Diagnostic applications of monoclonal antibodies
Monoclonal antibodies have been a great success as in vitro and diagnostic reagents. They are used widely in medicine and biology to measure hormones, to identify contaminants in the food industry, to purify proteins, and to identify pathogens in human, veterinary, or plant pathology, where they have largely replaced polyclonal antisera. The reaction between an antibody and its antigen can be directly observed in vitro when an immunoprecipitate is seen at equivalent concentrations this is the...
Sequencebased typing
Determination of HLA type, by analysing the DNA sequence of polymorphic regions of the HLA gene, has been made possible through the use of oligonucleotide probes and the specificity of PCR primers, as discussed above. These methods only consider a selection of motifs within the polymorphic region, as defined by the probe specificities in PCR-SSOP, or primer specificities in each PCR-SSP reaction. Both these assays define HLA specificities within the context of known HLA alleles. Although these...
Protocol 7 Preparation of wholecell lysates in SDS sample buffer
. PBS pH 7.4 . 1 M DTT optional 1. Wash the cells in PBS, pH 7.4, to remove proteins present in the culture medium. 2. For efficient solubilization ensure the cell pellet is well dispersed. If necessary, add a small volume e.g. 10 of the final volume of PBS. 3. Add 4 SDS sample buffer to a final concentration of 5-10 x 107 cells ml and boil for 10 min. Reduce samples, if necessary, by adding a 10 volume of 1 M dithiothreitol DTT before SDS-PAGE. 12 Biochemical characterization of lymphocyte...
Cytokines and growth factors for culturing T cells
The survival and long-term culture of T cells is dependent on autocrine or exogenous IL-2 or IL-4. Furthermore, exogenous cytokines or anti-cytokine antibodies can be used for the selective stimulation and expansion of distinct T-cell subtypes 10 . Prior to the cloning of the IL-2 gene, crude supernatants of mitogen-activated T cells were used as a source of 'T-cell growth factor' for culturing murine T cells. Alternatively, certain transformed T-cell lines such as Gibbon MLA can constitutively...
Protocol 10 Preparation of murine peritoneal macrophages Equipment and reagents
Cold PBS or serum-free medium 5 ml syringe and a 23-gauge needle Sterile scissors and forceps 1. Kill a mouse by cervical dislocation. 2. Cut the skin on the abdomen and peel it back. 3. Inject 5 ml cold PBS or serum-free medium intraperitoneal using a 5 ml syringe fitted with a 23-gauge needle and withdraw fluid. 5. Centrifuge the cells at 300 g for 5 min at 4 C and perform a cell count. Expect to obtain 5-7 x 106 cells mouse. 6. Purify macrophages 60-70 of peritoneal lavage cells , if...
Applications of hybridoma technology 31 Bcell hybridomas
The first B-cell hybridomas were produced in an attempt to study immunoglobulin gene expression, and have led to a much greater understanding of the genetic mechanisms of the generation of antibody diversity. The first antibodies with a particular application those to histocompatibility antigens were made in 1977 28 . The potential use of monoclonal antibodies as reagents was soon widely appreciated leading to a rapid growth in the field, and a whole area of the biotechnology industry developed...
Principles of lymphocyte handling and culture
4.1 Short- and long-term storage freezing After isolation, lymphocytes and PAPCs can, if necessary, be stored. Human PBMCs can be stored in a constant pH, physiological medium supplemented with nutrients e.g. R 10 at 37 C in a humid C02 incubator for up to 2 days. There is no apparent loss in their ability to secrete lymphokines such as IL-4 or IFN-y , proliferate, or become activated for lysis by in vitro restimulation. Store them at 106-107 ml, but preferably at 2-5 X 106 ml. Since mouse...
Fusion partners
A number of myeloma or T-cell tumour lines are available for fusion. In the mouse, where there is the greatest choice and experience for antibody production, there is no apparent reason, apart from ready availability, for using a line that retains the ability to code for its own Ig light or heavy chains, since this might complicate the process of monoclonal hybridoma selection. However, both non-secretor and secretor myelomas have been used successfully. Some commonly used mouse myeloma lines...
