The last 25 years – the most highly cited papers

Journal of Immunological Methods 216 Ž1998. 7]36

The last 25 years ] the most highly cited papers The relative importance of scientific articles is always of interest. As part of the look back over the last 25 years, the Institute for Scientific Information ŽISI. was asked to provide a list of the most highly cited papers published in the Journal of Immunological Methods during that time. Below are listed the highest ranked 100 papers, with abstracts Žif provided. of the top 50 papers. The abstracts of all of the 100 papers may be found at the Journal of Immunological Methods Web-site, the URL of which is http:rrwww.elsevier.nlrlocaterjimweb. The ranking is based on the total of all of the citings, totalled over the period since publication. The most highly cited paper, by Tim Mosmann, for example, received almost 4000 citations. On the JIM Web-site, the number of citations are listed, alongside the ranking of the papers. The information provided by ISI, can be provided for any paper with more than 100 citations, and is copyright 1998.

Rank: 1. J. Immunol. Methods 65, 55]63 (1983) RAPID COLORIMETRIC ASSAY FOR CELLULAR GROWTH AND SURVIVAL: APPLICATION TO PROLIFERATION AND CYTOTOXICITY ASSAYS Tim Mosmann DNAX Research Institute of Molecular and Cellular Biology, Inc., Palo Alto, CA 94304, USA Abstract: A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. The method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer ŽELISA reader. and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the

lack of any radioisotope. We have used the assay to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.

Introduction: Many biological assays require the measurement of surviving andror proliferating mammalian cells. This can be achieved by several methods, e.g. counting cells that includerexclude a dye, measuring released 51 Cr-labeled protein after cell lysis, and measuring incorporation of radioactive nucleotides Žw 3 H xthymidine or w 125 Ixiododeoxyuridine. during cell proliferation. The radioactive method can be partially automated and can handle moderately large numbers of samples, but even with these methods, it is difficult to process thousands of assay points per day. In our current research we assay many samples of various lymphokines that induce cell proliferation, and so we required a rapid and quantitative assay capable of handling large numbers of samples. Viable cells could be measured by using any of several staining methods, but we wished to avoid

0022-1759r98r$19.00 Q 1998 Elsevier Science B.V. All rights reserved. PII S0022-1759Ž98.00068-4

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Abstracts r Journal of Immunological Methods 216 (1998) 7]36

any washing steps that would increase processing time and sample variation. Multiwell scanning spectrophotometers ŽELISA readers. can measure large numbers of samples with a high degree of precision, and so we investigated the possibility of using a color reaction as a measure of viable cell number. Ideally, a colorimetric assay for living cells should utilize a colorless substrate that is modified to a colored product by any living cell, but not by dead cells or tissue culture medium. Tetrazolium salts are attractive candidates for this purpose, since they measure the activity of various dehydrogenase enzymes ŽSlater et al., 1963.. The tetrazolium ring is cleaved in active mitochondria, and so the reaction occurs only in living cells. We have developed a rapid colorimetric assay, based on the tetrazolium salt MTT Ž3-Ž4,5-dimethythiazol-2-yl . -2,5-diphenyl tetrazolium bromide. wSigma a M2128x, that measures only living cells and can be read on a scanning multiwell spectrophotometer ŽELISA reader.. This assay is versatile and quantitative, and we consider it a significant advance over traditional techniques for several commonly used proliferation and cytotoxicity assays. References: Baehner, R.L., L.A. Boxter and J. Davis, 1976. Blood 48, 309. Farrar, J.J., J.F. Farrar, P.L. Simon, M.L. Hifliker, B.M. Stadler and W.L. Farrar, 1980. J. Immunol. 125, 2555. Kappler, J.W., B. Skidmore, J. White and P. Marrack, 1981. J. Exp. Med. 153, 1198. Lachman, L.B., M.P. Hacker, G.T. Blyden and R.E. Handschumaker, 1977. Cell. Immunol. 34, 416. Slater, T.F., B. Sawyer and U.D. Strauli, 1963. Biochim. Biophys. Acta 77, 383. Keywords: Lymphokine assays; Proliferation assays; Colorimetric assay; Tetrazolium; TCGF. A comment from Tim Mosmann The MTT assay was developed during the early stages of the work at DNAX on the characterisation and molecular cloning of cytokines. We were

Professor Tim Mosmann, author of the most highly cited papers published in the Journal of Immunological Methods.

investigating several different cytokines, but many of the cytokine assays in use at the time involved stimulation of proliferation, measured by thymidine incorporation. As we planned to assay large numbers of samples, we wished to develop a faster method for analysing proliferation. Remembering an undergraduate project in which seeds were painted with a tetrazolium salt solution to measure viability, we ordered all the tetrazolium salts in the Sigma catalog, and tested whether some common assay cell lines could cleave these to colored products. One compound, MTT, was cleaved by all cell lines that we tested, and so we developed a simple colorimetric assay for live cells. In the original paper, we described the use of this assay for cytotoxicity and proliferation } more cells produce a larger signal, and proliferating cells are normally larger and give a higher signal than non-proliferating or dying cells. The original criteria for the design of the MTT assay were that it should be rapid and accurate,

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

so that we could reproducibly assay large numbers of samples. For this purpose, the number of steps was kept to a minimum, and all steps were addition steps, which could be accurately controlled. The resulting MTT assay provided us with large amounts of data, fulfilling the original purposes. As a bonus, this large volume of data also allowed us to investigate the bioactivities of T cell supernatants in greater detail, revealing that there were qualitative differences between T cell supernatants with regard to activities such as T cell growth factor and mast cell growth factor. In turn, this allowed us to see that there were two types of T cell clone, Th1 and Th2, producing similar bioactivities that were nevertheless due to different cytokines. The subsequent popularity of the MTT assay may be due to some of the same features that were the driving force for its invention } rapidity, a small number of steps, no radioactivity, and applicability to several cell types. April 1998 Professor Tim Mosmann Department of Medical Microbiology and Immunology Uni¨ ersity of Alberta Edmonton, Alberta Canada T6G 2R3

Rank: 2. J. Immunol. Methods 43, 349]350 (1981) SHORT COMMUNICATION: A SIMPLE METHOD OF REDUCING THE FADING OF IMMUNOFLUORESCENCE DURING MICROSCOPY G.D. Johnson and Gloria M. de C. Nogueira Araujo Bone and Joint Research Unit, The London Hospital Medical College, London E1, UK The phenomenon of fading of stained preparations on exposure to fluorescence-stimulating radiation has been an accepted feature of the immunofluorescent ŽIF. procedure since the defini-

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tive description of the technique by Coons and Kaplan Ž1950.. Exposure to short-wave Žultraviolet. excitation as provided by high-pressure mercury vapour burners in common use results in very rapid fading, especially of fluorescein staining. The blue light emitted by quartz]halogen bulbs produces less rapid fading, but although these low-powered sources are entirely satisfactory Žwith appropriate microscope accessories . for viewing gross staining as seen for example, with tissue sections in screening tests for antibodies, more recent applications of IF involving the staining of specific determinants on cell surfaces and precise localisation of intracellular components and inclusions require maximum excitation by the more powerful light source. We have found that the addition of p-phenylenediamine to the buffered glycerol used for mounting the stained preparations has a marked effect in retarding fading during microscopy. This is now routinely employed in this laboratory in the test for antibody to rheumatoid arthritis nuclear antigen ŽRANA. ŽAlspaugh and Tan, 1976. which involves the recognition of very fine speckled nuclear staining of EB virus-infected B lymphocytes ŽRaji or Wil 2 cells.. It enables prolonged inspection of individual fields under a fluorescence microscope ŽZeiss. equipped for incident illumination with an HBO 50 mercury burner, the installation having a direct short light-path in order to produce maximum excitation. Only minimal fading of the fluorescent staining is discernible after 20 min continuous exposure, whereas marked bleaching occurs almost instantly with preparations mounted in conventional buffered glycerol. The improved mountant has also proved to be satisfactory in other systems involving cellular reactions and for tissue selections examined with mercury light excitation, and appears to have general applicability. An obvious further advantage is in the photographic recording of results. Preparation of Mountant: Add 10 ml of phosphate-buffered saline Ž0.01 M PO4 pH 7.4 in 0.15 M NaCl. containing 100 mg of p-phenylenediamine ŽHopkin and Williams, Romford, UK. to 90 ml of glycerol. The final

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Abstracts r Journal of Immunological Methods 216 (1998) 7]36

pH should be adjusted to approximately 8.0 with 0.5 M carbonate]bicarbonate buffer pH 9.0. Solutions containing p-phenylenediamine rapidly become brown on standing at room temperature and are then unsatisfactory for use in critical microscopy. This problem is overcome by storing the mixture in the dark at y208C. Acknowledgement: The possibility of using p-phenylenediamine to reduce bleaching was suggested to us by Mr KC McNamee of Carl Zeiss ŽOberkochen. Ltd. References: Alspaugh, M.A. and E.M. Tan, 1976. Arthr. Rheum. 19, 711. Coons, A.H. and M.H. Kaplan, 1950. J. Exp. Med. 91, 1.

Rank: 3. J. Immunol. Methods 35, 1]21 (1980) REVIEW ARTICLE: PRODUCTION OF MONOCLONAL ANTIBODIES: STRATEGY AND TACTICS

ment of standard trays by microtrays resulted in a higher frequency of surviving hybrids. By using a feeder layer, the spleen cell input can be reduced 50-fold. At such low multiplicities the positive cultures arise predominantly from single hybrids, eliminating the need for subsequent cloning. The hybrids can be labelled and will yield in serum-free medium. Since at least a third of them inherit the fast growth rate of their myeloma parent and keep producing over 2000 antibody molecules per second, readaptation to ascitic growth is also superfluous. A simplified technique of producing monoclonal antibodies is given in detail, together with the experimental evidence prompting modifications of the classical method of Kohler and Milstein Ž1975, ¨ 1976..

Rank: 4. J. Immunol. Methods 5, 239]247 (1974) SEPARATION OF MOUSE SPLEEN CELLS BY PASSAG E TH R O UG H C O LUM N S O F SEPHADEX G-10

S. Fazekas de St.Groth and Doris Scheidegger Basel Institute for Immunology, 4005 Basel, Switzerland

Iris A. Ly and Robert I. Mishell Dept. of Bacteriology and Immunology, Uni¨ ersity of California at Berkeley, Berkeley, CA 94720, USA

Abstract: A myeloma line has been developed which produces no globulin chains of its own, has a duplication time of 8.7 h, fuses effectively with Blymphoblasts and produces stable hybrids. An enhancing effect of macrophages on hybridoma yields has also been observed. Among the fusing agents tested, PEG of mol.wt. 4000 gave the best results, 208C being the optimum working temperature. The maintenance medium of choice has been found to be Iscove’s with 10% FCS. Direct exposure of fusion cultures to a selective medium with hypoxanthine, aminopterine and thymidine reduced the labor involved and increased the yield. A mechanical device for changing the medium has been designed. The replace-

Abstract: A convenient simple method for preparing sterile subpopulations of mouse spleen cells is described. Passage of mouse spleen cells through a column of Sephadex G-10 beads results in the selective retention of antibody-forming cells and of accessory cells required for in vitro induction of primary and secondary humoral immune responses to sheep red blood cells. Restoration experiments with peritoneal cells and 2mercaptoethanol show that normal and antigenprimed T lymphocytes with helper function and B lymphocytes which are precursors of antibody-forming cells are present in substantial quantity in the effluent population. In several restoration experiments, the effects

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

of 2-mercaptoethanol and peritoneal cells were synergistic.

