Bovine colostrum or milk as a serum substitute for the cultivation of a mouse hybridoma

A mouse-mouse hybridoma was grown in serum-free medium supplemented with bovine milk or colostrum. Bovine colostrum supported growth of the hybridoma whereas bovine milk alone did not support cellular proliferation. For growth in medium supplemented with colostrum, the maximum cell concentration achieved was 1.4 x 10(6) cells/mL in 2.2% colostrum, which is 44% of that obtained in 9% serum. When cells were grown in media containing milk and low amounts of serum (<1%) the maximum cell concentration in 2.2% milk with 0.4% serum was 2 x 10(6) cells/ml, whereas it was only 0.2 x 10(6) cells/ml and 1.3 x 10(6) cells/ml in 2.2% milk alone and 0.4% serum alone, respectively. Similar behavior was observed for growth in media containing colostrum and low amounts of serum. The monoclonal antibody production in media containing combinations of serum and milk or colostrum was comparable to that obtained in media with higher serum concentrations. Experiments performed with conditioned media suggest that the rapid decrease in viability, after the maximum cell concentration has been reached, is partially due to the presence of some inhibitory components generated during the cell culture rather than due to depletion of some serum components.     

Characterisation of whey proteins of camel (Camelus dromedarius) milk and colostrum

Variation in the composition of whey proteins from camel (Camelus dromedarius) colostrum and milk was recorded over a 192 h period following parturition. Whey proteins were separated by cation-exchange fast protein liquid chromatography and identified by polyacrylamide gel electrophoresis. The main components of whey proteins in camel milk and colostrum were similar to that in bovine, except for the lack in β-lactoglobulin. Serum albumin was the major whey protein present in camel milk, with an average concentration of 10.8 g/l. Camel colostrum was rich in immunoglobulins G, which consist of IgG1, and the enzyme inhibitory antibodies IgG2 and IgG3. The concentration of these proteins decreased rapidly 48 h post partum. Lactophorin (proteose peptone-component 3) and basic whey protein were detected only within 48 h after parturition, reaching a level of 4.9 and 3.1 g/l at 192 h post partum, respectively. The maximum level of lactoferrin (2.3 g/l) was observed at 48 h after parturition. Camel milk and colostrum were shown to be rich in protective proteins, especially IgG2 and IgG3, which revealed to be a potential source of inhibitory antibodies.     

IgG1 variations in the colostrum of Holstein dairy cows

High-immune quality colostrum (IgG1 concentration ⩾50 g/l) is crucial for the health and development of the young calf. Studies on colostrum quality tend to focus on external factors such as breed, parity or dry period length, but few have focused on within-cow variations. Here we ran experiments to gain a deeper insight into within-cow variation in IgG1 concentrations in dairy cow colostrum. Trials were performed in an experimental farm, located in the Western part of France. Colostrum from each quarter and a composite sample (mix of four quarters) were concomitantly collected on 77 Holstein dairy cows just after calving to assess the influence of sample type on IgG1 concentrations. Variation in IgG1 concentrations during the first milking was studied on samples from nine cows collected every minute from the start of milking. Repeatability of colostral IgG1 concentration was estimated from 2009 and 2010 data on 16 healthy cows. IgG1 concentrations were tested using a radial immunodiffusion method. Sensitivity and specificity were similar regardless of sample type tested (individual quarter or composite milk). Mean average IgG1 concentration was 54.1 g/l in composite colostrum, and was significantly higher in hind quarter teats (56.2 g/l) than front quarter teats (53.1 g/l). Average IgG1 concentration did not change significantly during colostrum milking, and the variations observed (15% or less) were likely due to the laboratory method (CV 15%). IgG1 concentrations in dam colostrum increased slightly from 2009 to 2010 due to BW and parity effects. In 56% of cases, colostrum quality could have been assessed on either individual or composite colostrum samples collected at any time during the first milking without affecting the reliability of the measurement. However, in other cases, differences were significant enough to mean that estimates of average IgG1 concentration in colostrum from any one quarter would not be reliable. It is concluded that colostrum quality, from an IgG1 concentration point of view, could be assessed with a composite sample taken at any time during the first milking.     

