实用淋巴细胞培养技术

采自人体的外周静脉血液,用淋巴细胞分离液进行分析,收集分离得到淋巴细胞,同时回收血液的血清成分,此血清不经灭活可直接用于培养。本实验对影响淋巴体外激少在、增殖的相关因子进行了详细的统计分析,实验结果表明,在基础培养液ＲＰＭＩ１６４０中添加１００μｇ／ｍｌ的ＰＨＡ、１００ｉｕ／ｍｌ的ＩＬ－２和１０％的自体或胎血清,可以有产地激活淋巴细胞并在营养条件允许的情况下长期处于增鱼的状态。     

Separation of lymphokine-activated killer cell precursors and T-cells: differential growth characteristics and apparent lack of a T-cell suppressor of LAK-cell induction

Lymphokine activated killer (LAK) cell clinical effectiveness may be limited by the total cell dose and cytotoxic activity. We have, therefore, examined methods to expand the number of LAK-cells by serial passage of unfractionated and fractionated peripheral blood lymphocytes. Human purified lymphocytes were obtained by Ficoll Hypaque gradients followed by exposure of resultant mononuclear cells to phenylalanine methyl ester to remove monocytes. Lymphocytes were then fractionated on a six-step Percoll gradient (50%, 47.5%, 45%, 42.5%, 40%, and 37.5% Percoll). Unfractionated cells and fractions were cultured in standard media (RPMI-1640, 10% human sera, antibiotics and 10 mM HEPES) containing 10 nM of recombinant Interleukin-2 (rIL-2). Lymphocytes were cultured at 1 X 10(6)/ml and recultured every 3 to 4 days in fresh standard media and rIL-2. Utilizing unfractionated and fractionated lymphocytes from seven donors we made the following observations: (1) Continued passage of unfractionated lymphocytes resulted in a loss of LAK-cell activity by greater than or equal to 14 days (e.g., percent lysis of Raji at 10:1 effector:target ratio on days 0, 4, 7, and 21 was 0.38 +/- 11, 41 +/- 17, and 8 +/- 1, n = 4, respectively). (2) LAK-cell functional precursors were predominantly confined to the lymphocytes in the upper (0-4) Percoll fractions (e.g., on day 4, the percent lysis by pooled fractions 1-4 was 63 +/- 5 vs. pooled fraction 5 plus pellet, 18 +/- 7%). (3) As expected, the upper fractions (0-4) were enriched for Leu 19 positive cells (approximately 40%) and large granular lymphocytes (LGL) by morphology (approximately 30%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Spontaneous interferon production and growth of lymphoblastoid cells in serum-free medium

Numerous lymphoblastoid cell lines were established from human adult peripheral blood and cord blood lymphocytes, using Epstein Barr virus, and most cell lines from cord blood lymphocytes spontaneously produced abundant interferon without induction with Sendai virus, whereas lymphoblastoid cells from adult peripheral blood lymphocytes did not. These potential cells grow well in a newly developed serum-free culture medium based on Dulbecco's modified Eagle medium supplemented with non-essential amino acid, vitamins, nucleic acid derivatives, metal compounds, human transferrin, insulin and bovine or human serum albumin (Chon Fr.V). In serum-free medium, as well as in serum-containing conventional medium (RPMI-1640), the cells could also spontaneously produce interferon. The cells in the serum-free, culture could produce about 10 000 U/ml of interferon every day, harvesting the culture fluid and refeeding the cells with the fresh medium at the saturation cell density (10⁷ cells/ml). The interferon proved to be α-type interferon on the basis of its physico-chemical and antigenic properties.     

Genotoxic effect of formocresol pulp therapy of deciduous teeth

OBJECTIVE: To investigate whether formocresol, in Buckley's original formulation, used for pulp therapy of deciduous teeth, can have a genotoxic effect. Genotoxicity was tested in lymphocyte cultures from the peripheral blood of children aged 5-10y, in Recife, Pernambuco, Brazil. This was a case-control study. The sample comprised 40 children who had primary teeth with non-vital pulps. Two venous blood samples (6-8ml) were collected from each child, the first prior to pulp therapy (control group) and the second 24h after pulp therapy (experimental group). Lymphocyte cultures were grown in 78% RPMI 1640 medium, 20% fetal bovine serum, 2% phytohemagglutinin. The lymphocytes were assessed for chromosomal aberrations; each sample involved analysis of 100 metaphases. There was a statistically significant difference between the control and treated groups for the isochromatid gap (p<0.001), chromatid break (p<0.009), isochromatid break (p<0.046), other chromosomal alterations (p<0.001), and for total aberrations. In view of these results, caution in the use of formocresol in pediatric dentistry is recommended.     