Rank: 5. J. Immunol. Methods 5, 131]135 (1974)

AN IMPROVED ROSETTING ASSAY FOR DETECTION OF HUMAN T LYMPHOCYTES Manuel E. Kaplana and Connie ClarkU a Dept. of Medicine, VA Hosp., Minneapolis, MN U 55417, USA and Uni¨ ersity of Minnesota School of Medicine, Minneapolis, MN 55455, USA

Abstract: An improved procedure for the detection of human T lymphocytes using sulfhydryl-treated sheep erythrocytes is described. Major advantages of this procedure compared with previously described methods are: 1. a highly purified sulfhydryl reagent, AET, readily available, inexpensive, and has an indefinite shelf life at room temperature; 2. AET-treated sheep erythrocytes, once prepared, are stable and usable over a 5 day period; 3. the rosetting procedure is rapid, and highly reproducible, minimizing differences due to sheep cell age and source; 4. the rosettes formed are very stable, being quite resistant to disruption by routine handling procedures; 5. the percentage of rosette-forming cells in normal peripheral blood is significantly and reproducibly increased; 6. the data reported confirm the presence of ‘double marker’ lymphocytes in all normals and chronic lymphocytic leukemia patients studied.

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Michael G. Mage, Louise L. McHugh and Thomas L. Rothstein Laboratory of Biochemistry, Di¨ ision of Cancer Biology and Diagnosis, National Cancer Institute, Bethesda, MD 20014, USA

Abstract: Mouse spleen cells could be preparatively separated into immunoglobulin positive ŽIg q . and immunoglobulin negative ŽIg y . populations by incubating as many as 2 = 10 8 cells per 100 mm diameter petri plate coated with specifically purified goat anti-mouse immunoglobulin. The nonadherent population was 95% or more Ig y , and possessed graft versus host and cytotoxic effector activities, as would be expected for T cells. They could also give a mixed lymphocyte reaction and generate cytotoxic effector activity on culture in vitro. The adherent cells could not be released undamaged from plates coated with undiluted anti-Ig, but they could be released from plates coated with 1r4 or 1r10 dilution of anti-Ig in an irrelevant antibody. The released cells were over 90% viable by trypan-blue staining, and 94% or more of the viable cells were Ig q .

Rank: 7. J. Immunol. Methods 20, 241]253 (1978)

USE OF STAPHYLOCOCCAL PROTEIN A AS AN IMMUNOLOGICAL REAGENT

J. Immunol. Methods 15, 47]56 (1977)

James W. Goding Walter and Eliza Hall Inst. of Medical Research, Royal Melbourne Hospital, Park¨ ille VIC 3050, Australia

MOUSE LYMPHOCYTES WITH AND WITHO U T SU R FAC E IM M U N O G LO BU LIN : PREPARATIVE SCALE SEPARATION IN POLYSTYRENE TISSUE CULTURE DISHES COATED WITH SPECIFICALLY PURIFIED ANTI-IMMUNOGLOBULIN

Abstract: This brief review summarises the major uses of staphylococcal protein A in immunology. Protein A is covalently linked to the cell wall of most strains of Staphylococcus aureus, and binds immunoglobulin molecules with high affinity. The

Rank: 6.

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Abstracts r Journal of Immunological Methods 216 (1998) 7]36

principal molecule bound is IgG, although in many cases binding is restricted to certain IgG subclasses. Some IgM and IgA binds in certain species. This property allows rapid, simple and economical methods for the purification and analysis of immunoglobulins, and the fractionation of subclasses which are difficult to separate by other means. Fractionation on protein A affinity columns is a simple and efficient way of separating immunoglobulin FŽab. and FŽab9. 2 from Fc fragments. Intact staphylococci are useful as a solid phase adsorbent for isolating antigen]antibody complexes, membrane antigens and receptors, and to replace ‘second antibody’ in radioimmunoassay. Finally, protein A has proven useful for the study of antigens and receptors on the surface of intact cells, and for the detection of antibody-secreting cells. Thus, the use of protein A is now the method of choice for many preparative and analytical purposes in immunology.

Rank: 8. J. Immunol. Methods 13, 215]226 (1976) CONJUGATION OF ANTIBODIES WITH FLUOROCHROMES: MODIFICATIONS TO THE STANDARD METHODS James W. Goding Walter and Eliza Hall Inst. of Medical Research, Royal Melbourne Hospital, Park¨ ille VIC 3050, Australia Abstract: A number of modifications to the standard procedures for coupling of fluorochromes to antibodies are described. The suggested procedures result in economies of time, labour and materials, and allow the reliable production of high quality conjugates. The modifications include the use of staphylococcal protein A-Sepharose for Ža. a simple onestep procedure for preparation of IgG from whole serum, Žb. removal of undigested IgG after pepsin treatment, and Žc. concentration of dilute solutions of IgG. Many other details of coupling procedures are discussed, and modifications sug-

gested. Optimally coupled antibodies were separated by linear salt gradient elution from DEAESephadex, and the effects of fluorescein and tetramethyl rhodamine on the antibody isoelectric point studied.

Rank: 9. J. Immunol. Methods 95, 99]105 (1986) A HIGHLY SENSITIVE CELL LINE, WEHI 164 CLONE 13, FOR MEASURING CYTOTOXIC FACTOR r TUMOR NECROSIS FACTOR FROM HUMAN MONOCYTES Terje Espevik and Jon Nissen-Meyer Institute of Cancer Research, Regionsykehuset, Uni¨ ersity of Trondheim, Trondheim, Norway Abstract: By limiting dilution of WEHI 164 mouse fibrosarcoma cells we have isolated a cell line, WEHI 164 clone 13, which is extremely sensitive to cytotoxic factor ŽCF. derived from human monocytes. By using WEHI 164 clone 13 in a MTT tetrazolium cytotoxicity assay it was found that CF supernatants from activated monocytes had to be diluted 10 5 ]10 6 times to reach the dose which produced 50% dead cells ŽLD50 .. By comparing the LD50 of different target cells, WEHI 164 clone 13 cells were found to be approximately 10 3 times more sensitive for CF-induced cytotoxicity as compared to WEHI 164 parental cells and approximately 10 2 times more sensitive as compared to actinomycin D-treated L929 cells. Treatment of the WEHI 164 clone 13 cells with actinomycin D did not increase their sensitivity for CF-induced cytotoxicity. Recombinant tumor necrosis factor ŽrTNF. also mediated high cytotoxicity towards WEHI 164 clone 13 cells, with an LD50 of 2 = 10y3 ngrml. Neutralizing CF antiserum completely inhibited the toxic activity of rTNF. WEHI 164 clone 13 cells were highly sensitive to monocyte-mediated cytotoxicity in that 1]2 monocytes were able to kill at least 5000 of these target cells. Neutralizing TNF antiserum completely inhibited monocyte-mediated cytotoxicity. These results indicate that the high level of cyto-

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

toxicity mediated by CF supernatants and monocytes on WEHI 164 clone 13 cells is due to TNF as the effector molecule. Keywords: WEHI 164; Cytotoxicity; Monocytemediated; Tumor necrosis factor; Bioassay; MMT.

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vening years, there has been an explosive increase in applications of these techniques, as well as numerous attempts to improve on the original protocols. We will attempt to review the various strategies and also summarize some applications Žfor an earlier review on this subject cf. Gershoni and Palade, 1983..

Rank: 10. J. Immunol. Methods 72, 313]340 (1984)

Keywords: Immunoblotting; Dot immunoblotting; Western blotting.

REVIEW ARTICLE: IMMUNOBLOTTING AND DOT IMMUNOBINDING — CURRENT STATUS AND OUTLOOK

Rank: 11.

H. TowbinU and J. GordonUU Ciba-Geigy Ltd., Pharmaceuticals Research UU Laboratories, Friedrich Miescher-Institut, 4002 Basle, Switzerland

FADING OF IMMUNOFLUORESCENCE DURING MICROSCOPY: A STUDY OF THE PHENOMENON AND ITS REMEDY

U

Introduction: In recent years, solid-phase immunoassay techniques have found wide application in basic research and clinical applications. The high sensitivity which can be obtained with labelled ligands, and the simple separation of bound from unbound reagents make this type of immunoassay particularly attractive. However, the methodology on its own yields no information on the nature of the reacting antigen. To determine homogeneity or identity of unlabelled antigens the classical methodologies of immunodiffusion and immunoelectrophoresis relying on precipitation are available. More recently, as an extension of gel overlay techniques, the development of methods for transferring proteins from the gel phase of electrophoresis to a solid phase for immunoreaction permitted the optimum combination of high resolution gel electrophoresis with the sensitivity and simplicity of solid-phase assays. The first practical methods for transfer of proteins from gel electropherograms to a suitable solid phase were published in 1979. Renart et al. Ž1979. introduced transfer by diffusion to activated cellulose to provide covalent binding, and we ŽTowbin et al., 1979. described the transfer in an electric field and non-covalent binding to nitrocellulose. Diffusional transfer to nitrocellulose was reported by Bowen et al. Ž1980.. In the inter-

J. Immunol. Methods 55, 231]242 (1982)

G.D. Johnson, R.S. DavidsonU , K.C. McNameeUU , G. Russell, D. GoodwinU and E.J. Holborow The Bone and Joint Research Unit, The London U Hospital Medical College, London E1, UK; Dept. of Chemistry, The City Uni¨ ersity, London EC1, UU UK; Zeiss (Oberkochen) Ltd, London W1, UK Abstract: We have previously reported that the addition of para-phenylenediamine ŽPPD. to the glycerol used for mounting preparations stained for immunofluorescence with FITC conjugates results in a marked retardation of the fading that occurs during fluorescence microscopy. Studies of the kinetics of this photo-bleaching performed by microfluorimetry and on FITC conjugate in solution and the effects of a range of additives incorporated in the mountant suggest that the fading is due to a destructive reaction with protein of the dye in its excited singlet state, and that retarding additives suppress this. They also indicate the non-participation of singlet oxygen in the process. PPD was found to be the most efficient retarding agent in terms of effective molar concentration of the substances tested, but promising results were obtained with other materials, in particular 1,4-diazobicyclo-Ž2,2,2.-octane. Keywords: Immunofluorescence fading; Photobleaching; Fluorescence microscopy.

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Abstracts r Journal of Immunological Methods 216 (1998) 7]36

Rank: 12 J. Immunol. Methods 33, 239]247 (1980) A 48-WELL MICRO CHEMOTAXIS ASSEMBLY FOR RAPID AND ACCURATE MEASUREMENT OF LEUKOCYTE MIGRATION

phocytes previously shown to be the principal NK cells in human peripheral blood. Percoll density gradient centrifugation provides a useful tool for analysis of human NK cells and their relationship to other blood mononuclear cells.

Rank: 14. Werner Falk, Richard H. Goodwin Jr. and Edward J. Leonard Immunopathology Section, Lab. of Immunobiology, National Cancer Institute, NIH, Bethesda, MD 20205, USA Abstract: We designed a 48-well chemotaxis chamber to minimize manipulation time and amount of material required by the larger blindwell or Boyden chemotaxis chamber. Cell and chemoattractant dose]response curves showed that results were comparable to or better than those obtained with blindwell chambers. The volume of chemoattractant per well is 25 m l; the number of cells can be as low as 10,000. The time needed for setting up this multiwell unit and for staining the membrane filter sheet is negligible. Combined with the use of an image analyzer to count the number of migrated cells, the method is suitable for clinical research on the functional state of monocytes in large groups of patients.