无牛血清IgG细胞培养基（—GFCS培养基）的制备及其在杂交瘤细胞体外培养中的应用

为了制备不含牛血清IgG的细胞培养基（－GFCS培养基）,并研究其在杂交瘤细胞体外培养中的应用,采用蛋白G亲和层析的方法,将含有血清的细胞培养基中的牛血清IgG去除,以制备无IgG的培养基。使用该培养基体外培养杂交瘤细胞后,监测细胞生长和上清抗体浓度。对培养上清中的IgG类单克隆抗体可以采用蛋白G亲和层析进行纯化。与示去除牛血清IgG的培养基相比,－GFCS培养基培养的杂交瘤细胞的生长状况及上清抗体浓度均无明显变化;从－GFCS培养上清中成功纯化出不被血清IgG污染的IgG类单克隆抗体,本文结果表明,采用－GFCS培养基体外培养分泌IgG类单抗的杂交瘤细胞,可以简化上清抗体的纯化工艺。     

Methods for increasing monoclonal antibody production in suspension and entrapped cell cultures: biochemical and flow cytometric analysis as a function of medium serum content.

The growth and antibody production of the SP2/0-derived hybridoma HB124 (ATCC) grown in media containing varying amounts of fetal bovine serum (FBS) were monitored using biochemical and flow cytometric methods. Hybridomas grown in 100 ml spinner flasks with RPMI-1640 containing varying amounts of serum demonstrated that cell growth, viability and IgG production show significant changes when serum content is decreased from 10.0 to 5.5 to 1.0 and 0.5%. A longer lag phase resulted when the lower serum content media were used. Cellular rates of glucose uptake showed a significant increase as serum levels were lowered. Similarly, exponential phase IgG production rates increased as the amount of serum was decreased, probably as a result of the decreased rate of exponential growth. Flow cytometric analysis showed a similar increase in cellular IgG content as medium serum levels declined. In contrast, the maximum IgG concentrations were found in flasks containing 1% FBS or above with the lowest concentration in the 0.5% FBS flask being due to the lower numbers of viable cells. Cells grown in microporous hollow fiber reactors were fed with medium containing serum which was decreased stepwise with time. Decreasing medium serum content stepwise from 10 to 2.5% resulted in increased antibody production. However, complete removal of serum from the medium resulted in a significant drop in antibody productivity. Cumulative antibody production was equivalent for cells grown entirely in medium containing 10% FBS and for those which experienced a drop to 2.5% FBS. To compare a defined serum-free medium preparation with medium containing 10% FBS, cells were again grown in batch suspension culture and analyzed. The growth rates were similar but there was a significant difference in IgG production rates. The serum-free culture exhibited both higher cellular production rates and higher IgG concentrations. These results indicate that decreasing medium serum content can adversely affect antibody yield because of lower cell viabilities, not because of lower production rates. Use of a defined serum-free medium, as done in this study, results in higher yields because of a higher IgG production rate as well as good cell growth and viability.     

Influence of growth supplements on lactic acid production in whey ultrafiltrate by Lactobacillus helveticus

Summary Investigations have been carried out on lactic acid production by Lactobacillus helveticus CNRZ 303 in whey ultrafiltrate. Addition of beet molasses was investigated with good results, although yeast extract proved to be more effective. The size of inoculum and the preculture medium also played a significant role in determining the amount of lactic acid produced during the fermentation process. High lactose consumption (94.09%), together with good lactic acid production (26.09 g/l) and yield (0.90%), were obtained in whey ultrafiltrate supplemented with 1% (w/v) beet molasses (WUM), with a 10% (w/v) inoculum and peptonized milk as preculture medium. Although these results were similar to those obtained when yeast extract was used as supplement, the maximum volumetric productivities proved to be quite different, and were definitely higher with yeast extract. Offprint requests to: L. Chiarini     

Preparation of a high-immunoglobulin product from bovine colostrum

An effective colostrum management programme is the most important factor in determining the health and survival of the neonatal calf. Commercially available colostrum replacers (CR) and colostrum supplements (CS) are an alternative to colostrum on farms that do not have an adequate, high-quality colostrum supply, or those farms that want to prevent transmission of disease from cow to calf. The present study aimed to obtain a high immunoglobulin (Ig), dried bovine colostrum product that could be used as a CR or CS for dairy calves. Dried whey was made from 6 batches of colostrum and an additional 6 batches were used to produce dried whey and curds. Dried whey had higher IgG concentration (P<0.05) and lactose (P<0.05), and less fat (P<0.05) compared to curds or the original colostrum. There was a strong linear relationship between initial colostrum IgG concentration and whey (R² = 0.86) with approximately 0.45 of the initial colostral IgG residing in curd. The high IgG and the composition of colostrum whey powder suggests it could be an effective CS product for use with dairy calves. The high fat and IgG content of the curd by-product indicate that it might be a potential weaning supplement in piglets or even a product for human consumption.     