Lycium barbarum polysaccharides regulate phenotypic and functional maturation of murine dendritic cells

Lycium barbarum polysaccharides (LBPs) have been known to have a variety of immunomodulatory functions including activation of T cells, B cells and NK cells. Dendritic cells (DC) are potent antigen-presenting cells that play pivotal roles in the initiation of the primary immune response. However, little is known about the immunomodulatory effects of LBPs on murine bone marrow derived dendritic cells (BMDC). In the present study, the effects of LBPs on the phenotypic and functional maturation of murine BMDC were investigated in vitro. Compared to the BMDC that were only subjected to treatment with RPMI1640, the co-expression of I-A/I-E, CD11c and secretion of IL-12 p40 by BMDC stimulated with LBPs (100 microg/ml) were increased. In addition, the endocytosis of FITC-dextran by LBPs-treated BMDC (100 microg/ml) was impaired, whereas the activation of proliferation of allogenic lymphocytes by BMDC was enhanced. Our results strongly suggest that LBPs are capable of promoting both the phenotypic and functional maturation of murine BMDC in vitro.     

The effect of interleukin-15 on the expression of killer-cell immunoglobulin-like receptors on peripheral natural killer cells in human

Interleukin (IL)-15 has emerged as a key regulator of both natural killer (NK) cell differentiation and activation. The aim of the present study was to investigate the expansion of the population of cells expressing killer-cell immunoglobulin-like receptors (CD158a and CD158b) in human peripheral lymphocytes by treatment with IL-15. One million peripheral lymphocytes were cultured in RPMI1640 medium alone or in medium containing IL-2 at 100 U/ml or IL-15 at 0.1, 1.0, or 10.0 ng/ml for 48 h. After each incubation, we assessed the natural killing activity and the population of CD16(+)CD158a(+)/b(+) cells and CD8(+)CD158a(+)/b(+) cells. IL-15 increased the NK activity and expanded the populations of CD16(+)CD158a(+)/b(+) cells and CD8(+)CD158a(+)/b(+) cells. These actions were dose dependent, and the effects of IL-15 at 1.0 ng/ml were close to those of IL-2 at 100 U/ml. These findings suggest that IL-15 induces the effector functions of resting NK cells throughout the body, and thereby plays a critical role in the activation of tissue-associated immune responses.     

Studies of serum-free medium for the generation of lymphokine-activated killer cells

We examined a serum-free medium (designated as TYI 101) for the generation of lymphokine-activated killer (LAK) cells from human lymphocytes, regional lymph node lymphocytes (RLNL) and peripheral blood lymphocytes (PBL). TYI 101 medium consisted of, in addition to nutrient mixture, transferrin, insulin, fetuin, sodium selenite, 2-mercaptoethanol, o-phosphorylethanolamine, chick egg yolk and porcine kidney extract. These hormones were effective for supporting RLNL proliferation as assessed by (³H)-thymidine uptake. When human lymphocytes from two different sources were cultivated with recombinant interleukin 2 (rIL-2) in TYI 101 medium, LAK activity was generated. In cultures of PBL from a healthy donor, LAK cells were generated in TYI 101 medium as efficiently as in RPMI 1640 medium supplemented with 10% human AB-type serum (RPMI-AB). In cultures of RLNL from lung cancer patients, LAK activity obtained in TYI 101 medium was about sixty-five percent of that in RPMI-AB. However, the addition of a small amount of AB-type serum improved the generation of LAK activity, LAK cell expansion, and cell viability in TYI 101 medium. We conclude that TYI 101 medium can be used for the generation of LAK cells from human lymph node lymphocytes with supplementation of none or only a reduced amount of human serum.Abbreviations IMDM Iscove's Modification of Dulbecco's Medium - rIL-2 recombinant Interleukin - LAK Lymphokine-Activated Killer - RLNL Regional Lymph Node Lymphocytes - PBL Pheripheral Blood Lymphocytes - PBS Phosphate-Buffered Saline - RBC Red Blood Cells - RPMI-AB RPMI 1640 medium supplemented with 10% human AB-type serum Address for offprints: Tsukuba Research Laboratory, Japan Synthetic Rubber Co., Ltd., 25 Miyukigaoka, Tsukuba-shi, Ibaraki, 305 Japan     