Rank: 13. J. Immunol. Methods 36, 285]291 (1980) ISOLATION OF HUMAN NK CELLS BY DENSITY GRADIENT CENTRIFUGATION T. Timonen and E. Saksela Dept. of Pathology, Uni¨ ersity of Helsinki, and I-II Depts. of Gynecology and Obstetrics, Helsinki Uni¨ ersity Central Hospital, Helsinki, Finland Abstract: Sedimentation characteristics of human NK cells in discontinuous Percoll density gradients were studied. NK activity against the leukaemic cell line K-562 peaked in a single low density fraction mainly consisting of large granular lym-

J. Immunol. Methods 39, 285]308 (1980) REVIEW ARTICLE: Antibody production by hybridomas James W. Goding Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia Introduction: Conventional antisera contain the products of hundreds or thousands of different antibodysecreting clones. Many of these clonal products may be directed against undesired antigens, such as impurities in the immunising antigen, or molecules, which are closely related to it. Until recently, these considerations have limited what could be achieved by conventional serology. In 1975, Kohler and Milstein demonstrated that ¨ individual clones of normal antibody-secreting cells could be immortalised by fusion with myeloma cells. The resulting ‘hybridoma’ approach has moved serology into a new era of precision. In one blow, the major problems of specificity and reproducibility were solved. Virtually unlimited quantities of homogeneous, exquisitely specific antibodies could be produced, even if the immunising antigen was not pure. In the ensuing 5 years, the use of hybdridoma antibodies has moved into almost every area of biology ŽMelchers et al., 1978; Kennett et al., 1980; Secher, 1980; Staines and Lew, 1980.. It has even penetrated areas where a serological approach had not previously been considered feasible. Monoclonal antibodies have been used for probes for the fine structure of proteins, radioimmunoassay of hormones and drugs, histocompatibility testing, tumour localisation and classification, immunotherapy, purification of molecules by

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

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affinity chromatography, microbial and parasitic disease, neurochemistry and embryology. They have also proven invaluable as a means of ‘fishing’ for previously unidentified molecules, such as new antigens on the surface of subpopulations of lymphocytes. While hybridoma technology is now quite firmly established, many different strategies have been used. In this review, I have summarized the main approaches and tried to give a basis for choice between them. Of course, there is no ‘best’ approach, and success will usually depend on adaptation of the methods to each individual problem.

Rank: 16.

Rank: 15.

Abstract: Two simple semiautomated microassays for the . and hydrogen measurement of superoxide ŽOy 2 peroxide ŽH 2 O 2 . production by cultured macrophages ŽMPs. are described. The measurement of Oy 2 is based on the reduction of ferricytochrome c as assayed by the increase in its absorbance at 550 nm. Quantitation of H 2 O 2 is based on the horseradish peroxidase ŽHRPO.-dependent oxidation of phenol red which is assayed by its increased absorbance at 600 nm. MPs are cultured in monolayers in 96-well flat-bottom tissue culture plates and covered with 100 m l amounts per well of either a ferricytochrome c solution or a solution containing phenol red and HRPO. Following the addition of an agent eliciting an oxidative burst ŽOB. and incubation of the plates at 378C for various time intervals, the changes in the absorbance of ferricytochrome c and phenol red, respectively, are measured directly in the wells of the tissue culture plates with the cells in situ, by using an automatic 8-channel photometer which reads absorbances vertically through individual wells. This instrument, which was originally designed for reading enzyme immunoassays in microtitration plates, can be easily adapted for use in the above tests, when fitted with interference filters with wave lengths of 550 . and 600 nm Žfor the nm Žfor the assay of Oy 2 assay of H 2 O 2 .. The principal advantages of this technique are: the ability to perform the assays directly in the culture plates with cells in situ; the small amounts of cells and reagents needed; its sensitivity and reproducibility; the ease with which

J. Immunol. Methods 29, 17]25 (1979) PRETREATMENT OF PLASTIC PETRI DISHES WITH FETAL CALF SERUM. A SIMPLE METHOD FOR MACROPHAGE ISOLATION Katsuo Kumagai, Kyogo Itoh, Shuhi Hinuma and Masato Tada Dept. of Microbiology, Tohoku Uni¨ ersity School of Dentistry, Sendai, Japan Abstract: We authors have developed a simple method which can recover the highly purified macrophages or monocytes in suspension from mouse peritoneal exudate cells and human peripheral blood mononuclear cells. Plastic Petri dishes coated overnight with heat-inactivated fetal calf serum ŽFCS. selectively bind macrophages and monocytes. The adherent macrophages and monocytes are easily removed by incubation in phosphatebuffered saline containing 0.2% ethylenediamine tetraacetate ŽEDTA. and 5% FCS, and recovered as a cell suspension with greater than 95% purity. A small number of isolated cells can restore the mitogenic response to phytohemagglutinin ŽPHAP. of macrophage-depleted lymphocytes and can lyse 51 Cr-labeled target cells in an antibody-dependent cell-mediated cytotoxicity system. Thus, the method should be valuable for studies of various functions of macrophages and monocytes from different immune tissues of man and animals.

J. Immunol. Methods 46, 211]226 (1981) RAPID MICROASSAYS FOR THE MEASUREMENT OF SUPEROXIDE AND HYDROGEN P E R O X ID E P R O D U C T IO N BY MACROPHAGES IN CULTURE USING AN AUTOMATIC ENZYME IMMUNOASSAY READER Edgar Pick and Diane Mizel Lab. of Microbiology and Immunology, Nat. Inst. of Dental Research, NIH, Bethesda, MD 20205, USA

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Abstracts r Journal of Immunological Methods 216 (1998) 7]36

kinetic experiments can be done; the large number of samples which can be tested in parallel, and especially the speed and convenience offered by the automated reading and printout of absorbance values.

individual stained proteins could be excised and assessed for bound antibody in an indirect radioimmunoassay. Keywords: Chlamydial proteins; Antigenic analysis; Immunoblot; Indirect radioimmunoassay.

Rank: 17 J. Immunol. Methods 55, 297]307 (1982)

Rank: 18. J. Immunol. Methods 16, 165]183 (1977)

THE USE OF TWEEN 20 AS A BLOCKING AGENT IN THE IMMUNOLOGICAL DETECTION OF PROTEINS TRANSFERRED TO NITROCELLULOSE MEMBRANES Byron BatteigerU , Wilbert J. Newhall V U and Robert B. JonesUU U UU Dept. of Medicine, Dept. of Microbiology and Immunology, Indiana Uni¨ ersity School of Medicine, Indianapolis, IN 46223, USA Abstract: The determination of the immunoreactivity of protein antigens in complex mixtures has been greatly facilitated by combining their separation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis Ž SDS ] PAG E . with electrophoretic transfer to nitrocellulose membrane ŽNCM., and probing of bound proteins with specific antisera. Methods using various buffers and blocking agents have been published, but no studies have been published which compare these methods with each other or with others of potential merit. We have performed such a comparative study using protein antigens from Chlamydia trachomatis and Neisseria gonorrhoeae. In addition, we describe a method that blocks unoccupied protein binding sites on NCM with the nonionic detergent Tween 20, rather than proteins. This system proved to be equivalent or superior to other methods evaluated in the detection of immunoreactive proteins, and permitted staining of the NCM for protein after immunological probing. Such staining allowed precise identification of immunoreactive proteins. In addition,

DETECTION OF CIRCULATING IMMUNE COMPLEXES IN HUMAN SERA BY SIMPLIFIED ASSAYS WITH POLYETHYLENE GLYCOL M. Digeon, M. Laver, J. Riza and J.F.Bach INSERM U-25, Hopital Necker, 75730 Paris, France ˆ Abstract: The search for circulating immune complexes by precipitation tests using polyethylene glycol ŽPEG. was performed on a series of normal and pathological sera. Various factors affecting PEG precipitation were studied. Immunoglobulins and complement factors precipitated by PEG Ž3.5%. were quantified and their significance was discussed in relation to serum levels. The PEG test was compared to labeled C1q binding test with a fairly good correlation. The direct evaluation of the amount of C4 precipitated with IgG by 3% PEG ŽC4 test. provided a simpler routine assay than the C1q binding test for detecting complement-fixing immune complexes. The direct PEG test and the C4 test gave positive results in patients with diseases generally presumed to be associated with immune complexes including systemic lupus erythematosus, acute glomerulonephritis, bacterial subacute endocarditis and chronic active hepatitis. The demonstration of HBs antigen and antibody after acid dissociation of PEG precipitates from hepatitis B seronegative sera illustrated the fact that PEG does precipitate and thus concentrates circulating immune complexes.

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

Rank: 19. J. Immunol. Methods 5, 249]252 (1974) SINGLE-STEP SEPARATION OF RED BLOOD CELLS, GRANULOCYTES AND MONONUCLEAR LEUKOCYTES ON DISCONTINUOUS DENSITY GRADIENTS OF FICOLL-HYPAQUE Denis English and Burton R. Andersen Veterans Administration West Side Hospital and The Departments of Medicine and Microbiology, Abraham Lincoln School of Medicine, Uni¨ ersity of Illinois Medical Center, Chicago, Illinois, USA

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m M Ž1]60 nmolrml. range. Due to the non-toxic character of phenol red and HRPO, the assay permits measurement of H 2 O 2 production and release by macrophages for time intervals 5]60 min under regular tissue culture conditions. Using this assay, the ability of a number of agents to induce H 2 O 2 release by guinea pig peritoneal macrophages was demonstrated. These agents were: phorbol myristate acetate ŽPMA., opsonized zymosan, concanavalin A ŽCon A., wheat germ agglutinin ŽWGA., N-formyl-methionylleucyl-phenylalanine ŽFMLP. and A23187.

Rank: 21. Abstract: Centrifugation of heparinized human blood on discontinuous gradients of Ficoll-Hypaque resulted in the simultaneous separation of mononuclear leukocytes, granulocytes, and erythrocytes. Nearly complete separation of cells was achieved with high recovery of each cell type. Leukocytes isolated by this method retained the ability to exclude Trypan blue dye and granulocytes retained their chemotactic responsiveness

Rank: 20. J. Immunol. Methods 38, 161]170 (1980) A SIMPLE COLORIMETRIC METHOD FOR THE MEASUREMENT OF HYDROGEN PEROXIDE PRODUCED BY CELLS IN CULTURE Edgar Pick and Yona Keisari Section of Immunology, Dept. of Human Microbiology, Sackler School of Medicine, Tel-A¨ i¨ Uni¨ ersity, Tel-A¨ i¨ , Israel Abstract: A simple, rapid and inexpensive method for the measurement of hydrogen peroxide ŽH 2 O 2 . produced by cells in culture is described. The assay is based on the horseradish peroxidase ŽHRPO.mediated oxidation of phenol red by H 2 O 2 which results in the formation of a compound demonstrating increased absorbance at 610 nm. A linear relationship between absorbance at 610 nm and concentration of H 2 O 2 was found in the 1]60

J. Immunol. Methods 102, 259]274 (1987) STRATEGIES FOR EPITOPE ANALYSIS USING PEPTIDE SYNTHESIS H. Mario Geysen, Stuart J. Rodda, Tom J. Mason, Gordon Tribbick and Peter G. Schoofs Department of Molecular Immunology, Commonwealth Serum Laboratories, Park¨ ille, Vic 3052, Australia Abstract: A recently developed approach to the synthesis and ELISA screening of large numbers of peptides is described. The method has created the opportunity to tackle questions about the sites and specificity of antigenic determinants which were formerly thought to be too difficult to answer. The various strategies for application of this method are described along with examples of their successful use. They include a procedure for locating all the continuous antigenic peptides of a protein antigen, and the identification of non-replaceable amino acid residues within an antigenic peptide. An approach to the determination of amino acid residues involved in the epitope for any monoclonal antibody is also described. These strategies open up the prospect of rapid mapping of the antigenic properties of hitherto poorly understood antigens. Keywords: Peptide synthesis; ELISA; Enzyme linked immunosorbent assay; Epitope; Mimotope.

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Abstracts r Journal of Immunological Methods 216 (1998) 7]36

Rank: 22. J. Immunol. Methods 7, 291]300 (1975) A PROCEDURE FOR REMOVING RED CELLS AND DEAD CELLS FROM LYMPHOID CELL SUSPENSIONS Wendy F. Davidson and C.R. Parish Dept. of Microbiology, John Curtin School of Medical Research, Australian National Uni¨ ersity, Canberra ACT, Australia Abstract: A procedure is described for simultaneously removing red cells and dead cells from lymphoid cell suspensions, based on the observation that when populations of lymphoid cells are centrifuged on a mixture of IsopaquerFicoll, dead cells and red cells sediment whereas viable cells float. The technique very efficiently removed red cells from a wide range of lymphoid cell suspensions and eliminated lymphocytes killed by mechanical stress, by antibody and complement and by prolonged tissue culture. The depletion of red cells was ) 99% and the recovery of viable lymphocytes usually ) 90%, the resulting cell suspensions being around 95]100% viable. The immunological activity of B cells, helper T cells and cytotoxic T cells was virtually unimpaired by the separation procedure.