Flow cytometry 101 Principle
The fluorescent tetramers are added to a cell population and the sample is analysed using a fluorescence activated cell sorter FACS . Protocol 8. Flow cytometry Equipment and reagents Cell population Fluorescent tetramer see Protocol 7 Fluorochrome-conjugated anti-CD8 Caltag PBS pH 7.3 FACS and tubes Becton Dickinson 1. Centrifuge the cell population s in FACS tube s , e.g. at 500 g for 5 min in 50-100 d of PBS. 2. Add the titrating amounts fluorescent tetramer to the required tubes.3 Incubate...
Deutsche Sammlung von Mikroorganismen und Zellkulturen DSMZ
Mascheroder Weg lb, D-38124 Braunschweig, Germany. Dynal Dynal UK Ltd, Thursby Rd, Croft Business Park, Bromborough, Wirral, Merseyside L62 3PW, UK. Dynal UK Ltd, Station House, 28 Grove Street, New Ferry, Wirral, Merseyside L62 5AZ. Dynal Inc., 475 Northern BLVD, Great Neck, NY 11021, USA. Dynal, P.O. Box 158, Sk0yen, N-0212, Oslo, Norway. Eppendorf Scientific Inc., 1 Cantiague Road, P.O. Box 1019, Westbury, NY11590-0207, USA. Eppendorf-Netherler-hinz GmbH, Barkhansenweg 1, D22339 Hamburg,...
Preparation of lymphoid cells and tissues for immunohistochemistry
Immunohistochemical procedures can be applied to cytological preparations cell smears, cytocentrifuge preparations, and cell imprints , frozen sections, paraffin sections, and resin sections. Procedures used on resin sections using light microscopy are adequately documented elsewhere 1 , as are all those used in electron microscopy 2 . Whichever type of preparation is made, the cells and tissues will be supported on a glass slide, but the very nature of the immunohistochemical procedure greatly...
Generation of CD4 Tcell lines and clones
CD4 T-cell lines specific for soluble proteins can only be initiated with lymphoblasts, previously activated in vivo. Bulk cultures are established by stimulating spleen or lymph node cells with soluble protein antigen. Spleen cells already contain a source of APCs however, lymph node cells may require additional APCs in the form of irradiated autologous spleen cells. Viable cells, recovered from the bulk cultures, are repeatedly stimulated with antigen and APCs to establish T-cell lines, and...
Analysis of light scatter by flow cytometry size and granularity
One of the characteristic features of apoptosis is cell shrinkage. The plasma membrane becomes convoluted and acquires a blebbed appearance. In addition to cell shrinkage, the apoptotic cell finally develops chromatin condensation and nuclear fragmentation. Interaction of the cell with the laser beam in the flow cytometer results in light scatter. Light scattered in the forward direction forward scatter correlates with the cell size, and light measured at 90 to the laser beam side scatter...
Detection by oligonucleotide probing PCRSSOP
Detection of polymorphism by oligonucleotide probing has been a well-established method for HLA typing 13 . The method was quickly adopted in response to the poor resolution and technical difficulties met in performing HLA class II typing by serology. PCR-SSOP for class II 44-46 marked the first real and popular use of PCR for HLA typing. The strategy for PCR-SSOP is straightforward see Figure 4A . A specific PCR product usually based on a gene locus-specific reaction is blotted on to a...
Measuring the antigenspecific release of cytokines by CTLs
Most CTLs release a variety of cytokines and chemokines on antigen-specific contact 26, 40-44 . The most commonly produced are IFN-7 26, 40, 45, 46 and tumour necrosis factor-alpha TNF-a 42,47,48 , but other soluble factors include TNF-p 41 , granulocyte-macrophage colony-stimulating factor GM-CSF , IL-2, IL-3, and IL-4 42 , and the CC chemokines macrophage-inflammatory protein-l-alpha MIP-la , MIP-1 5, and RANTES 43, 44, 49 . It is therefore possible to use the antigen-specific release of...
Protocol 4 Microlymphocytotoxicity assay
Frozen Terasaki plates containing antisera including negative and positive controls and light mineral oil see Protocol 1 . Lymphocytes or PMBCs at 106 ml . PBS 50 til ml Acridine Orange in PBS 1. Thaw the stored Terasaki plates containing antisera see above . 2. Stain the required number of lymphocytes or PBMCs at a con- Frozen complement see Section 4.1.2 100 ng ml ethidium bromide in PBS Ink solution ethidium bromide black India ink 1 4 v v , respectively centration of 10 cells ml by adding...