Effects of culture media on spontaneous incidence of mitotic index, chromosomal aberrations, micronucleus counts, sister chromatid exchanges and cell cycle kinetics in peripheral blood lymphocytes of male and female donors

The spontaneous incidence of mitotic index (MI), chromosomal aberrations (CA), micronucleus counts (MNC), sister chromatid exchanges (SCE) and cell cycle kinetics (CCK) were studied in human peripheral blood lymphocytes grown in M199 and RPMI-1640 culture media. Lower frequencies of CAs, MNC and SCEs were observed in lymphocytes cultured in medium RPMI-1640. The reduction of the MI and the replicative index in M199 medium showed delayed cell cycle kinetics.     

Age-related alterations in cell division and cell cycle kinetics in control and trimethyltin-treated lymphocytes of human individuals

Trimethyltin chloride induced age-related suppression of cell division and cell cycle kinetics in human peripheral blood lymphocytes cultured in RPMI 1640 culture medium supplemented with human AB serum, phytohemagglutinin and bromodeoxyuridine. A high frequency of M₁ (first metaphase) cells was seen in cultures treated with a high dose (C ₁ = 1.0 g per culture) and in lymphocytes from donors in the age range 40–70 years. The delay in cell division and cell cycle kinetics may indicate a longer duration in DNA synthesis induced by trimethyltin chloride in aged lymphocytes.     

In vitro effects of pure microcystin-LR on the lymphocyte proliferation in rainbow trout (Oncorhynchus mykiss)

The aim of this study was to determine the in vitro influence of microcystin-LR on the viability and mitogenic response of rainbow trout (Oncorhynchus mykiss) lymphocytes since few data are available in the literature on the influence of cyanotoxins on fish immunocompetent cells. Lymphocytes were isolated from blood and haematopoietic organs (pronephros and spleen) and cultivated in RPMI 1640 medium with different concentrations of the toxin (1, 5, 10, 20, 40 mg ml-1 of cell suspension). Dose-dependent effects of microcystin-LR on the lymphocyte viability were shown. The lymphocyte proliferation was inhibited after application of microcystin at a concentration of 40 mg ml-1 but significantly increased at a concentration of 1mg ml-1 in comparison to the control group. The results suggest the modulatory effects of microcystin-LR dependent on the applied concentration.     

Immune oxidative injury induced in mice exposed to normobaric O2: effects of thiol compounds on the splenic cell sulfhydryl content and Con A proliferative response

In vivo exposure of mice to normobaric O2 depresses the cellular immune response by a mechanism that remains unknown. In vitro oxidative injury leads to decreased sulfhydryl groups (SH) in lymphocytes. To determine whether in vivo exposure to O2 would have similar effects, we measured the SH content in spleen cells both from mice that had been exposed to normobaric O2 (O2 SC) and from controls exposed to ambient air (Air SC). The SH content of the fresh O2 SC was slightly decreased, whereas after 48 hr of culture, the SH content and the proliferative response of these cells were found to vary with the type and concentration of thiol or disulfide compounds added to the culture medium. Under standard culture conditions, i.e., RPMI 1640 medium containing 0.41 mM half-cystine, the SH content in O2 SC decreased sharply to about 10 and 20% that of Air SC in the absence or presence of Con A (2 micrograms/ml), respectively. Under these culture conditions, the proliferative response of O2 SC was 20.5% +/- 3.2 of Air SC. In cystine-free RPMI 1640 medium supplemented with various concentrations of L-cystine, L-cystine and 2-mercaptoethanol (2-ME), L-cysteine, or reduced glutathione (GSH), the proliferative response to Con A and the SH content of the O2 SC varied in parallel and were correlated (p less than 0.01). Half-cystine (0.41 mM) plus 2-ME (5 X 10(-5) M) or L-cysteine alone (4 mM) completely protected the SH content of O2 SC and induced a proliferative response 82% +/- 6 that of the controls. In cystine-free RPMI 1640 medium supplemented with GSH (4 mM), the SH content and proliferative response of O2 SC were 79 and 67.5% of Air SC, respectively. Other concentrations of these compounds were less effective. Oxygen scavengers such as SOD, catalase, mannitol, and vitamin E did not protect against the decrease of the O2 SC. The induced oxidative cellular damage might be related in part to a membrane lipid peroxidative process. These data show that in vivo exposure of mice to normobaric O2 induced lesions in splenic cells manifested under standard culture conditions by a decrease in both SH content and Con A proliferative response. The extent of these alterations could be modulated by variations of the thiol environment. Protection of the SH content correlated with protection of the proliferative response of the O2 SC.     