Rank: 23. J. Immunol. Methods 68, 167]175 (1984) COMPARISON OF IN VITRO CELL CYTOTOXIC ASSAYS FOR TUMOR NECROSIS FACTOR David A. Flick and George E. Gifford Department of Immunology and Medical Microbiology, Uni¨ ersity of Florida College of Medicine, Gaines¨ ille, FL 32610, USA Abstract: Four published in vitro assays which measure cell cytotoxicity were compared utilizing murine tumor necrosis factor. These included determination of residual cell number by crystal violet staining in the presence and absence of actinomycin

D, lack of viability as determined by neutral red uptake, and w 3 Hxthymidine release in cytotoxin treated L929 cells. Treatment of cells with actinomycin D followed by crystal violet staining was the most sensitive method measured. However, addition of actinomycin D to the neutral red uptake assay could be shown to be even more sensitive. Additionally, it was shown how actinomycin D dosage, cell seeding density and the time of incubation affect TNF titer. Keywords: Actinomycin D; Crystal violet assay; Neutral red assay; w 3 Hxthymidine release assay; Tumor necrosis factor.

Rank: 24. J. Immunol. Methods 14, 267]269 (1977) RAPID IDENTIFICATION OF MONOCYTES IN A MIXED MONONUCLEAR CELL PREPARATION S.B. Tucker, R.V. Pierre and R.E. Jordon Depts. of Dermatology, Immunology and Hematology, Mayo Graduate School., Medical School and Foundation, Rochester, Minn. 55901, USA Abstract: A method for identifying monocytes by the ‘non specific esterase’ stain is described. This method is particularly applicable to mononuclear cell suspensions obtained by Ficoll-Hypaque density gradient separations and allows rapid as well as accurate determinations.

Rank: 25. J. Immunol. Methods 12, 285]288 (1976) SINGLE STEP SEPARATION OF HUMAN T AND B CELLS USING AET TREATED SRBC ROSETTES Andrew Saxon, Joanne Feldhaus and R. Adrian Robins Immunobiology Group, Dept. of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90024, USA

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

Abstract: A technique for the single step separation of human thymus derived ŽT. and Bursa equivalent ŽB. lymphocytes was developed. Density separation of B lymphocytes and T lymphocyte sheep red blood cell ŽSRBC. rosettes on Ficoll-Hypaque was modified by pretreatment of the SRBC with 2 aminoethylisothiouronium bromide hydrobromide. This modification yielded significantly purer populations of T and B cells. Up to 15 = 10 7 were separated without increasing contamination allowing for the recovery of B lymphocytes as well as T lymphocytes in sufficient numbers to use in functional assays.

Rank: 26. J. Immunol. Methods 78, 143]153 (1985) DETECTION OF MONOCLONAL ANTIBODIES SPECIFIC FOR CARBOHYDRATE EPITOPES USING PERIODATE OXIDATION M.P. WoodwardU , W.W. Young Jr.UU and R.A. BloodgoodU Department of AnatomyU and PathologyUU , Uni¨ ersity of Virginia School of Medicine, Charlottes¨ ille, VA 22908, USA Abstract: A method is described for determining whether particular monoclonal antibodies are specific for carbohydrate or non-carbohydrate antigenic determinants. In a model system consisting of the Lewis A human blood group determinant attached to either protein or lipid, mild periodate oxidation destroyed the carbohydrate determinant without altering protein or lipid epitopes. The technique was readily applied to antigens bound to plastic wells for ELISA, to nitrocellulose sheets for Western blots, and to thin layer chromatography ŽTLC. plates for TLC immunostaining. Mild periodate oxidation can prove useful during the early stages of hybridoma screening in order to select for or against anti-carbohydrate antibodies. Keywords: Monoclonal antibody; Carbohydrate; Periodate; Glycoprotein; Glycolipids.

19

Rank: 27. J. Immunol. Methods 1, 273]287 (1972) THE SEPARATION OF DIFFERENT CELL CLASSES FROM LYMPHOID ORGANS. V. SIMPLE PROCEDURES FOR THE REMOVAL OF CELL DEBRIS, DAMAGED CELLS AND ERYTHROID CELLS FROM LYMPHOID CELL SUSPENSIONS Ken Shortman, Neil Williams and Peter Adams The Walter and Eliza Hall Institute of Medical research, Melbourne, Vic, Australia Abstract: A range of techniques for efficient removal of damaged cells, cell debris and erythroid cells from lymphoid cell suspensions is presented. They are simple, reproducible, and give almost complete recovery of intact lymphoid cells with maintenance of biological activity. The procedures are a zonal setting and spinning procedure for removal of very fine and very course debris; density separations in albumin media for elimination of damaged cells Žat pH 5.1. or damaged cells and erythroid cells ŽpH 7.2.; NH 4 Cl treatment for elimination of erythroid cells alone. Other applications are discussed including a simple procedure for separation of cells of any given density.

Rank: 28. J. Immunol. Methods 89, 271]277 (1986) RAPID COLORIMETRIC ASSAY FOR CELL GROWTH AND SURVIVAL — MODIFICATIONS TO THE TETRAZOLIUM DYE PROCEDURE GIVING IMPROVED SENSITIVITY AND RELIABILITY Franc¸ois Denizot and Rita Lang The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic. 3050, Australia Abstract: A convenient way to estimate the number of viable cells growing in microtitre tray wells is to use a colorimetric assay and an automatic microplate scanning spectrophotometer. One such assay, developed by Mosmann, depends on the

20

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

reduction by living cells of tetrazolium salt, MTT, to form a blue formazan product. However, the original technique has several technical limitations, namely a less than optimal sensitivity, a variable background due to protein precipitation on adding an organic solvent to dissolve the blue formazan product, and a low solubility of the product. These problems have been overcome by the following modifications: avoidance of serum in the incubation medium, thus overcoming precipitation problems in the organic solvent; avoidance of phenol red in the incubation medium, thus avoiding the use of acid in the final solvent which altered the spectral properties of the formazan; elimination of the medium containing MTT after the reaction and subsequent use of pure propanol or ethanol to rapidly solubilize the formazan; use of a higher concentration of MTT; use of half-area microtitre trays to increase the spectrophotometer readings from a given amount of formazan; use of a more judicious reference wavelength in a dual wavelength spectrophotometer. With these modifications the reliability and sensitivity of the test have been increased to the point where it can in many cases replace the w 3 Hxthymidine uptake assay to measure cell proliferation or survival in growth factor or cytotoxicity assays. Examples of its use in IL-2 assays are given. Keywords: Colorimetric assay; Tetrazolium dye; Cell growth assay; Cell viability assay; Lymphokine assay.

Rank: 29. J. Immunol. Methods 77, 305]319 (1985) MEASUREMENTS OF THE TRUE AFFINITY CONSTANT IN SOLUTION OF ANTIGEN–ANTIBODY COMPLEXES BY ENZYME-LINKED IMMUNOSORBENT ASSAY Bertrand Friguet, Alain F. Chaffotte, Lisa Djavadi-Ohaniance and Michel E. Goldberg Unite` de Biochimie des Regulations Cellulaires, ´ Departement de Biochimie et Genetique Molecu´ ´´ laire, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France

Abstract: A simple, general procedure is described for the determination of the dissociation constant ŽK D . of antigen]antibody equilibria in solution. First the monoclonal antibody is incubated in solution with the antigen until the equilibrium is reached; then the proportion of antibody which remains unsaturated at equilibrium is measured by a classical indirect ELISA. The experimental values of K D found by this ELISA procedure for 2 monoclonal antibodies are shown to be very close to those obtained by conventional methods Žimmunoprecipitation of the radiolabeled antigen, or fluorescence transfer .. Moreover, it is shown that, provided the measurements are made under conditions where the total antigen concentration is in large excess over the total antibody concentration, the dissociation constant of antibody]antigen complexes can be determined even with crude preparation of monoclonal antibody. The sensitivity of the ELISA use permits the detection of very small concentrations of antibody and the determination of K D values as small as 10y9 M. This method also offers the great advantage of dealing with unmodified molecules since no labeling of either the antigen or the antibody is required. Keywords: Affinity constant; Monoclonal antibody; ELISA; Tryptophan synthase.

Rank: 30. J. Immunol. Methods 36, 109]117 (1980) OPTIMAL CONDITIONS FOR SIMULTANEOUS PURIFICATION OF MONONUCLEAR AND POLYMORPHONUCLEAR LEUCOCYTES FROM HUMAN BLOOD BY THE HYPAQUEFICOLL METHOD A. Ferrante and Y.H. Thong Dept. Paediatrics, Uni¨ ersity of Adelaide, The Adelaide Children’s Hospital, North Adelaide, SA, Australia Abstract: Data on the optimal conditions for the simulta-

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

neous purification of mononuclear ŽMN. and polymorphonuclear ŽPMN. leucocytes from human blood on Hypaque-Ficoll medium of one density is presented. The efficiency of separation of MN and PMN leucocytes by the Hypaque-Ficoll method was dependent on both the Ficoll concentration and the density of the medium. Best results were obtained with a medium consisting of a Ficoll concentration of 8.2% and a density of 1.114 grml. The separation procedure was much more efficient if carried out at room temperature than at 48C. Storing human blood markedly affected the efficiency of separation and best results were obtained using fresh blood. Data are also presented on the variability in the separation with respect to 40 adults and 80 children.

Rank: 31. J. Immunol. Methods 57, 301]309 (1983) A SOLID-PHASE IMMUNOENZYMATIC TECHNIQUE FOR THE ENUMERATION OF SPECIFIC ANTIBODY-SECRETING CELLS J.D. Sedgwick and P.G. Holt Clinical Immunology Research Unit, Princess Margaret Hospital for Children, Subiaco, WA 6008, Australia Abstract: A sensitive immunoassay is described for the detection of idiotype- and isotype-specific antibody-secreting cells ŽASC., based upon the well established principles of ELISA. Single cell suspensions containing ASC are incubated on solid phase to which specific antigen has been chemically conjugated. Antibody attaches to the latter within the immediate microenvironment of the ASC, producing localized zones of bound antibody which are subsequently developed as visual ‘spots’ in the ELISA. This assay system has sensitivity and specificity at least equivalent to haemolytic plaque assays. Keywords: Antibody-secreting cells; Immunoenzymatic technique; Haemolytic plaque assay.

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Rank: 32. J. Immunol. Methods 119, 203]210 (1989)

RE-EXAMINATION AND FURTHER DEVELOPMENT OF A PRECISE AND RAPID DYE METHOD FOR MEASURING CELL GROWTH r CELL KILL

Morten Bagge Hansen, Svend Erik Nielsen and Kurt Berg The Interferon Laboratory, Institute of Medical Microbiology, Uni¨ ersity of Copenhagen, 2100 Copenhagen, Denmark

Abstract: The tetrazolium salt ŽMTT. method involving conversion of MTT to coloured formazan by cells serving as indirect measurements of cell growthrcell kill has been reported by several groups, although technical problems have been encountered. The present investigation was undertaken in order to delineate what laboratory variables have direct influence on the sensitivity and reproducibility of the method. The pH of the extraction buffer was of the utmost importance, since it was demonstrated that a pH ) 5 would give rise to false signals. Furthermore, modifying the composition of the extraction buffer, all formazan dye grains were solubilised, totally. A direct comparison with published methods demonstrated that only the modified method would yield 100% higher signals without increasing the background. In contrast to previous reports, it was shown that phenol red does not interfere with the measurements and no washing steps are required since all ingredients can be added subsequently. Serum proteins at concentrations up to 25% have no influence on the result. All samples can be measured in an ELISA scanner at 570 nm with little intra-assay variation.

Keywords: Tetrazolium salt; Cell growth; Cell kill.