Demonstration of viral episomes and virion DNA
To demonstrate and distinguish persistent non-integrated episomal slowly migrating or lytical linear higher mobility viral DNA, in situ lysis gel electrophoresis can be used. The method was originally developed by Gardella 64 , and a simplified version is now used modified after ref. 22 . Typical examples for 'Gardella gels' are shown in various reports 11, 22, 36, 64, 65 . The electrophoresis is done in a vertical 1 agarose gel in 1 X TBE 89 mM Tris, 89 mM boric acid, 20 mM EDTA with gel size...
Virus titration
The limiting dilution method is the simplest way to estimate virus titres. OMK cells are trypsinized, split into 24-well plates, and incubated at 5 C02. The cell-free virus suspension is serially diluted in DMEM at liT4 to 10 9 and 1 ml of each dilution is added to each well of the 24-well plate the following day. It is important to perform the assay in triplicate at least, and to run control cultures. The plates are observed for a minimum of 14 days and the progress of the cytopathic effect...
Considerations in the choice of method and label
All the staining methods and labels described in Sections 3.1 and 3.2 can be applied to all the preparations shown in Table 1 from all lymphoid cell and tissue specimens. There is no single optimal combination of staining method and label. The optimum is determined by a number of important factors that should be considered at an early stage a Blocking endogenous peroxidase in cytological preparations and frozen sections can be damaging. In paraffin sections of bone marrow and spleen, the...
Immunoblotting 41 Introduction
Blotting is the term used for transferring molecules to a matrix on which they are immobilized. It was originally described for DNA 22 , but subsequently a method for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets was devised 23 . This method is called Western blotting. Immunoblotting describes the technique of probing a Western blot with polyclonal or monoclonal antibodies to identify specific antigens 24 . The principle of immunoblotting is the same...
Cloning hybridomas
Once the screening assay indicates that a well contains a hybridoma of interest, the contents of the well should be cloned as soon as possible. There are several reasons for cloning, the main one being to ensure that a monoclonal reagent is produced. However, it is also important to clone positive wells to prevent them being overgrown by non-specific cells. The most common cloning method is that of limiting dilution, in which wells are plated out from a starting culture into a number of...
lines
ATCC TIB-152 DSMZ ACC-282 ECACC 88042803 ECACC 88052401 Fusion partner for Thybridomas 8-azaguanine resistant ATCC CRL-1582 DSMZ ACC-362 ECACC 85011413 ATCC CRL-1552 DSMZ ACC-84 ECACC 90021901 Uses TNF-a as growth factor Secretes IL-2 Source abbreviations ATCC, American Type Culture Collection DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen ECACC, European Collection of Animal Cell Cultures Source abbreviations ATCC, American Type Culture Collection DSMZ, Deutsche Sammlung von...
Immunoprecipitation using antibody directly conjugated to Sepharose beads
A further improvement to using Protein G-Sepharose is to covalently bind the antibody directly to CNBr-activated Sepharose beads Pharmacia . Between 1 and 10 mg of antibody can be bound adequately to 1 ml of beads. Antibody-Sepharose is used in exactly the same way as described in the protocol for Gammabind Protein G-Sepharose see Protocol 4 . The immunoprecipitation shown in Figures 1 and 2 were performed using antibody coupled to beads. The quickest immunoprecipitation method, and that which...
Protocol 8 Transfer of proteins to membrane
Electroblotting apparatus Bio-Rad or Hoefer Scientific Instruments Whatman 540 paper or equivalent Transfer membrane nitrocellulose, PVDF, or nylon Hybond series from Amersham 1. Separate proteins by SDS-PAGE, include one lane of pre-stained molecular weight markers to indicate the membrane orientation and protein transfer. 2. Wear gloves during the following procedures as oil from hands can block transfer. 3. After electrophoresis remove the stacking gel and place the separating gel in...