Observations consistent with autocrine stimulation of hybridoma cell growth and implications for large-scale antibody production

Existence of autocrine growth factors (aGFs) may influence the serum requirement for growth of hybridoma cells and thus significantly influence process economics. For the murine hybridoma cell line S3H5/2bA2, critical inoculum density (cID) and serum requirement for growth were inversely related for cultivation in both T flasks and spinner flasks. In spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 10³ cell/ml was necessary in RPMI 1640 medium with 10% serum. In T flasks, where the local cell density is higher than in spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 1 cell/ml was also necessary in RPMI 1640 medium with 10% serum. Further, immobilized cells at high local cell density could grow under conditions where cells in T flasks at corresponding overall cell density could not grow. The cells at high inoculum density were less sensitive to shear induced by mechanical agitation than the cells at low inoculum density. Taken together these observations support the existence of secreted aGF(s) by the hybridoma cell line used. Since the specific MAb production rate was independent of cultivation method and inoculum density, the existence of autocrine growth factors would suggest that the use of immobilized cells should improve the economics of MAb production.     

Functional activities of isolated lymphocytes following drying by sublimation of ice in vacuo: I. Rosette formation,stimulation by plant lectins (mitogens), and the mixed lymphocyte reaction

The following activities of isolated human lymphocytes were used for evaluating the effects of freezing and thawing and freeze-drying and rehydration on these cells: (a) spontaneous rosette formation, (b) responses to plant lectins (mitogens), and (c) the one-way mixed lymphocyte reaction. The successes achieved in drying of isolated lymphocytes by sublimation of ice in vacuo and rehydration with water with retention of the functions above, all of which appear to require living cells, were dependent upon a freeze-drying apparatus of unique design and the ability to freeze-dry suspending media containing dimethylsulfoxide. Best results were obtained when lymphocytes were: (a) isolated from blood collected in citrate-phosphate-dextrose (CPD); (b) suspended in Roswell Park Memorial Institute Medium-1640 (RPMI-1640) in sufficient amount to make 100%, 20% fetal calf serum, 8% serum albumin, 5% dimethylsulfoxide, and 1% L-glutamine; (c) cooled at approximately 1 °C/min from +4 to ?25 °C and approximately 5 °C/min from ?25 to ?70 °C, and (d) rehydrated at low temperatures.     

Human T lymphocyte differentiation antigens: effects of blood sample storage on Leu antibody binding

Current studies of human T lymphocytes and their subsets often use quantitative immunofluorescence analysis with monoclonal antibodies against cell surface antigens. With storage of whole blood or separated mononuclear cells for more than a few hours we have found marked changes in lymphocyte analysis using a fluorescence activated cell sorter (FACS). Experiments were done to determine if these lymphocyte changes were influenced by storage temperature and if lymphocytes could be made more stable by addition of culture media RPMI 1640 to whole blood. Optimal conditions found for blood storage were with with addition of 50% RPMI 1640 and with samples held at room temperature (22 degrees C). With these storage conditions, delay on FACS analysis up to 24 hours did not result in spurious results. When blood samples are collected in places remote from the laboratory or when batch analysis of serially collected samples is desirable, excessive storage times should be avoided.     