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

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Rank: 33. J. Immunol. Methods 139, 271]279 (1991) A RAPID AND SIMPLE METHOD FOR MEASURING THYMOCYTE APOPTOSIS BY PROPIDIUM IODIDE STAINING AND FLOW CYTOMETRY I. NicolettiU , G. MiglioratiUU , M.C. PagliacciU , F. GrignaniU and C. RiccardiUU

This new rapid, simple and reproducible method should prove useful for assessing apoptosis of specific cell populations in heterogeneous tissues such as bone marrow, thymus and lymph nodes. Keywords: Apoptosis; Thymocyte; DNA fragmentation; Flow cytometry; Dexamethasone.

Rank: 34. J. Immunol. Methods 24, 389]393 (1978)

U

Istituto di Clinica Medica 1 and Istituto di FarmacologiaUU , Uni¨ ersita di Perugia, 06100 Perugia, Italy Abstract: Corticosteroids, calcium ionophores and antiCD3 monoclonal antibodies kill mouse thymocytes incubated in vitro. Cell death is preceded by extensive DNA fragmentation into oligonucleosomal subunits. This type of cell death Žapoptosis., which physiologically occurs in the intrathymic process of immune cell selection, is usually evaluated by either electrophoretic or colorimetric methods which measure DNA fragmentation in the nuclear extracts. These techniques are unable to determine the percentage of apoptotic nuclei or recognize the apoptotic cells in a heterogeneous cell population. We have developed a flow cytometric method for measuring the percentage of apoptotic nuclei after propidium iodide staining in hypotonic buffer and have compared it with the classical colorimetric and electrophoretic techniques using dexamethasone ŽDEX.-treated mouse thymocytes. Apoptotic nuclei appeared as a broad hypodiploid DNA peak which was easily discriminable from the narrow peak of thymocytes with normal Ždiploid. DNA content in the red fluorescence channels. When the DEX-induced apoptosis was inhibited by either low-temperature Ž48C. incubation or cycloheximide treatment, no hypodiploid DNA peak appeared. Similarly, thymocyte death induced by sodium azide, a substance with cell-killing activity through non-apoptotic mechanisms, did not result in any variation in the normal DNA peak. The flow cytometric data showed an excellent correlation with the results obtained with both electrophoretic and colorimetric methods.

SHORT COMMUNICATION: A RAPID ONESTEP PROCEDURE FOR PURIFICATION OF MONONUCLEAR AND POLYMORPHONUCLEAR LEUKOCYTES FROM HUMAN BLOOD U SIN G A M O D IFIC ATIO N O F TH E HYPAQUE-FICOLL TECHNIQUE A. Ferrante and Y.H. Thong Dept. of Paediatrics, The Uni¨ ersity of Adelaide, Adelaide Children’s Hospital, Adelaide SA 5006, Australia Abstract: A one-step procedure for purification of mononuclear and polymorphonuclear cells from human blood is described. It is modification of the Hypaque-Ficoll method with density of 1.095 grml. Centrifugation at 200 = g for 20]30 min resulted in the separation of mononuclear and polymorphonuclear cells into 2 distinct bands at the interface. The mononuclear cell fraction contained 83.9" 1.6% lymphocytes and 13.8" 2.3% monocytes, while the other consisted of highly purified neutrophils Ž96.4" 1.0%.. Leukocyte recovery by this method was always greater than 80% and viability exceeded 98%. Both cell fractions retained their immunological functions.

Rank: 35. J. Immunol. Methods 67, 379]388 (1984) MEASUREMENT OF CELL NUMBERS BY MEANS OF THE ENDOGENOUS ENZYME HEXOSAMINIDASE. APPLICATIONS TO DETECTION OF LYMPHOKINES AND CELL SURFACE ANTIGENS

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

Ulf Landegren Department of Immunology, Biomedical Center, Uppsala Uni¨ ersity, 751 23 Uppsala, Sweden Abstract: By using a chromogenic substrate for an ubiquitous lysosomal enzyme, hexosaminidase to estimate cell numbers, a sensitive and simple procedure has been developed in which microtiter reaction wells are directly scanned in a spectrophotometer. This method has been adapted to several cell biological assays. Quantitation of the biological activities of T cell growth factor and interferon can be performed on large numbers of samples. Adhesion of dispersed solid tissue cells to fibronectin coated substrates may be quantitated with little expenditure of reagents. By use of a panning procedure in microtiter plates a sensitive and very simple assay for the binding to monoclonal antibodies to cell surface antigens has been developed. Keywords: Cell quantitation; Growth factor assay; Cell surface antigens; Cell adhesion; Endogenous enzyme assay.

Rank: 36.

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culture media they stimulate individual hemopoietic progenitor cells to form colonies of granulocytes andror macrophages ŽPluznik and Sachs, 1965; Bradley and Metcalf, 1996.. They are believed to regulate granulocyte and macrophage production in intact animals although their in vivo effects have not been elucidated. As a group, however, they display considerable variation in apparent molecular weights ŽM r S., physical properties and biological activities depending on the preparation. Two important findings have greatly simplified our understanding of the heterogeneity of CSFs as a group; the existence of at least 4 separable CSF subclasses with different physical properties and target cell specificities; and the characterization of several of these subclasses as sialic acid-containing glycoproteins Žreviewed in Stanley, 1981..

Rank: 37. J. Immunol. Methods 72, 77]89 (1984) DETERMINATION OF THE IMMUNOREACTIVE FRACTION OF RADIOLABELED MONOCLONAL ANTIBODIES BY LINEAR EXTRAPOLATION TO BINDING AT INFINITE ANTIGEN EXCESS

J. Immunol. Methods 42, 253]284 (1981) REVIEW ARTICLE: METHODS FOR THE PURIFICATION, ASSAY, CHARACTERIZATION AND TARGET CELL BINDING OF A COLONY STIMULATING FACTOR (CSF-1) E.R. Stanley and L.J. Guilbert Depts. of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA Introduction: The production of granulocytes and macrophages from immature hemopoietic progenitor cells in tissue culture is dependent on the presence of a group of specific growth factors ŽMetcalf and Moore, 1971; Metcalf, 1977; Stanley, 1981.. These factors have been termed the colony stimulating factors ŽCSFs. because in semi-solid

T. Lindmo, E. Boven, F. Cuttitta, J. Fedorko and P.A. Bunn, Jr. National Cancer Institute, Na¨ al Medical Oncology Branch, Na¨ al Hospital, Bethesda, MD 20814, USA Abstract: Conjugates of monoclonal antibodies with radioactive isotopes, drugs or toxins have great potential for specific radiolocalization and inactivation of tumour cells. Because the conjugation procedure may adversely alter the antibody, quality control procedures must be applied to determine important characteristics of the conjugated antibody. One such property is how much of the conjugated antibody is able to bind to the relevant antigen. Based on theoretical considerations, we have developed a binding assay for radiolabeled monoclonal antibodies in which the fraction of im-

24

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

munoreactive antibody is determined by linear extrapolation to conditions representing infinite antigen excess. This ensures that the true value of the immunoreactive fraction is obtained, as opposed to the apparent immunoreactive fraction determined under conditions of limited antigen excess. The described assay is based on a doubleinverse plot of the binding data which may be considered a modification of the Lineweaver] Burk plot. We established the method using 125 Iand 111 In-labeling of the 2 monoclonal antibodies T101 and 9.2.27 which currently are undergoing radioimaging trials at the National Cancer Institute. For properly performed conjugation procedures, immunoreactive fractions of about 0.9 were obtained, but a prolonged chloramine-T reaction for 125 I-labeling resulted in an immunoreactive fraction of only 0.6. Due to its principle of determining binding at infinite antigen excess, the present method is quite insensitive to variation in the actual amounts of cells and antibody used, as well as the incubation time. We therefore recommend it as a quality control procedure for radiolabeled antibodies. Under certain conditions, this procedure is also applicable for quality control of drug- and toxinconjugated monoclonal antibodies. Keywords: Radiolabeled monoclonal antibodies; Immunoreactive fraction; Radioimaging; Antibody-mediated radiotherapy; Nuclear medicine quality control.

Rank: 38. J. Immunol. Methods 53, 313]319 (1982) ONE-STEP PURIFICATION OF MOUSE MONOCLONAL ANTIBODIES FROM ASCITIC FLUID BY DEAE AFFI-GEL BLUE CHROMATOGRAPHY C. BruckU , D. PortetelleU , C. Glineur UU and A. BollenUU U UU Lab. de Chimie Biologique and Lab. de Gene´´ tique, Dept. de Biologie Moleculaire, Uni¨ ersite´ Li´ bre de Bruxelles, 1640 Rhode-St-Genese, ` Belgium

Abstract: Monoclonal antibodies can be purified directly from ascitic fluids by chromatography on a DEAE Affi-gel blue column. Optimal conditions were determined for the recovery of immunoglobulins free of contaminating protease and nuclease activities. Keywords: Affi-gel blue chromatography; Monoclonal antibody purification.

Rank: 39. J. Immunol. Methods 96, 271]278 (1987) A SIMPLE, NON-CHROMATOGRAPHIC PRO C ED URE TO PURIFY IM M UN O GLOBULINS FROM SERUM AND ASCITES FLUID Michella McKinney and Andrew Parkinson Department of Pharmacology, Toxicology and Therapeutics, Uni¨ ersity of Kansas Medical Center, Kansas City, KS 66103, USA Abstract: A simple, two-step procedure to purify the immunoglobulin G ŽIgG. fraction from mammalian sera and ascites fluid is described. In the first step, albumin and other non-IgG proteins are precipitated with caprylic acid Žoctanoic acid.. In the second, the IgG fraction is precipitated with ammonium sulfate. Factors influencing the precipitation of serum proteins by caprylic acid are described, as are procedural modifications to purify the IgG fraction from sera with a high lipid content. The procedure can be used to purify the IgG fraction of serum from rabbit, sheep, goat, horse, rat and mouse, as well as monoclonal antibodies from mouse ascites fluid. Greater than 80% of the IgG in rabbit serum could be isolated by this procedure, with a purity equal to rabbit IgG purified by anion-exchange chromatography. In addition to its simplicity and low cost, the procedure described offers several advantages over other methods to purify IgG. Keywords: Caprylic acid; IgG purification; Monoclonal antibody purification.

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

Rank: 40. J. Immunol. Methods 65, 109]121 (1983) A SOLID-PHASE ENZYME-LINKED IMM U N O SPO T ( ELISPO T ) ASSAY FO R ENUMERATION OF SPECIFIC ANTIBODYSECRETING CELLS U

U

˚ Nilsson , Hakan Cecil C. Czerkinsky , Lars Ake ˚ UU ¨ Nygrena , Orjan Ouchterlony and Andrej UU Tarkowski U Department of Medical Microbiology, aDepartment UUU of Histology, Uni¨ ersity of Goteborg, and De¨ partment of Rheumatology, Sahlgrenska Hospital, Goteborg, Sweden ¨ Abstract: A solid-phase enzyme-linked immunosorbent assay ŽELISPOT. is described for enumeration of cells secreting specific antibody. Spleen cells from immunized mice are incubated in antigen-coated polystyrene plates. After removal of the cells, bound antibodies are demonstrated by means of immunoenzyme procedure in which enzyme]substrate reactions are performed in agarose. Darkbrown circular zones Žspots., localized in areas of the dish where antibody production has occurred, are enumerated with the naked eye. Spectrophotometric estimation of enzyme-bound activity may be performed by substituting the gel for a liquid buffer, allowing accurate estimation of the total amount of secreted antibody. Versatile, sensitive and very easy to perform, this new assay provides a useful alternative to conventional plaque-forming cell assays. Keywords: Immunospot ŽELISPOT.; Antibodysecreting cells; ELISA antibody secretion.