Formation of tetramers 91 Principle
Fluorescent streptavidin is added in a 1 4 molar ratio streptavidin refolded complex . The use of deglycosylated streptavidin may reduce non-specific binding of the complex to cells. The fluorescent markers we have tested include fluorescein isothiocyanate FITC , phycoerythrin PE , and allo-phycocyanin APCN . The bright fluorescence obtained with PE often means that it is the marker of choice. Streptavidin is added slowly to ensure complete saturation of all biotin-binding sites. Protocol 7....
Definition of apoptosis
Apoptosis is a distinct mode of cell death that is responsible for the deletion of cells during development, and the deletion of superfluous cells from normal tissue sometimes it also occurs in pathological situations. Apoptosis was originally detected because of the distinctive morphology of apoptotic cells, which allows them to be easily distinguished from healthy cells and from those dying by necrosis. Although different cell types do not always display all the hallmarks of apoptosis, cell...
Antigen Concentration gml
Figurel. Coupling of antigen to latex microspheres amplifies the proliferative T-cell response. Response of the poliovirus-specific T-cell clone 3N2s5.1 against disrupted poliovirus type 3 disrupted by boiling in Tris buffer with 0.2 SDS and 0.5 2-mercaptoethanol coupled to latex microspheres , or uncoupled disrupted virus O , or FCS-coupled microspheres as a control. The nominal antigen concentration represents the estimated final protein concentration of uncoupled disrupted virus protein and...
Primer design 1
The design of primers is based upon the amplification refractory mutation system ARMS principle 42 . This principle holds the key to specificity within the PCR-SSP assay. In the PCR amplification, extension takes place from the 3' end of the primer. Hence, if the polymorphism against which the primer holds specificity is located at the 3' end of the primer sequence, under the appropriate levels of stringency, extension will only be permitted when the primer and template are matched at this...
Inclusion body purification 41 Principle
The proteins are synthesized as inclusion bodies, which can be rapidly purified by either chemical enzymatic or physical lysis methods. Protocol 2. Inclusion body purification by physical lysis Triton wash 0.5 Triton X-100, 50 mM Tris pH 8, 100 mM NaCI, 0.1 azide, 1 mM EDTA, 1 mM DTT Resuspension buffer 50 mM Tris pH 8, 100 mM NaCI, 1 mM EDTA, 1 mM DTT . Urea buffer 8 M urea, 0.1 M NaH2PO 0.01 M Tris pH 8, 0.1 mM EDTA, 0.1 mM DTT Duolite indicator resin BDH 1. If necessary thaw the bacterial...
SBT strategies
As with other molecular strategies, HLA typing by SBT requires an initial PCR to generate a specific template containing the regions of polymorphism which define the HLA alleles. This HLA specific template is then sequenced using sequencing primer sites found within the template. These sequencing primer sites can be nested within the PCR template, or introduced as 'tails' incorporated in the PCR primers. There are a number of strategies for generating template by PCR. A single PCR may be...
Generation of CD8 Tcell lines and clones
Spleen cell preparations, which contain CTL memory cells from primed mice previously infected with the virus or immunized with viral antigen in an appropriate live vector or adjuvant system, are restimulated in vitro by antigen in bulk cultures. These bulk cultures provide sources of effector cells for establishing virus-specific CTL lines see Protocol 22 or for the immediate detection of CTL responses see Protocol 23 . The murine spleen cells are restimulated in vitro with target cells see...
Growthtransformation of human T ceils
Infection of primary human T lymphocytes with HVS subgroup C strains notably strain C488 , but not with strains of subgroups A or B, yields continuously proliferating, mature T-cell lines 11 reviewed in refs 12 and 13 . Permanently growing T-cell lines have been obtained from primary cells of various sources. Polyclonal preparations of mononuclear cells from adult peripheral blood or from cord blood, from thymus or bone marrow, as well as characterized T-cell clones, or flow cytometry-sorted T...
AnnexinV
Vital cells maintain a degree of plasma membrane asymmetry, and phos-phatidylserine PS is maintained on the inner plasma-membrane leaflet. Apoptotic lymphocytes expose PS on the outer membrane leaflet early after the onset of apoptosis, even though the integrity of the membrane has not been compromised at this stage. PS exposure seems to last from the early execution phase of apoptosis until the final stage, in which the cell is broken up into apoptotic bodies. Annexin V has been shown to...
