression of neprilysin CD10 on granulocytes. QuantiCALC (BD Biosciences) is an automated quantitation analysis software, which automatically converts the FL2 axis to PE...

Fixation and Permeabilization

Intraprep permeabilization reagent (Beckman Coulter, Miami, FL). 2. Phosphate-buffered saline (flow PBS) prepare with 5.6 g sodium phosphate dibasic anhydrous, 35.48 g sodium chloride, 2.8 g bovine albumin, and 4 g sodium azide. Add water to 4 L and adjust to pH 7.4 with HCl. 3. Fluorochrome-labeled surface markers CD3-FITC, CD19-PerCP, and CD34-APC (BD Bioscience, San Jose, CA). 4. Fluorochrome-labeled isotype controls IgG-FITC, IgG-PerCP, and IgG-APC (BD Bioscience). 5. Primary monoclonal...

Cloning by Limiting Dilutions Cell Propagation and Archiving

Every primary hybridoma clone requires at least three consecutive rounds of recloning. 2. Examine 96-well plates containing hybridoma clones on an inverted microscope (low magnification). Reclone all positive wells after primary fusion and only the largest single colonies (5-10 colonies per one primary hybridoma clone) in subsequent rounds of cloning. 3. Reserve two flat-bottom 96-well plates per each well to be recloned add 100 p,L of complete hybridoma medium 1 h prior to the recloning...

Buffer for Basophil Histamine Release

10X PIPES buffer, pH 7.4 250 mM PIPES (Sigma), 1.10 M NaCl, and 50 mM KCl. 2. PAG buffer 10 10X PIPES, 0.003 human serum albumin (Calbiochem Bering Corp., La Jolla, CA), and 0.1 D-glucose. 3. PAG-EDTA buffer PAG containing 4 mM EDTA. 4. PAGCM buffer PAG containing 1 mM CaCl2 and 1 mM MgCl2. 5. Isotonic Percoll nine parts Percoll (Pharmacia, Piscataway, NJ) plus one part 10X PIPES. In the last decade, basophiles have been found to upregulate cell surface markers (CD45, CD63, CD69, and CD203)....

Huai En Huang Chan Iman Jilani Richard Chang and Maher Albitar

As the signaling pathways involved in leukemogenesis are being elucidated, several proteins have emerged as potential targets for therapy. Downstream from those targets are numerous intracellular factors that are constantly modulated. Monitoring those factors could provide insight into the potential efficacy of therapies by predicting which patients will respond to them and by determining the optimal dosage that will inhibit the target protein. We describe a flow cytometry method for...

Production of MAbs in Protein Free Medium

In this section, we describe our experience of selecting a hybridoma clone capable of secreting high MAb concentrations when growing in a serum-, protein-free medium. See Chapter 13 for a detailed description of the uses of this MAb for the detection of MDR1 P-glycoprotein expression and functional activity. 2. A subclone of the UIC2 hybridoma, termed UIC2 A (ATCC cat. no. HB11287), was developed by gradually replacing the original growth medium (DMEM supplemented with 10 FBS and...

Detection of Chromosome Translocations by Bead Based Flow Cytometry

Huai En Huang Chan, Iman Jilani, Richard Chang, and Maher Albitar Summary Chromosome translocations resulting in fusion genes have been implicated in leukemo-genesis. The paradigm involves the fusion of the genes encoding BCR and ABL, leading to a constitutively active tyrosine kinase. The detection of BCR-ABL has been limited to fluorescence in situ hybridization analysis, reverse transcription-polymerase chain reaction, of mRNA, and Western blot of analysis downstream effectors in the BCR-ABL...

Specific Antibodies and Their Diagnostic Combinations

Because of the multitude of IHC stains available to characterize lymphoid neoplastic processes, a preliminary differential diagnosis based on the histo-logical assessment typically drives the selection of markers to be applied in a particular case. Next, several specific antibodies and their combinations are discussed in the context of lymphoid proliferations, as well as specific diagnoses. Fig. 17. Comparison of follicular hyperplasia and follicular lymphoma. (A) H& E and (B) Ki-67...

Where Have We Been and Where Are We Going

About 40 yr ago, two groups of investigators identified a new class of immunoglobulins, IgE. By exchanging their results and reagents, they proved that the immunoglobulin responsible for immediate hypersensitivity was IgE. From that day forward the science of allergy was greatly advanced. Within a few years of the IgE discovery, an assay for IgE was developed. This test was named the radio allergosorbent test. The specific IgE testing methodology has matured in the last four decades. Different...

Recombinant Antibody Engineering

Scfv Antibody

The major types of recombinant antibody fragments that are usually expressed in E. coli are named Fv, dsFv, scFv, and Fab (Fig. 3). Fv (fragment variable) fragments consists of only the variable domains of the heavy and light chains (VH and VL) and are the smallest units that retain binding properties to a given antigen (10). VH and VL expressed in E. coli assemble spontaneously into Fv fragments through noncovalent interactions. However, the VH-VL interaction is usually weak, and therefore...