Rank: 41. J. Immunol. Methods12, 117]124 (1976) PREPARATION AND USE IN IMMUNOHISTOLOGY OF ANTIBODIES SPECIFIC FOR TYPE I AND TYPE III COLLAGEN AND PROCOLLAGEN

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Hans Nowack, Steffen Gay, Georg WickU , Udo Becker and Rupert Timpl Max-Planck-Institut fur ¨ Biochemie, Abt.UBindegewebsforschung, Martinsreid, Germany and Institut fur ¨ allgem eine und experim entelle Pathologie, Uni¨ ersitat ¨ Innsbruck, Innsbruck, Austria Abstract: Antibodies to bovine type I and type II collagen and their precursor form procollagen were produced in rabbits and rendered specific for the immunizing antigen by immunoadsorption. These purified antibodies showed distinct immunofluorescence staining on frozen sections of both bovine and human connective tissue at concentrations as low as 1]10 m grml. Antibodies to type III collagen and procollagen reacted with reticulin in liver and spleen, with fascicles around tendons and with the upper portion of the dermis. Antibodies to type I collagen and procollagen reacted with skin and fiber bundles in tendon but did not stain reticulin. No reaction was observed with cartilage collagen or with kidney glomerular basement membrane.

Rank: 42. J. Immunol. Methods 93, 157]165 (1986) AN IMPROVED COLORIMETRIC ASSAY FOR INTERLEUKIN 2 Hiroko Tada, Osamu Shiho, Ken-ichi Kuroshima, Masura Koyama and Kyozo Tsukamoto Biotechnology Laboratories, Central Research Di¨ ision, Takeda Chemical Industries, Ltd., Yodogawaku, Osaka 532, Japan Abstract: Mosmann’s method for measuring the number of viable cells with a tetrazolium salt, 3-Ž4,5-dim ethylthiazol-2-yl . -2,5-di-phenyltetrazolium bromide ŽMTT., was modified to make it possible to measure a large number of interleukin 2 ŽIL-2. samples at one time with less labor and more accuracy. Each step of the method was examined in detail and modified Žthe modified MTT method.. Ž1. An IL-2-dependent mouse natural

26

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

killer cell line, NKC3, was used as an indicator cell line. The incubation period before adding MTT was reduced to 24 h, Ž2. A solution of 10% sodium dodecyl sulfate-0.01 N HCl was used to dissolve the MTT formazan produced. We have compared the values obtained by the modified MTT method and the conventional w 3 Hxthymidine method Ž 3 H-TdR method., and confirmed that the estimates of IL-2 content were almost equal. The variation of IL-2 content measured by both methods was within 5% in terms of the standard error.

lymphotoxins produced by human lymphocytes. The data indicate that the L929 cell lines was 10]50-fold more sensitive than the ESH-5L line to the lytic activity of cytotoxins produced by human phytohemagglutinin-P-activated T lymphocytes, or the cytotoxins produced by peripheral blood lymphocytes stimulated with various tumor cell lines. This assay system was also useful in detecting antibodies capable of neutralizing lymphotoxin activity and thus should be a suitable method to aid in the molecular characterization of these lymphokines.

Keywords: Interleukin 2; Colorimetric assay; Tetrazolium salt.

Keywords: Colorimetric assay; Cytotoxic growth inhibition assay; Lymphotoxins; Tetrazolium dye; Cell viability.

Rank: 43. J. Immunol. Methods 70, 257]268 (1984)

Rank: 44. J. Immunol. Methods 53, 133]173 (1982)

RAPID COLORIMETRIC ASSAY FOR CELL VIABILITY: APPLICATION TO THE QUANTITATION OF CYTOTOXIC AND GROWTH INHIBITORY LYMPHOKINES Lora M. Green, Jeanne L. Reade and Carl F. Ware Di¨ ision of Biomedical Sciences, Uni¨ ersity of California, Ri¨ erside, CA 92521, USA Abstract: A rapid colorimetric microtiter assay has been developed to detect cytotoxic lymphokines produced by human lymphocytes activated with lectins or tumor cells. The viability of lymphotoxin-treated target cells was detected using a tetrazolium dye that is reduced to a blue formazan by living but not dead cells. The amount of dye formed was quantitated using a microplate spectrophotometer ŽELISA plate reader. and visual observations confirmed the amount of formazan dye produced was directly proportional to the number of viable target cells. The advantages of using this colorimetric methods are that it requires no washing steps or radioisotopes and its precision and rapidity. Optimal conditions were established using the murine L929 and human ESH-5L cell lines as target cells for detecting

REVIEW ARTICLE: MONOCLONAL ANTIBODIES: PURIFICATION FRAGMENTATION AND APPLICATION TO STRUCTURAL AND FUNCTIONAL STUDIES OF CLASS I MHC ANTIGENS Peter Parham, Matthew J. Androlewicz, Frances M. Brodsky, Nicholas J. Holmes and Judy P. Ways Dept. of Structural Biology, Stanford Uni¨ ersity School of Medicine, Stanford, CA 94305, USA Abstract: Characterization of panels of polymorphic antibodies clearly shows there are a minimum of 2 spatially separate polymorphic regions of HLA antigens, A2, A28, B7, B27, B4O ŽParham, 1981.. Study of rat class I antigens ŽGalfre et al., 1977; Howard et al., 1979. and H-2 antigens ŽLemke et al., 1979; Lemke and Hammerling, 1981; Ozato et al., 1980. resulted in similar conclusions. Perhaps they correlate with the 2 major regions of variability seen in the primary sequence ŽOrr et al., 1979.? Four monomorphic determinants of HLA also define 2 spatially separate regions of the molecule: one more closely associated with b 2microglobulin, and the other confined to the heavy

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

chain. For HLA-A2, each of the polymorphic regions is more closely associated with one of the 2 monomorphic regions: the A2 specific site with the b 2-m region, the A2,A28 common site with the heavy chain region. This juxtaposition of monomorphic and polymorphic determinants is not identical for all gene products. The position andror orientation of the B7 specific determinant is different from either the A2 or A2,A28 determinants. The serological difference between A2 and A28 has been analyzed. It appears as though A2 is more highly evolved than A28. That part of the molecule which is A2 specific in A2 has a structure in A28 shared by many gene products, e.g., B7,40. This reflects the fundamental relationship between highly specific and broadly polymorphic antigenic sites ŽBrodsky and Parham, 1982b.. Analogous relationships are seen in the primary sequences. At most positions where A28 differs from A2 it is identical to B7 ŽStrominger, 1980.. Although A2 has long been considered a single gene product, the rate at which variants are being discovered and similar mutants produced indicate that it encompasses a large family of molecules. It has been puzzling that A2 often behaved differently from other HLA antigens and the known primary sequences reflect this. Characterization of the finer details of variation within the A2 family molecules will establish whether a gradual or punctuated molecular evolution of class I molecules has occurred in human populations. Keywords: Monoclonal antibody; MHC antigens; Fragmentation; Affinities; Antigenic determinants.

Rank: 45. J. Immunol. Methods 11, 273]279 (1976) ISOLATION OF HUMAN T AND B LYMPHOCYTES BY ROSETTE FORMATION WITH 2AMINOETHYLISOTHIOURONIUM BROMIDE (AET) — TREATED SHEEP RED BLOOD CELLS AND WITH MONKEY RED BLOOD CELLS

27

M.A. Pellegrino, S. Ferrone and A.N. Theofilopoulos Depts. of Molecular Immunology and Immunopathology, Scripps Clinic and Research Foundation, La Jolla, Calif. 92037, USA Abstract: Purified human B and T lymphocytes were obtained by rosetting HPL with AET-SRBC or MRBC and separating the non-rosetted from the rosetted cells on Ficoll-Hypaque gradient, 92 " 3% of the purified B cells were fluorescent positive for MBIg and 95 " 2% of the purified T cells rosetted with AET-SRBC. 69 " 5% of the B cells and 61 " 8% of the T cells present in the unfractionated HPL were recovered in the purified fractions.

Rank: 46. J. Immunol. Methods 37, 39]45 (1980) RAPID QUANTITATION OF NEUTROPHIL C H E M O T A X IS : U S E O F A P O LYVINYLPYRROLIDONE-FREE POLYCARBONATE MEMBRANE IN A MULTIWELL ASSEMBLY Liana Harvath, Werner Falk and Edward J. Leonard Immunopathology Section, Lab. of Immunobiology, National Cancer Institute, NIH, Bethesda, MD 20205, USA Abstract: A neutrophil chemotaxis assay was developed which permits rapid, quantitative assessment of migration across a membrane filter. The critical factor in the assay was the use of a 10 m m thick polycarbonate membrane without the usual polyvinylpyrrolidone coating. Migrated neutrophils remain adherent to the uncoated membrane, whereas 20]50% fall off polyvinylpyrrolidonecoated membranes. A major advantage of the method is that neutrophil chemotaxis can be readily quantified, since the migrated cells adhere to the membrane surface and are in one optical plane for counting. A 25 mm= 80 mm membrane

28

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

sheet was used in a 48-well micro chemotaxis assembly, which requires only 20,000 neutrophils and 25 m l of attractant per assay well. Neutrophil chemotaxis was complete with 10]20 min at 378C, with 20]30% of the cells migrating to N-formylmethionyl-leucyl-phenylalanine and 40]50% migrating to complement derived C5a.

Rank: 47. J. Immunol. Methods 43, 95]101 (1981) A RAPID METHOD FOR PURIFICATION OF HUMAN GRANULOCYTES USING PERCOLLW . A COMPARISON WITH DEXTRAN SEDIMENTATION Rolf Hjorth, Anna-Karin Jonsson and Per Vretblad Cell Biology Group, Pharmacia Fine Chemicals AB, 751 04 Uppsala, Sweden Abstract: A simple one-step method for purification of human granulocytes, based on the use of a discontinuous gradient of Percoll, is described. The cell yield is 55% and the purity of the granulocyte fraction is 97%. The cells’ ability to exclude trypan blue and phagocytic function are not altered during the purification process. It is concluded that centrifugation in Percoll is superior to conventional dextran sedimentation; it is also less time-consuming.

Abstract: A method has been developed for studying quantitatively the separate processes of bacterial opsonization, phagocytosis, and killing by human polymorphonuclear leukocytes using w 3 Hxthymidine labeled Staphylococcus aureus. Phagocytosis is determined by assaying for leukocytes-associated radioactivity after differential centrifugation and washing the leukocytes. Opsonization is studied by incubating bacteria with an opsonic source for varying durations and then adding leukocytes. By treatment of samples with the muralytic enzyme, lysostaphin, the attachment and ingestion phases of phagocytosis can be separated. Sampling for colony forming units after disruption of the leukocytes permits the measurement of bacterial killing. Using this method, differences in the kinetics of staphylococcal opsonization by normal and C2 deficient sera were defined, opsonic influences on the attachment and ingestion phases of phagocytosis were delineated, and the influences of different opsonins and leukocyte populations on killing were determined.

Rank: 49. J. Immunol. Methods 10, 61]66 (1976) THE CHROMIC CHLORIDE METHOD OF COUPLING ANTIGENS TO ERYTHROCYTES: DEFINITION OF SOM E IM PORTANT PARAMETERS

Rank: 48. J. Immunol. Methods 14, 303]311 (1977) KINETICS OF STAPHYLOCOCCAL OPSONIZATION, ATTACHMENT, INGESTION AND KILLING BY HUMAN POLYMORPHONUCLEAR LEUKOCYTES: A QUANTITATIVE ASSAY USING [ 3 H ] THYM IDINE LABELED BACTERIA Jan Verhoef, Phillip K. Peterson and Paul G. Quie Depts. of Medicine and Pediatrics, Uni¨ ersity of Minnesota School of Medicine, Minneapolis, Minn. 55455, USA

James W. Goding The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Melbourne Vic., Australia Abstract: Factors affecting the efficiency of the chromic chloride method of coupling antigen to erythrocytes were studied using a radioactive tracer. The amount of chromic chloride required for optimal coupling was related to the concentration of protein in the reaction mixture. Coupling was considerably more efficient when the chromic chlo-

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

ride solution was allowed to ‘age’ prior to use. In the system studied, the addition of piperazine buffer made little difference to the degree of coupling. The reaction was shown to proceed very rapidly } 50% of the final uptake of antigen took place within seconds, and the reaction was essentially complete 5 min after initiation. The importance of adequate mixing during the addition of the chromic chloride solution was emphasised.