Biochemical analysis of antigens in conjunction with immunoprecipitation
The glycosylation status of antigens, purified by immunoprecipitation, can be analysed with different enzymes that cleave different carbohydrate moieties, prior to SDS-PAGE analysis. For example, peptidyl-A -acetylglucosaminidase F PNGase F cleaves most N-linked carbohydrate moieties from the protein 15 and O-glycosidase cleaves mature O-linked carbohydrate moieties from glycoproteins 16 . Removal of carbohydrate from the antigen is detected as a reduction in apparent molecular weight see...
PCR cycling parameters
An example of PCR cycling parameters is 95 C for 20 sec, 62 C for 45 sec, 72 C for 2 min for 30 cycles. At 95 C, the double-stranded DNA is denatured to single strands. At 62 C, the primers anneal to the single strands, and at 72 C, the enzyme using the dNTPs, extends from the primer to produce a second DNA strand. One approach used for thermal cycling is the 'step-down' or 'touch-down' PCR, in which the first few cycles of amplification are of high annealing stringency. Although the...
Contributors
Department of Immunology, Imperial College School of Medicine at St Mary's, Norfolk Place, London W2 IPG, UK. Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK. Department of Paediatric Pathology, Womens' Centre, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK. MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, UK. Department of Microbiology and Immunology, University of Leicester, Medical Sciences...
The Practical Approach Series
Department of Biochemistry and Molecular Biology University of Leeds, Leeds LS2 9JT, UK See also the Practical Approach web site at http www.oup.co.uk PAS indicates new and forthcoming titles Affinity Chromatography Affinity Separations Anaerobic Microbiology Animal Cell Culture 2nd edition Animal Virus Pathogenesis Antibodies I and II Antibody Engineering Antisense Technology Applied Microbial Physiology Basic Cell Culture Behavioural Neuroscience Bioenergetics Biological Data Analysis...
Protocol 2 Lymphocyte isolation Note
a It is usually advisable to perform the isolation under sterile conditions, particularly if the cells will be required for transformation into a cell line, or are required for functional studies. b This procedure should yield approximately 106 PMBCs per 10 ml of blood processed. Sodium citrate or preservative free sodium Heparin, 1000 U aI 0.3 ml per 20 ml blood For defibrination by sodium citrate step 2a 250 ml Erlenmeyer conical flask, 30 glass beads, 1 M CaCI2 Orbital shaker Innova 2000...
Transformation of macaque B cells with herpesvirus papio
B cells from macaques like Rhesus monkeys Macaca mulata and cynomol-gus monkeys Macaca fascicularis can be transformed by herpesvirus papio, an EBV-like virus of baboons Papio hamadryas 71-73 . The herpesvirus papio-producer cells S594 71 are seeded at 2 X 105 ml in complete RPMI and cultivated for 10 d at 37 C. The supernatant is then filter-sterilized and stored in aliquots at -80 C. Transformation of fresh macaque mononuclear cells is performed in the same way as for the human EBV protocol....
Protocol 11 Irradiated splenic APCs
Spleen cells see Protocol3 . PBS with 10 FCS Irradiation source Cobalt-60, Caesium-137, or X-ray Complete medium RPM-1640 supplemented with 10 FCS RPM 1-2 supplemented with 2 FCS 1. Prepare spleen cells see Protocol 3 and suspend in PBSa with 10 FCS in a 10 ml plastic tube. 2. Place the tube within the chamber of a cobalt-60, caesium-137, or X-ray source and expose to 15-30 Gy irradiation.fa 3. Wash the cells with RPM 1-2 and centrifuge at 300 g for 5 min at 4 C and resuspend them in complete...
Quantitation at the clonal level using limiting dilution analysis LDA
To quantify the number of lymphocytes that have a defined functional property e.g. antigen-specific cytotoxicity within a mixed population of cells, one needs an assay that is capable of detecting the presence of a single responding cell. Because the cytotoxic activity of an individual T cell is too small to be measured accurately with existing methods, it is necessary to amplify the response by stimulating the single responding cell to undergo clonal proliferation in vitro to generate a...