Inhibition of lymphocyte activation by cyclosporin A: interference with the early activation of the membrane phospholipid metabolism in rabbit lymphocytes stimulated with concanavalin A, anti-rabbit immunoglobulin or the Ca2+ ionophore A 23187

Rabbit lymphocytes from the mesenteric lymph nodes were stimulated with concanavalin A, goat anti-rabbit immunoglobulin, or the Ca2+ ionophore A 23187. The stimulated incorporation of labeled uridine into RNA as well as of labeled thymidine into DNA was suppressed within a dose range of 40-1000 ng/ml cyclosporin A in both Con A-stimulated T lymphocytes and in anti-immunoglobulin-stimulated B lymphocytes, without affecting the resting cells. A 23187-stimulated rabbit lymphocytes proved to be more sensitive to cyclosporin A. At 40 ng/ml the immunosuppressive drug was effective in inhibiting elevated incorporation of labeled nucleosides into macromolecules in ionophore-stimulated cells. Cyclosporin A, at the same concentrations that were effective in inhibiting stimulated RNA and DNA synthesis, suppressed one of the earliest events occurring in stimulated lymphocytes, i.e., enhanced incorporation of unsaturated fatty acids into membrane phospholipids. Whereas cyclosporin A significantly inhibited the incorporation of arachidonic acid into phosphatidylcholine and phosphatidylethanolamine in concanavalin A-, anti-immunoglobulin-, and A 23187-stimulated cells, it proved to be ineffective in inhibiting the incorporation of arachidonate into phosphatidylinositol. The data indicate that cyclosporin A inhibits both T- and B-cell stimulation by interfering with a common target, e.g., the early activation of membrane phospholipid metabolism of rabbit lymphocytes.     

Dynamic characteristics of the fluorescence of human peripheral blood neutrophilic polymorphonuclear leukocytes and lymphocytes intravitally fluorochrome-stained with acridine orange]

Neutrophil segmentonuclear leukocytes and lymphocytes of the human peripheral blood vitally stained with Acridine Orange (AO) in concentrations of 250 and 330 mcg/ml show different fluorescence dynamics. The number of neutrophil segmentonuclear granulocytes with green fluorescence of nuclei decreases, whereas the number of cells with red fluorescence of nuclei increases. As a criterion of this process, time T1/2 is taken during which the number of green-fluorescent cells decreases twofold. With AO concentrations of 250 and 330 mcg/ml, T1/2 is equal to 40 or 5 minutes, resp. The nuclei of lymphocytes within a 60 minutes observation show green fluorescence. This effect is likely to be due to structural-functional peculiarities of neutrophil segmentonuclear granulocytes and lymphocytes.     

Nickel compound-induced DNA single-strand breaks in chromosomal and nuclear chromatin in human blood lymphocytes in vitro: role of oxidative stress and intracellular calcium

The effects of nickel sulfate, and soluble forms of nickel carbonate hydroxide (NiCH), nickel subsulfide, and nickel oxide on delayed induction of DNA single-strand breaks (DNA SSBs) in chromosomal and nuclear chromatin of human blood lymphocytes in culture were studied. After 46 h of initial culture in supplemented RPMI-1640 media at 37 degrees C, human whole blood lymphocytes in culture were exposed to low concentrations (0-15 microM) of different nickel (Ni) compounds for 2 h, whereas only RPMI-1640 medium served as control. Immediately after 2 h of such exposure, both control and Ni-treated cells were washed with the same medium and incubated further in fresh complete RPMI-1640 culture medium for another 24h. After a total 70 h of incubation, cells were then arrested at metaphase. Two hours later, the induction of DNA SSBs involving both metaphase chromosomal and interphase nuclear chromatin was measured using the method of electron microscopy in situ end-labeling. The metaphase chromosomal chromatin showed significantly higher DNA SSBs (as measured by an increase in immunogold particles per microm2 chromatin) due to 15 microM NiCH and NiO when compared to the corresponding control value. Both NiCH and nickel oxide produced significantly higher induction of DNA SSBs than those of nickel subsulfide and nickel sulfate in chromosomal chromatin. The DNA SSBs in chromosomal chromatin were found to be significantly higher than those in nuclear chromatin due to different Ni compounds. Overall, the genotoxic potency seems to be decreased as follows: NiCH>nickel oxide>or=nickel subsulfide>nickel sulfate. Pretreatment of human blood lymphocytes with either catalase (a H2O2 scavenger), or superoxide dismutase (a scavenger of O2- radical) or dimethylthiourea (a hydroxyl radical scavenger), or N-acetylcysteine (GSH precursor) significantly reduced DNA SSBs in both chromosomal and nuclear chromatin induced by NiCH, suggesting the involvement of different types of oxidative stress in such genotoxicity. Deferoxamine (a highly specific iron chelator) pretreatment prevented NiCH-induced DNA SSBs in both chromosomal and nuclear chromatin suggesting a role of iron-mediated oxidative stress generating hydroxyl radical in such genotoxicity. Simultaneous treatment with either verapamil (an inhibitor of Ca 2+ through plasma membranes), or dantrolene (an inhibitor of mobilization of [Ca2+]i from endoplasmic reticulum), or BAPTA (a Ca2+ chelator) significantly reduced Ni compound-induced DNA SSBs in both chromosomal and nuclear chromatin, suggesting that Ni compound-induced destabilization of calcium homeostasis may also involved in the induction of such DNA SSBs.     