Effects of bovine colostrum, foal serum immunoglobulin concentration and intravenous plasma transfusion on chemiluminescence response of foal neutrophils

Summary. The effects of bovine colostrum, absorption of equine colostral immunoglobulins and age on phagocytic and serum opsonizing activity of nine clinically healthy foals were examined. Cells and serum were collected prior to suckling and at 7, 14 and 28 days of age. Seven foals had serum IgG concentrations >600mg/dl whereas two foals had <350mg of IgG/dl. Phagocytic and serum opsonic activity of eight clinically ill foals with <400mg of IgG/dl of serum were also examined before and after plasma transfusion. Phagocytic and serum opsonizing activities were evaluated by an assay for chemiluminescence (CL) after addition of opsonized streptococci. Results showed that bovine colostrum stimulated CL of foal neutrophils. Preliminary characterization of opsonins in bovine colostrum by ammonium sulphate fractionating and heat inactivation indicated that opsonins generating CL were mainly associated with immunoglobulin G. Chemiluminescence generated by foal neutrophils varied with age with foal neutrophils collected at day 14 producing more CL than adult neutrophils ( P <0.05). Foal serum opsonizing activity was similar to adult opsonizing activity if serum IgG concentrations were >600mg/dl but it was less if IgG concentration was <350mg/dl ( P <0.05). Chemiluminescence generated by foal and adult neutrophils was higher when post-transfusion foal serum was used as the source of opsonin than when pre-transfusion foal serum was used ( P <0.05). When adult serum was the opsonin, chemiluminescence of foal neutrophils collected before and after plasma transfusion did not differ. The increase in CL following plasma transfusion was probably due to an increase in serum opsonizing activity.     

Factors associated with the concentration of immunoglobulin G in the colostrum of dairy cows

Transfer of sufficient immunoglobulin G (IgG) to the neonatal calf via colostrum is vital to provide the calf with immunological protection and resistance against disease. The objective of the present study was to determine the factors associated with both colostral IgG concentration and colostral weight in Irish dairy cows. Fresh colostrum samples were collected from 704 dairy cows of varying breed and parity from four Irish research farms between January and December 2011; colostral weight was recorded and the IgG concentration was determined using an ELISA method. The mean IgG concentration in the colostrum was 112 g/l (s.d. = 51 g/l) and ranged from 13 to 256 g/l. In total, 96% of the samples in this study contained >50 g/l IgG, which is considered to be indicative of high-quality colostrum. Mean colostral weight was 6.7 kg (s.d. = 3.6 kg) with a range of 0.1 to 24 kg. Factors associated with both colostral IgG concentration and colostral weight were determined using a fixed effects multiple regression model. Parity, time interval from calving to next milking, month of calving, colostral weight and herd were all independently associated with IgG concentration. IgG concentration decreased (P < 0.01) by 1.7 (s.e. = 0.6) g/l per kg increase in the colostral weight. Older parity cows, cows that had a shorter time interval from calving to milking, and cows that calved earlier in spring or in the autumn produced colostrum with higher IgG concentration. Parity (P < 0.001), time interval from calving to milking (P < 0.01), weight of the calf at birth (P < 0.05), colostral IgG concentration (P < 0.01) and herd were all independently associated with colostral weight at the first milking. Younger parity cows, cows milked earlier post-calving, and cows with lighter calves produced less colostrum. In general, colostrum quality of cows in this study was higher than in many previous studies; possible reasons include use of a relatively low-yielding cow type that produces low weight of colostrum, short calving to colostrum collection interval and grass-based nutritional management. The results of this study indicate that colostral IgG concentration can be maximised by reducing the time interval between calving and collection of colostrum.     

Effect of a bovine colostrum whey supplementation on growth performance, faecal Escherichia coli population and systemic immune response of piglets at weaning

This study examined the effect of a bovine colostrum whey supplementation on growth performance, feed intake, faecal Escherichia coli population and systemic immune response of piglets at weaning. A total of 96 piglets weaned at 26 ± 2 days of age were assigned for 4 weeks to one of the two treatments: (1) the control (commercial diet with bovine milk whey powder) and (2) the colostrum (commercial diet with freeze-dried bovine colostrum whey) treatments. The two supplements were incorporated in the diet at a level of 20 g/kg during the first 2 weeks after weaning and lowered to a level of 10 g/kg for the next 2 weeks. BW and feed intake were measured weekly. Faecal E. coli counts were determined weekly on specific culture media. Blood samples were collected weekly and submitted to a cell counter analyser for their main components (red and white blood cells, platelets) and flow cytometry was used to determine the lymphocyte population (B, T, Th and Tc). Finally, total seric immunoglobulin (IgM, IgG and IgA) concentrations were determined by the ELISA method. During the first week of the trial, the piglets from the colostrum treatment had improved average daily gain (170 g/day v. 81 g/day, P < 0.001), average daily feed intake (346 g/day v. 256 g/day, P = 0.03) and feed efficiency (BW gain/feed intake) (0.48 v. 0.31, P = 0.04). The pigs fed the colostrum treatment had also a 25% increase in circulating IgA (P = 0.03) compared with the control treatment the first week. It is concluded that a distribution of bovine colostrum whey (20 g/kg diet) during the first week post-weaning induces a systemic IgA response and has a beneficial action on growth performances and feed efficiency.     