Rank: 50. J. Immunol. Methods 32, 297]304 (1980) HIGH FREQUENCIES OF ANTIGEN-SPECIFIC HYBRIDOMAS: DEPENDENCE ON IMMUNIZATION PARAMETERS AND PREDICTION BY SPLEEN CELL ANALYSIS Christian Stahli, Theophil Staehelin, Vincenzo ¨ Miggiano, Jorg Schmidt and Pal Haring ¨ ¨

29

Pharma Research Dept., F. Hoffmann-La Roche and Cie. AG., 4002 Basel, Switzerland Abstract: Hybridomas producing antibodies against soluble antigens have in most cases been difficult to establish. After fusion of myeloma cells with spleen cells obtained from mice immunized with a soluble protein hybridomas secreting specific antibodies have been observed to occur very rarely among non-specific hybridomas. We found that the frequency of specific hybridomas correlates directly with the increase over background of the frequency of blast andror plasma cells in the spleen Žmeasured by cell size analysis . after antigenic stimulation. High yields of specific hybridomas were obtained simply by following a novel immunization technique consisting of several conventional preimmunization courses followed by 4 very high doses of antigen in saline on each of the last 4 days before fusion.

NB: The abo©e abstracts, plus abstracts of the remaining fifty papers may be r jimweb found on the JIM Web site: www.else©ier.nl r locater Rank: 51.

Rank: 53.

J. Immunol. Methods 7, 283]290 (1975)

J. Immunol. Methods 18, 165]182 (1977)

REFINEMENTS IN THE AUTOMATED FLUOROMETRIC HISTAMINE ANALYSIS SYSTEM

ANTIBODIES TO DISTINCT TYPES OF COLLAGENS AND PROCOLLAGENS AND THEIR APPLICATION IN IMMUNOHISTOLOGY

Reuben P. Siraganian Clinical Immunology Section, Laboratory of Microbiology and Immunology, NIDR, NIH, Bethesda, MD 20014, USA

Rupert Timpl, Georg WickU and Steffen Gay Max-Planck-Institute for Biochemistry, Martinsried, U Germany and Institute for General and Experimental Pathology, Uni¨ ersity of Innsbruck, Austria

Rank: 52.

Rank: 54.

J. Immunol. Methods 15, 279]289 (1977)

J. Immunol. Methods 2, 293]301 (1973)

USE OF ANTIBODY-COATED RED CELLS FOR THE SENSITIVE DETECTION OF ANTIGEN AND IN ROSETTE TESTS FOR CELLS BEARING SURFACE IMMUNOGLOBULINS

The separation of different cell classes from lymphoid organs. IX. A simple and rapid method for removal of damaged cells from lymphoid cell suspensions

N.R. Ling, S. Bishop and R. Jefferis Dept. of Experimental Pathology, Uni¨ ersity of Birmingham, Birmingham, UK

Harald Von Boehmer and Ken Shortman The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic., Australia

30

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

Rank: 55.

Rank: 59.

J. Immunol. Methods 51, 269]277 (1982)

J. Immunol. Methods 56, 329]339 (1983)

REVIEW ARTICLE: ISOLATION OF HUMAN AND RAT NATURAL KILLER CELLS

UTILIZATION OF THE BIOTINr r AVIDIN SYSTEM TO AMPLIFY THE SENSITIVITY OF THE ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

Tuomo Timonen, Craig W. Reynolds, John R. Ortaldo and Ronald B. Herberman Lab. of Immunodiagnosis, National Cancer Institute, NIH, Bethesda, MD 20205, USA

Rank: 56. J. Immunol. Methods 118, 245]250 (1989) A SENSITIVE SANDWICH-ENZYME IMMUNOASSAY FOR HUMAN ENDOTHELIN Nobuhiro Suzuki, Hirokazu Matsumoto, Chieko Kitada, Tomoh MasakiU and Masahiko Fujino Tsukuba Research Laboratories, Takeda Chemical U Industries, Ltd., and Institute of Basic Medical Science, Uni¨ ersity of Tsukuba, Tsukuba, Ibaraki, Japan

Rank: 57.

Cynthia Kendall, Irina Ionescu-Matiu and Gordon R. Dreesman Dept. of Virology and Epidemiology, Baylor College of Medicine, Houston, TX 77030, USA

Rank: 60. J. Immunol. Methods 72, 219]227 (1984) SERUM-FREE MEDIUM FOR GENERATION AND PROPAGATION OF FUNCTIONAL HUMAN CYTOTOXIC AND HELPER T CELL CLONES Hans Yssel, Jan E. De Vries, Marcel Koken, Wim Van Blitterswijk and Hergen Spits Di¨ isions of Immunology and Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands

J. Immunol. Methods 3, 147]164 (1973)

Rank: 61. A MINIATURIZED MOUSE MIXED LEUKOCYTE CULTURE IN SERUM-FREE AND MOUSE SERUM SUPPLEMENTED MEDIA Ammon B. Peck and Fritz H. Bach Dept. of Medical Genetics, School of Medicine, Uni¨ ersity of Wisconsin, Madison, Wis. 53706, USA

Rank: 58. J. Immunol. Methods 102, 127]141 (1987)

J. Immunol. Methods 62, 1]13 (1983) REVIEW ARTICLE: BINDING OF IMMUNOGLOBULINS TO PROTEIN A AND IMMUNOGLOBULIN LEVELS IN MAMMALIAN SERA U Roger Lindmark, Kerstin Thoren-Tolling and ´ John Sjoquist ¨ Dept. of Medical and Physiological Chemistry, UppU sala Uni¨ ersity, and Dept. of Clinical Chemistry, Veterinary Faculty, Uni¨ ersity of Agricultural Sciences, Uppsala, Sweden

EXPANSION OF HUMAN TUMOR INFILTRATING LYMPHOCYTES FOR USE IN IMMUNOTHERAPY TRIALS

Rank: 62.

Suzanne L. Topalian, Linda M. Muul, Diane SolomonU and Steven A. Rosenberg U Surgery Branch, and Laboratory of Pathology, National Cancer Institute, Bethesda, MD 20892, USA

A RADIOIMMUNOASSAY OF CELLULAR SURFACE ANTIGENS ON LIVING CELLS USING IODINATED SOLUBLE PROTEIN A FROM STAPHYLOCOCCUS AUREUS

J. Immunol. Methods 7, 237]250 (1975)

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

G. DorvalU , K.I. WelshUU and H. WigzellUUU Dept. of Tumor Biology, Karolinska Institute, UU McIndoe Research Unit, Stockholm, Sweden; Queen Victoria Hospital, East Grinstead, Sussex, UUU UK; Dept. of Immunology, Uppsala Uni¨ ersity, Uppsala, Sweden

U

Rank: 63. J. Immunol. Methods 76, 171]183 (1985) IMMUNOASSAY FOR THE DETECTION AND QUANTITATION OF INFECTIOUS HUMAN RETROVIRUS, LYMPHADENOPATHY-ASSOCIATED VIRUS (LAV) J.S. McDougal, S.P. Cort, M.S. Kennedy, C.D. Cabridilla, P.M. Feorino, D.P. Francis, D. Hicks, V.S. Kalyanaraman and L.S. Martin Immunology Branch, Centers for Disease Control, Atlanta, GA 30333, USA

31

Gerald SchiffmanU , Robert M. Douglas, Mary Jo Bonner, Miriam Robbins and Robert Austrian John Herr Musser Dept. of Research Medicine, Uni¨ ersity of Pennsyl¨ ania School of Medicine, U Philadelphia, PA 19104, and Dept. of Microbiology and Immunology, Downstate Medical Center, SUNY, Brooklyn, NY 11203, USA

Rank: 66. J. Immunol. Methods 30, 1]10 (1979) DISCONTINUOUS DENSITY GRADIENT SEPARATION OF HUMAN MONONUCLEAR LEUCOCYTES USING PERCOLLW AS GRADIENT MEDIUM A.J. Ulmer and H.-D. Flad Laboratory of Immunology, Dept. of Microbiology, Uni¨ ersity of Ulm, 7900 Ulm, Germany

Rank: 67. J. Immunol. Methods 47, 25]30 (1981)

Rank: 64. J. Immunol. Methods 109, 49]59 (1988) MONOCLONAL ANTIBODIES TO PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA) r CYCLIN AS PROBES FOR PROLIFERATING CELLS BY IMMUNOFLUORESCENCE MICROSCOPY AND FLOW CYTOMETRY P. Kurki, K. Ogata and E.M. Tan W.M. Keck Autoimmune Disease Center, Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, CA 92037, USA

Rank: 65. J. Immunol. Methods 33, 133]144 (1980) A RADIOIMMUNOASSAY FOR IMMUNOLOGIC PHENOMENA IN PNEUMOCOCCAL DISEASE AND FOR THE ANTIBODY RESPONSE TO PNEUMOCOCCAL VACCINES. I. METHOD FOR THE RADIOIMMUNOASSAY OF ANTICAPSULAR ANTIBODIES AND COMPARISON WITH OTHER TECHNIQUES

PARAFORM ALDEHYDE FIXATION OF HEMATOPOIETIC CELLS FOR QUANTITATIVE FLOW CYTOMETRY (FACS) ANALYSIS L.L. Lanier and N.L. Warner Immunobiology Laboratory, Dept. of Pathology, Uni¨ ersity of New Mexico School of Medicine, Albuquerque, NM 87131, USA

Rank: 68. J. Immunol. Methods 49, R11]R23 (1982) REVIEW ARTICLE: THE EVALUATION OF LIMITING DILUTION ASSAYS S. Fazekas de St. Groth Basel Institute for Immunology, 4058 Basel, Switzerland

Rank: 69. J. Immunol. Methods 62, 31]37 (1983) USE OF GELATINr r PLASMA COATED FLASKS FOR ISOLATING HUMAN PERIPHERAL BLOOD MONOCYTES

32

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

Bruce Freundlich and Nebojsa Avdalovic Rheumatology Section, Dept. of Medicine, Uni¨ ersity of Pennsyl¨ ania School of Medicine, Philadelphia, PA 19104, USA

Rank: 70. J. Immunol. Methods 20, 173]183 (1978) A SENSITIVE ROSETTING METHOD FOR DETECTING SUBPOPULATIONS OF LYMPHOCYTES WHICH REACT WITH ALLOANTISERA C.R. Parish and I.F.C. McKenzie Dept. of Microbiology, John Curtin School of Medical Research, Australian National Uni¨ ersity, Canberra, ACT, and Dept. of Medicine, Austin Hospital, Heidelberg, Vic., Australia

Rank: 71. J. Immunol. Methods 51, 241]249 (1982) Q U A N T IT A T IV E E L E C T R O PH O R E T IC TRANSFER OF POLYPEPTIDES FROM SDS POLYACRYLAMIDE GELS TO NITROCELLULOSE SHEETS: A METHOD FOR THEIR REUSE IN IMMUNOAUTORADIOGRAPHIC DETECTION OF ANTIGENS Paul F. Erickson, Lee N. Minier and Robert S. Lasher Dept. of Anatomy, Uni¨ ersity of Colorado Health Science Center, Den¨ er, CO 80262, USA

Rank: 72. J. Immunol. Methods 27, 61]74 (1979) A METHODOLOGICAL STUDY OF EROSETTE FORMATION USING AET-TREATED SHEEP RED BLOOD CELLS M. Madsen and H.E. Johnsen Tissue Typing Laboratory, Blood Bank and Blood Grouping Laboratory, Aarhus Kommunehospital, 8000 Aarhus C, Denmark

Rank: 73. J. Immunol. Methods 109, 277]285 (1988)

MONOCLONAL ANTIBODIES TO PHOSPHOTYROSINE John R. Glenney Jr., Liza Zokas and Mark P. Kamps Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies, San Diego, CA 92138, USA

Rank: 74. J. Immunol. Methods 76, 157]169 (1985) ONE-STEP PURIFICATION OF MOUSE MONOCLONAL ANTIBODIES FROM ASCITES FLUID BY HYDROXYLAPATITE CHROMATOGRAPHY Larry H. Stanker, Martin Vanderlaan and Hector Juarez-SalinasU Lawrence Li¨ ermore National Laboratory, Biomedical Sciences Di¨ ision, Uni¨ ersity of California, Li¨ U ermore, CA 94550, and Life Sciences Research Group, Chromatography Business Unit, Bio-Rad Laboratories, Richmond, CA 94804, USA

Rank: 75. J. Immunol. Methods 33, 1]9 (1980) SEPARATION OF HUMAN BLOOD MONOCYTES AND LYMPHOCYTES ON A CONTINUOUS PERCOLL GRADIENT F. Gmelig-Meyling and T.A. Waldmann Metabolism Branch, National Cancer Institute, NIH, Bethesda, MD 20014, USA

Rank: 76. J. Immunol. Methods 14, 381]390 (1977) ANTIGEN AND ANTIBODY DETECTION BY IN VIVO METHODS; A REEVALUATION OF PASSIVE CUTANEOUS ANAPHYLACTIC REACTIONS Naohiro Watanabe and Zoltan Ovary Dept. of Pathology, New York Uni¨ ersity School of Medicine, New York, NY 10016, USA

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

Rank: 77.