Effects of arachidonic acid and other unsaturated fatty acids on mitogenesis in human lymphocytes.

The effect of fatty acids and other lipids on mitogenic responses in cultured human peripheral blood lymphocytes was studied. Several-fold enhancement of tritiated thymidine incorporation was observed at 0.1 to 5.0 micrograms/ml concentrations of arachidonic acid. Other unsaturated fatty acids produced less marked changes. Increased responsiveness was demonstrable in a variety of media including RPMI 1640 supplemented with 10% fetal calf serum. Changes were also observed in uridine incorporation, total cell number, and blast transformation, indicating that the effect was not on thymidine transport or pool size per se. Arachidonic acid failed to affect PHA binding, indicating that the lectin-cell interaction was not altered. Higher concentrations of fatty acids were inhibitory.     

Relative ranking of culture media based on proliferation kinetics and spontaneous chromosomal aberrations in PHA-responsive human peripheral blood lymphocytes.

Proliferation kinetics and spontaneous yield of chromosomal aberrations phytohemagglutinin (PHA)-responsive peripheral blood lymphocytes were studied from blood samples collected from 45 individuals in 4 different synthetic media. Except for a significant difference for Eagle's MEM and RPMI 1640, the other media did not show difference for the yield of chromatid or chromosome type of aberrations. Differences were however noticed in the proliferation kinetics (mitotic and proliferative rate indices) of cells among the media used. The study indicated that (i) the intrinsic properties of media which influence proliferation rate and yield of chromosomal aberrations are independent of each other as higher proportion of first division cells do not correspond with higher frequency of chromosomal aberrations, (ii) the amount of free-radical scavengers present in the medium, apart from the genetic make-up of the individuals, may contribute to the spontaneous yield of chromosomal aberrations and (iii) RPMI 1640 medium, which showed higher transformation and faster cycling rate for the lymphocytes, may be considered as medium of choice for analysing two main cytogenetic end-points, chromosomal aberrations and sister chromatid exchanges (SCEs).     

Comparison between aseric and seric culture-derived exoantigens of Babesia divergens in their ability to induce immunoprotection in gerbils

Babesia divergens Rouen 1987 was cultivated with a high percentage of parasitized erythrocytes (30–40%) in either RPMI 1640 supplemented by 10% human serum or in a serum-free medium consisting of RPMI 1640 supplemented with 5 g/l Albumax I®. Analysis of serum and Albumax culture supernatants, using polyacrylamide gel electrophoresis, revealed the presence of at least 10 parasitic exoantigens of B. divergens with molecular weights ranging between 27 and 200 kDa. The gerbils were injected twice, at 3-week intervals, with Albumax culture supernatants or seric culture supernatants. The vaccine doses ranged from 3 μl to 1.5 ml. The highest immunofluorescent antibody titers in gerbils (in 42 days) were obtained using Albumax supernatant and Quil A saponin as adjuvant. Analysis of the gerbil humoral response by immunoprecipitation showed that only three exoantigens were immunodominant: 92 kDa, 50 kDa and 37 kDa proteins. The gerbils were challenged 3 weeks after the last vaccine injection and the maximum protection was observed with vaccine doses ranging from 30 μl to 1.5 ml of culture supernatant and Quil A adjuvant. Albumax medium-derived antigens potentiated better protection at lower dose rates than that of serous medium-derived antigens (for example the gerbil mortality was 0% when they are immunized with 30 μl of Albumax supernatant and 100% with 30 μl of seric supernatant).