Results and Discussion

We used a standardized immune monitoring program based on recent advances in flow cytometry (exact quantification of surface marker expression) to investigate 54 patients with open heart surgery. All patients showed postoperative immunodepression, documented by decreased monocytic HLA-DR. During the postoperative follow-up, all markers investigated tended to recover. One day after open heart surgery, monocytic HLA-DR expression revealed a marked attenuation compared with values determined...

Reagent Summary Preparation and Storage see Note

All reagents are manufactured by Pharmacia and Upjohn Diagnostics, Uppsala, Sweden. Note date and initial all reagents upon opening. Each container should be labeled with substance name, lot number, date of preparation, expiration date, and any special storage instructions. 1. Anti-IgE ImmunoCAP (cat. no. 14-4417-01). Anti-IgE Mouse monoclonal antibodies. 16 ImmunoCAP carrier. Store at 2-8 C. 2. Allergen-specific ImmunoCAP (refer to catalog for specific allergen product number). Each ImmunoCAP...

Buffers and Diluents

Wash buffer 1X PBS solution, containing protein and detergent, used for wash steps and to resuspend beads for analysis. Store at 4 C. 2. Assay diluent 1X buffered solution used to dilute the BD CBA Human Soluble Protein Flex Set Standards and to dilute test samples. Store at 4 C. 3. Capture bead diluent for serum plasma samples 1X PBS solution containing protein used to resuspend capture beads prior to testing serum or plasma samples. Store at 4 C. 4. Capture bead diluent for cell culture...

IHC of Bone Marrow Bone Marrow

A comprehensive evaluation of BM involves examination of both marrow smears and tissue sections each is complementary to the other. With the growing battery of paraffin-reactive immunohistological reagents, coupled with newer techniques of antigen retrieval, new vistas have opened in the study of BM biopsies (BMB) (6). Normal BM consists of a heterogeneous population of cells proceeding along various differentiation pathways. Although most cell types can be easily distinguished on BM aspirate...

Meg L Flanagan Robyn S Arias Peisheng Hu Leslie A Khawli and Alan L Epstein

As a source of recombinant antigen, soluble constant fragment (Fc) fusion proteins have become valuable reagents for immunotherapy and laboratory investigations. Additional applications for these reagents include flow cytometry, immunohistochemistry, and in vitro activity assays. To aid investigators in the generation of these reagents, the materials and methods required for producing Fc fusion proteins are described. The investigator's protein moiety of interest is genetically linked to the...

Spleen

IHC is a valuable tool in the evaluation of splenic disorders. It provides unique challenges, however, because of the functional complexity of the spleen and the variety of its histological components. The purpose of this section is to provide general guidelines on evaluating the complex splenic microanatomy, stressing in particular the morphologic and the immunohistological characteristics of the white pulp and red pulp compartments (44). The lymphoid components will not be specifically...

AlaSTAT

The AlaSTAT IgE assay Diagnostic Products Corp., Los Angeles, CA employs biotin-labeled allergen extracts that bind in the fluid phase to specific IgE 5 . The biotin-IgE complexes are added to microtiter wells or plastic tubes where biotin-albumin is coated to the surface. Avidin is added to the solution and the IgE-biotin complexes are linked to the biotin solid phase. An enzyme-linked anti IgE is used to calibrate the specific IgE. Quantitative IgE calibrators are used to assess specific IgE...

PBS controlNormal control

Detection of BCR-ABL protein and its phosphorylation. Plasma samples prepared from peripheral blood plasma of a normal subject and an untreated chronic myeloid leukemia patient were incubated with anti-BCR coated beads, followed by incubation with antibody directed against ABL upper row or phosphorylated Tyr245 of ABL lower row . PBS served as a negative control. Fig. 3. Detection of BCR-ABL protein and its phosphorylation. Plasma samples prepared from peripheral blood plasma of a...

The Cytometric Bead Array System

Flex Set Dilution

Rudolf Varro, Roy Chen, Homero Sepulveda, and John Apgar Summary Analytical cytometry has significant potential beyond cellular analysis. The inherent capability of flow cytometers to efficiently discriminate between uniformly sized particles based on their intrinsic properties provides the foundation for multiplex bead assays. The technology can be exploited in designing immunoassays, Western blot-like antibody assays, and nucleic acid hybridization assays. This chapter focuses on immunoassay...

Detection of Human Antibodies Against Therapeutic Antibodies Materials

Absorption of Interfering Antiiso Allotypic Human Anti-Mouse Antibodies 1. Mouse IgG-agarose suspension polyclonal mouse IgG coupled to cyanogen bromide-activated agarose Sigma-Aldrich, Deisenhofen, Germany . Resuspend settled gel by repeated shaking immediately before filling the pipet see Note 1 . 2. Phosphate-buffered saline with Tween PBS-T prepare 10X stock with 0.027 M KCl, 1.37 MNaCl, 0.1 M Na2HPO4, 0.018 M KH2PO4, and 1 Tween-20. Store at 4 C. Prepare working solution by dilution...