Statistical optimization of a culture medium for biomass and poly(3-hydroxybutyrate) production by a recombinant Escherichia coli strain using agroindustrial byproducts.

A statistically based Plackett-Burman screening design identified milk whey and corn steep liquor concentrations as well as ionic strength (based on phosphate buffer concentration) as the three main independent components of the culture medium that significantly (p < 0.05) influenced biomass and poly(3-hydroxybutyrate) (PHB) production in recombinant cells of Escherichia coli. This strain carries a plasmid encoding phb genes from a natural isolate of Azotobacter sp. Response surface methodology, using a central composite rotatable design, demonstrated that the optimal concentrations of the three components, defined as those yielding maximal biomass and PHB production in shaken flasks, were 37.96 g deproteinated milk whey powder/l, 29.39 g corn steep liquor/l, and 23.76 g phosphates/l (r2 = 0.957). The model was validated by culturing the recombinant cells in medium containing these optimal concentrations, which yielded 9.41 g biomass/l and 6.12 g PHB/l in the culture broth. Similar amounts of PHB were obtained following batch fermentations in a bioreactor. These results show that PHB can be produced efficiently by culturing the recombinant strain in medium containing cheap carbon and nitrogen sources.     

Isolation and purification of lactoferrin and immunoglobulin G from bovine colostrum with serial cation-anion exchange chromatography

High-value dairy proteins such as lactoferrin (LF) and immunoglobulin (IgG) were separated from bovine colostrum. The whey was initially adjusted to pH 6.8 with 1 mol/L NaOH and then went through centrifugation, precipitation, and filtration to eliminate the fat and caseins in bovine colostrum. The treated whey was further ultra-filtrated to partially remove both other proteins and carbohydrates under 50 kD molecular weight. Then the ultra-filtrated whey was passed through cation and anion exchange columns in series. The LF and IgG were adsorbed on cation and anion exchanger, respectively, due to their different pI. Both the cation and anion exchange columns were washed with de-ionized water followed by successive elution with sodium chloride solutions of increasing molarities (0.27 and 0.85 mol/L; 17 and 51 mmol/L) in a stepwise manner, respectively. After desalted, the elution was freeze-dried. Finally, the LF and IgG with respective purities of 95.0% and 96.6% were obtained.     

IgG absorption by Santa Ines lambs fed Holstein bovine colostrum or Santa Ines ovine colostrum

The objective of this study was to evaluate the immunoglobulin G (IgG) absorption by Santa Ines lambs under two colostrum management systems usually used by producers. Twenty-seven Santa Ines newborn lambs received two meals of 250 ml of bovine colostrum from Holstein cows (BC group) or ovine colostrum from Santa Ines ewes (OC group) at 0 and 6 h of life. Pools of BC and OC were analyzed by radial immunodiffusion to quantify IgG. Results are expressed as least-square means and standard errors of mean (means ± s.e.m.). The concentration of IgG in bovine and ovine pools averaged 115.7 ± 20.5 and 48.1 ± 5.0 mg/ml, respectively, levels of concentration found in similar regular colostrum managements. The efficiency of IgG absorption was evaluated under two aspects, maximum apparent efficiency of absorption and total apparent efficiency of absorption (AEAmax and AEAtotal, respectively). The AEAmax was calculated taking into account the mass of IgG ingested just in the first meal of colostrum at birth and the serum IgG concentration at 6 h while the AEAtotal took into account the serum IgG concentration at 24 h of life that reflects the first colostrum offered at birth and the second meal at 6 h. The IgG and apparent efficiency of absorption results were transformed into the square root and log base 10, respectively, and were presented as geometric least-square means. In BC, lower (P < 0.05) AEAmax and AEAtotal were verified (14.2% and 15.6%, respectively), in relation to OC (23.6% and 24.4%, respectively). Serum IgG concentrations at 24 h were significantly higher (P < 0.05) in BC (31.4 mg/ml, respectively) compared with OC (22.2 mg/ml, respectively). The results in this study confirm that there is a limitation to the process of IgG absorption by the enterocytes of newborn lambs, which determined a nonlinear behavior of passive immunity acquisition. Similar values of AEAmax and AEAtotal for the two sources of colostrum reveal that the process of IgG absorption from the first and second meals during the first 6 h of life did not change and indicates that the ingestion of a second feeding of quality colostrum can enhance the acquisition of immune protection of newborn lambs.     