Refolding by dilution 51 Principle
The d naturant effect of the urea is removed by dilution in a refolding buffer. This allows HLA heavy chain, 32m, and epitope peptide to associate in a conformationally correct form which can be subsequently isolated. . Refolding buffer for HLA A201 and B 3501 use 100 mM Tris pH 8, 400 mM L-arginine HCI, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 2 mM EDTA 1. Pre-cool 1000 ml of the refolding buffer to 4 C before adding the glutathiones. Then add approximately 30 mg of p2m which is...
Methods of HLA typing
The remainder of this chapter will review some of the main methods currently applied to HLA typing. As already mentioned, it would be impossible to include full and detailed procedures due to the number of methods, and their complexity. However, this chapter describes the relevant aspects of each method discussed with regard to its performance and how it is best applied. References for each of the methods will provide more detailed information as to the performance of each assay. There are a...
Viable cell count
A variety of vital stains can be used for obtaining viable lymphoid cell counts. The combination of Ethidium Bromide and Acridine Orange is one of the simplest and least ambiguous for the discrimination of viable and non-viable cells, especially where cell preparations are contaminated with red cells e.g. ex-vivo, murine spleen-cell preparations . However, these compounds are carcinogenic and their use requires access to an UV-microscope. Alternatively, viable cells can be counted by Trypan...
PCR buffer
This is usually made, or provided, at a 10 X concentration. It includes salt, a buffer to maintain pH, and magnesium chloride. Components such as glycerol, albumin, detergent, DMSO, and other additives can be included to help stabilize the reaction. Many of the components of the buffer are dependent on the particular requirements of the enzyme used. The addition of DMSO can help reduce the formation of secondary DNA structures which may affect the efficiency of the PCR reaction. All the...
Alkaline phosphatase
This is the most widely used, alternative enzyme label to HRP. The major advantage is in its use as a label on preparations high in endogenous peroxidase or where blocking methods are harmful to the preparation. Thus, it is our method of choice for cytological specimens, bone-marrow aspirates trephines, spleen, and frozen sections. Endogenous alkaline phosphatase in bone, liver, and kidney is easily blocked by the addition of levamisole at the visualization stage. Intestinal and placental...
The microlymphocytotoxicity assay
As discussed, this assay detects HLA polymorphism, expressed on lymphocytes, through the specificity of a panel of antisera. Whether a particular antiserum has bound to the HLA antigen on the cell is detected by the addition of complement, which will cause cell lysis in the presence of bound antibody see Figure 3 . Cell lysis can be seen by the uptake of dye into the cell. If antibody is not bound to the HLA antigen, then complement will not be activated and the cell will remain viable. The...
Screening assays
It is of paramount importance to decide on, and test in good time, a suitable assay to screen hybridoma culture supernatants or T-cell hybridomas this really cannot be overemphasized. The need to test the animal sera before fusion provides the ideal opportunity to optimize the assay. It is important to include normal mouse sera as a control. The more specific and simple the screening test, the better the chance of quickly and easily obtaining the desired hybridoma, but the procedure for...
Assays for CD4 function 41 Principle of proliferation assay
Radiolabeled thymidine, 3H thymidine, provides an alternative nucleotide which can be incorporated into DNA. As cells grow and divide, DNA is synthesized and the amount of incorporation is directly proportional to the level of cell growth. Optimal lymphocyte growth can be achieved using a variety of different stimuli, notably anti-CD3 and the protein kinase C PKC activator PMA phorbol myristate acetate immobilized anti-CD3 and anti-CD28 or PMA in conjunction with the calcium ionophore,...
Protocol 4 Immortalization of human Tcells by HTLV1
HTLV-1 producer cells like MT-2 or HUT-102 Complete medium RPMI-1640, FCS 10 , 350 xg ml L-glutamine, and 100 ig ml supplemented with 10-100 U ml recombinant IL-2 24-well tissue-culture plates e.g. Nunc T-cells to be transformed, showing rapid growth 2-3 days after activation with PHA or specific antigen Equipment and reagents for PCR and flow cytometry 1. Seed rapidly proliferating T cells at 106 cells per well into 24-well plates, in complete medium supplemented with IL-2. 2. Irradiate...