33

J. Immunol. Methods 50, R85]R112 (1982)

Edmundo Lamoyi and Alfred Nisonoff Rosenstiel Resarch Center, Dept. Biology, Brandeis Uni¨ ersity, Waltham, MA 02254, USA

REVIEW ARTICLE: FLOW CYTOMETRY AS AN ANALYTICAL AND PREPARATIVE TOOL IN IMMUNOLOGY

Rank: 81.

Michael R. Loken and Alan M. Stall La Rabida-Uni¨ ersity of Chicago Institute and the Dept. of Microbiology, Uni¨ ersity of Chicago, Chicago, IL 60649, USA

Rank: 78. J. Immunol. Methods 145, 229]240 (1991) KINETIC ANALYSIS OF MONOCLONAL ANTIBODY–ANTIGEN INTERACTIONS WITH A NEW BIOSENSOR BASED ANALYTICAL SYSTEM Robert Karlsson, Anne Michaelsson and Lars Mattsson Pharmacia Biosensor AB, 751 82 Uppsala, Sweden

Rank: 79. J. Immunol. Methods 74, 353]360 (1984) S E N S IT IV E V IS U A L IZ A T IO N O F ANTIGEN–ANTIBODY REACTIONS IN DOT AND BLOT IMMUNE OVERLAY ASSAYS WITH IMMUNOGOLD AND IMMUNOGOLD r SILVER STAINING M. Moeremans, G. Daneels, A. Van Dijck, G. Langanger and J. De Mey Laboratory of Biochemical Cytology, Di¨ ision of Cellular Biology and Chemotherapy, Janssen Pharmaceutica Research Laboratories, 2340 Beerse, Belgium

J. Immunol. Methods 24, 269]285 (1978) [ 125 I]PROTEIN A: A TRACER FOR GENERAL USE IN IMMUNOASSAY John J. Langone Laboratory of Immunobiology, National Cancer Institute, NIH, Bethesda, MD. 20014, USA

Rank: 82. J. Immunol. Methods 78, 59]69 (1985) SYNTHETIC PEPTIDES AS ANTIGENS: PITFALLS OF CONJUGATION METHODS J.P. Briand, S. Muller and M.H.V. Van Regenmortel Institut de Biologie Moleculaire et Cellulaire du CNRS, 67000 Strasbourg, France

Rank: 83. J. Immunol. Methods 95, 157]168 (1986) REVIEW ARTICLE: AN ADJUVANT FORMULATION THAT SELECTIVELY ELICITS THE FORMATION OF ANTIBODIES OF PROTECTIVE ISOTYPES AND OF CELL-MEDIATED IMMUNITY Anthony C. Allison and Noelene E. Byars Institute of Biological Sciences, Syntex Research, Palo Alto, CA 94304, USA

Rank: 84. Rank: 80.

J. Immunol. Methods 151, 237]244 (1992)

J. Immunol. Methods 56, 235]243 (1983) PREPARATION OF F(AB9)2 FRAGMENTS FROM MOUSE IGG OF VARIOUS SUBCLASSES

AN IMMUNOCHEMICAL ANALYSIS OF THE HUMAN NUCLEAR PHOSPHOPROTEIN P53: NEW MONOCLONAL ANTIBODIES AND EPITOPE MAPPING USING RECOMBINANT P53

34

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

B. Vojtesek, ˇˇ J. Bartek ´ U , C.A. Midgley and D.P. Lane Cancer Research Campaign Laboratories, Dept. of Biochemistry, Uni¨ ersity of Dundee, Dundee DD1 U 4HN, UK and Institute of Haematology and Blood Transfusion, Prague 10, Czechoslo¨ akia

Rank: 85. J. Immunol. Methods 53, 261]291 (1982) THEORY AND METHODS FOR IMMUNIZATION IN CULTURE AND MONOCLONAL ANTIBODY PRODUCTION

DOUBLE-DECKER ROCKET IMMUNOELECTROPHORESIS FOR DIRECT QUANTITATIO N O F C O M PLEM EN T C 3 SPLIT PRODUCTS WITH C3D SPECIFICITIES IN PLASMA I. Brandslund, H.C. Siersted, S.-E. Svehag and B. Teisner Institue of Medical Microbiology, Odense Uni¨ ersity, 5000 Odense, Denmark

Rank: 89. J. Immunol. Methods 67, 353]369 (1984)

Christopher L. Reading Dept. of Tumor Biology, Uni¨ ersity of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, TX 77030, USA

Rank: 86. J. Immunol. Methods 98, 5]10 (1987) A SIMPLE NEW METHOD FOR USING ANTIGENS SEPARATED BY POLYACRYLAMIDE GEL ELECTROPHORESIS TO STIMULATE LYMPHOCYTES IN VITRO AFTER CONVERTING BANDS CUT FROM WESTERN BLOTS INTO ANTIGEN-BEARING PARTICLES

MURINE B CELL LYMPHOPOIESIS IN LONG TERM CULTURE Cheryl A. Whitlock, Debra Robertson and Owen N. Witte Department of Microbiology and Molecular Biology Institute, Uni¨ ersity of California, Los Angeles, CA 90024, USA

Rank: 90. J. Immunol. Methods 37, 97]108 (1980)

C. Abou-Zeid, E. Filley, J. Steele and G.A.W. Rook Dept. of Microbiology, School of Pathology, Middlesex Hospital Medical School, London W1, UK

DIRECT FLUORESCENT LABELING OF CELLS WITH FLUORESCEIN OR RHODAMINE ISOTHIOCYANATE. I. TECHNICAL ASPECTS

Rank: 87.

Eugene C. Butcher and Irving L. Weissman Laboratory of Experimental Oncology, Dept. of Pathology, Stanford Uni¨ ersity School of Medicine, Stanford, CA 94305, USA

J. Immunol. Methods 47, 129]144 (1981) REVIEW ARTICLE: IMMUNOHISTOCHEMISTRY WITH ENZYME LABELED ANTIBODIES: A BRIEF REVIEW Andrew G. Farr and Paul K. Nakane Dept. of Pathology, Uni¨ ersity of Colorado Health Sciences Center, Den¨ er, CO, USA

Rank: 88. J. Immunol. Methods 44, 63]71 (1981)

Rank: 91. J. Immunol. Methods 65, 147]157 (1983) THE PREPARATION OF DTPA-COUPLED ANTIBODIES RADIOLABELED WITH METALLIC RADIONUCLIDES: AN IMPROVED METHOD

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

D.J. Hnatowich, R.L. Childs, D. Lanteigne and A. Najafi Department of Nuclear Medicine, Uni¨ ersity of Massachusetts Medical School, Worcester, MA 01605, USA

Rank: 92. J. Immunol. Methods 2, 279]291 (1973) IN VITRO STIMULATION OF MURINE SPLEEN CELLS USING A MICROTITRE SYSTEM AND A MULTIPLE AUTOMATED SAMPLE HARVESTER Douglas M. Strong, Aftab A. Ahmed, Gary B. Thurman and Kennedy W. Sell Na¨ al Medical Research Institute, National Na¨ al Medical Center, Bethesda, Maryland 20014, USA

Rank: 93. J. Immunol. Methods 93, 201]206 (1986)

35

SEPHADEX G-10 COLUMN METHOD WITH OTHER COMMONLY USED TECHNIQUES Thomas R. Jerrells, Jack H. Dean, Gary L. Richardson and Ronald B. HerbermanU Dept. of Immunology, Biomedical Research Di¨ ision, Litton Bionetics Inc., Kensington, MD 20795, U and Laboratory of Immunodiagnosis, National Cancer Institute, Bethesda, MD 20014, USA

Rank: 96. J. Immunol. Methods 28, 187]192 (1979) ELISA METHODOLOGY FOR POLYSACCHARIDE ANTIGENS: PROTEIN COUPLING OF POLYSACCHARIDES FOR ADSORPTION TO PLASTIC TUBES Barry M. Gray Depts. of Pediatrics and Microbiology, Uni¨ ersity of Alabama, Birmingham, AL. 35294, USA

SERUM-FREE IN VITRO BIOASSAY FOR THE DETECTION OF TUMOR NECROSIS FACTOR

Rank: 97.

Susan Mukavitz Kramer and Monique E. Carver Department of Assay De¨ elopment, Genentech, Inc., South San Francisco, CA 94080, USA

125

Rank: 94.

John J. Langone, Michael D.P. Boyle and Tibor Borsos Lab. of Immunobiology, National Cancer Institute, NIH, Bethesda, MD 20014, USA

J. Immunol. Methods 33, 221]229 (1980) SEPARATION OF HUMAN MONOCYTES ON DENSITY GRADIENTS OF PERCOLLW

J. Immunol. Methods 18, 281]293 (1977) I PROTEIN A: APPLICATIONS TO THE QUANTATIVE DETERMINATION OF FLUID PHASE AND CELL-BOUND IGG

Rank: 98. H. Pertoft, A. Johnsson, B. Warmegard ¨ ˚ and R. SeljelidU Institute of Medical Physiological Chemistry., Uni¨ ersity of Uppsala, Biomedical Center, 751 23 UppU sala, Sweden, and Institute of Medical Biology, Uni¨ ersity of Tromsø, 9000 Tromsø, Norway

Rank: 95. J. Immunol. Methods 32, 11]29 (1980) DEPLETION OF MONOCYTES FROM HUMAN PERIPHERAL BLOOD MONONUCLEAR LEUKOCYTES: COMPARISON OF THE

J. Immunol. Methods 99, 235]241 (1987) DEVELOPMENT OF RADIOIMMUNOASSAYS FOR HUMAN ERYTHROPOIETIN USING RECOMBINANT ERYTHROPOIETIN AS TRACER AND IMMUNOGEN J.C. EgrieU , P. Mary CotesUU , J. LaneU , R.E. Gaines DasUUU and R.C. TamUU U UU Amgen, Thousand Oaks, CA 91320, USA, Clinical Research Centre, Harrow, Middlesex, and UUU National Institute for Biological Standards and Control, Hampstead, London, UK

36

Abstracts r Journal of Immunological Methods 216 (1998) 7]36

Rank 99. J. Immunol. Methods 116, 1]17 (1989) REVIEW ARTICLE: ASSAYS FOR TUMOUR NECROSIS FACTOR AND RELATED CYTOKINES Anthony Meager, Helen Leung and Jane Woolley Di¨ ision of Immunobiology, National Institute for Biological Standards and Control, South Mimms EN6 3QG, UK

Rank: 100. J. Immunol. Methods 46, 63]68 (1981)

EGGS: CONVENIENTLY PACKAGED ANTIBODIES. METHODS FOR PURIFICATION OF YOLK IGG

Jens Chr. JenseniusU a , Ingelise Andersena, Jann Hau $ , Monna Crone H and Claus Koch H U Institute of Medical Microbiology, Odense Uni¨ ersity, Odense, a The Finsen Institute, Copenhagen, $ Laboratory Animal Unit, Odense Uni¨ ersity, Odense and H Institute for Experimental Immunology, Uni¨ ersity of Copenhagen, Denmark