References

1 Kohler, G. and Milstein, C. 1976 Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur. J. Immunol. 6, 511-519. 2 He, Y., Honnen, W. J., Krachmarov, C. P., et al. 2002 Efficient isolation of novel human monoclonal antibodies with neutralizing activity against HIV-1 from transgenic mice expressing human Ig loci. J. Immunol. 169, 595-605. 3 Hoogenboom, H. R. and Chames, P. 2000 Natural and designer binding sites made by phage display technology. Immunol....

Patrick Schroeter Arnim Sablotzki and Dagmar Riemann Summary

HLA-DR expression on monocytes as a marker for the functioning of the immune system is known to be severely depressed in immunodeficiency. Up to now, other markers for the function of the immune system are scarce. In the peripheral blood of patients with open heart surgery the expression of the membrane peptidases neprilysin CD10 and aminopeptidase N CD13, was determined on granulocytes in comparison to the monocytic HLA-DR expression. We used the QuantiBRITE flow cytometry system, which yields...

Mariel Donzeau and Achim Knappik

Recombinant antibody technology is a rapidly evolving field that enables the study and improvement of antibody properties by means of genetic engineering. Moreover, the functional expression of antibody fragments in Escherichia coli has formed the basis for antibody library generation and selection, a powerful method to produce human antibodies for therapy. Because in vitro-generated antibodies offer various advantages over traditionally produced monoclonal antibodies, such molecules are now...

Expression and Screening of Soluble Fc Fusion Proteins

1. 1 L Hybridoma-SFM serum-free medium without L-glutamine Life Technologies, cat. no. 93-0247-170 . 2. Dialyzed fetal calf serum FCS Hyclone, cat. no. SH30079.03 . 3. 100X MEM nonessential amino acids solution Cellgro, cat. no. 25-025-Cl . 4. 100X Penicillin-streptomycin Pen-Strep solution Gemini BioProducts, cat. no. 400-109 . 5. 50X GSEM glutamine synthetase expression medium supplement Sigma, cat. no. G9785 . 6. NS0 myeloma cells included with pEE12 vector . 7. Characterized fetal bovine...

Principle of the Test

PE-labeled beads are commercially available in a kit, which contains one tube with a mixture of beads with four different predefined levels of PE. The PE-labeled antibodies used for staining of surface antigens on blood cells are at a 1 1 fluorochrome-to-antibody ratio. This allows determination of the ABC when beads are run under the same photomultiplier and compensation settings as blood cells see Notes 2 and 3 . All samples were analyzed on a FACS Calibur BD Biosciences using the software...

UIC Shift Assay in Drug Screening and Monitoring

Introduction of time- and cost-efficient tests for high-throughput MDR1 analysis is greatly needed to facilitate the development of new, safe, and clinically efficient Pgp modulators, as well as pharmaceuticals that do not interfere with patients' physiological MDR mechanisms. Information on drug resistance substrate specificity profiles for new drug candidates is critical for rational design of new drugs and for managing of clinical trials on drug resistance modulators, as well as other drug...

Testing of Cell Lines and Clinical Tumor Specimens

The UIC2 shift phenomenon was more pronounced in MDR1 cell lines with low levels of Pgp expression, reflecting lower molar Pgp drug ratio in these cells, as compared with high Pgp expressors 34 . This feature makes the UIC2 shift assay particularly attractive for Pgp detection in human tumors, the vast majority of which are characterized by low MDR1 expression levels 19-22 Table 2 . We used a series of MCF7 breast carcinoma-derived cell lines as a model of clinical breast cancer for...

Large Scale Production of Fc Fusion Proteins

For the following, prewarmed selective medium must be used in order to maintain the transfection vector in NS0 host cells. All incubations take place in a standard 37 C, 5 CO2 humidified incubator for cell culture. 1. Grow highest-producing clone to confluency in a T75 flask with selective medium. 2. Expand cells to a T225 flask until confluent 4-5 d . 3. Add entire T225 culture into an autoclaved 3-L spinner flask, followed by 1 L of selective medium. Incubate 3-4 d with continuous stirring....

Generating the pEEmuFc Master Expression Vector

Pee12 Sequencing Primers

To amplify murine Fc, the FWD and REV muFc primers Subheading 2.2. must be ordered as in Subheading 3.1.1., step 5. Fig. 2 is an example of expected results when muFc PCR products are run on a 1 agarose gel. Ligation of the resulting muFc PCR product into the pEE12 expression vector is depicted in Fig. 3. 3.2.1. Amplifying Murine Fc and Cloning Into pEE12 GS Expression Vector 1. Amplify, isolate, and purify murine Fc PCR product from the pSK muFc vector using FWD and REV muFc primers, as in...