Local production of IgG4 in human colostrum

Total IgG4 levels were determined in 27 colostrum and 27 plasma paired samples by using RIA techniques, and total IgG was determined on the same pairs by using radial immunodiffusion. In colostrum, the mean IgG4 level was 4.6 micrograms/ml (0.6 to 19.0), and in the plasma the mean IgG4 level was 170.5 micrograms/ml (30 to 920). IgG averaged 42.3 micrograms/ml (12 to 240) in colostrum and 7980.9 micrograms/ml (3250 to 16,000) in plasma. Of colostral IgG, 15.3% was IgG4, whereas only 3.5% of plasma IgG was IgG4. Specific IgG4 antibodies to beta-lactoglobulin, bovine serum albumin, bermuda grass, and alpha-gliadin were also assayed. In six patients, strong evidence was found for local mammary production of IgG4-specific antibodies.     

Continuous hybridoma growth and monoclonal antibody production in hollow fiber reactors-separators

Growth of a hybridoma culture, along with production of monoclonal antibody, was demonstrated over extended periods in polysulfone hollow fiber membrane modules. The molecular weight cutoffs of the membranes were 70,000, 50,000, and 100,000 daltons. The hybridoma cell line, designated 65/26, produced IgG (2b/kappa) directed at mouse thymus cell surface antigen, TL.1. Cell growth occurred in the shell space of the reactor, using supplemented RPMI 1640 (20% fetal bovine serum) supplied from a separate reservoir vessel through the hollow fiber lumen. The reservoir contained 125 mL media, which was changed every 4 days. Concentrations of immunoglobulin were determined by an enzyme immunoassay (using protein A and alkaline phosphatase-labeled antibody conjugate). For the 10K, 50K, and 100K hollow fiber membrane modules, the maximum IgG concentrations detected in the 2.5-mL shell space were 47.5-80, 510, and 740 mug/mL, respectively. In the 125-mL reservoir for the 100K hollow fiber membrane module, the IgG concentration was measured at 260 mug/mL These values compare with an IgG concentration of 1 mug/mL when grown in a standard tissue culture flask and 3.2-7.6 mug/mL when grown in 100 ml media in a spinner flask. In addition, 10K and 50K hollow fiber membrane modules were run in a mode that decreased the fetal bovine serum supplement with time. Differences between these systems suggest that it is possible to obtain high IgG accumulation rates, both during and after the exponential growth phase of the hybridoma population.     

Batch fermentation of whey ultrafiltrate by Lactobacillus helveticus for lactic acid production

Summary Cheese whey ultrafiltrate (WU) was used as the carbon source for the production of lactic acid by batch fermentation with Lactobacillus helveticus strain milano. The fermentation was conducted in a 400 ml fermentor at an agitation rate of 200 rpm and under conditions of controlled temperature (42° C) and pH. In the whey ultrafiltrate-corn steep liquor (WU-CSL) medium, the optimal pH for fermentation was 5.9. Inoculum propagated in skim milk (SM) medium or in lactose synthetic (LS) medium resulted in the best performance in fermentation (in terms of growth, lactic acid production, lactic acid yield and maximum productivity of lactic acid), as compared to that propagated in glucose synthetic (GS) medium. The yeast extract ultrafiltrate (YEU) used as the nitrogen/growth factor source in the WU medium at 1.5% (w/v) gave the highest maximum productivity of lactic acid of 2.70 g/l-h, as compared to the CSL and the tryptone ultrafiltrate (TU). L. helveticus is more advantageous than Streptococcus thermophilus and Lactobacillus delbrueckii for the production of lactic acid from WU. The L. helveticus process will provide an alternative solution to the phage contamination in dairy industries using Lactobacillus bulgaricus.     

Human colostral whey M-1 glycoproteins and their L. bifidus var. Penn. growth promoting activities

A total of seven M-1 glycoprotein fractions were isolated from human colostrum whey and all showed L. bifidus var. Penn. growth promoting activities using well defined chemical medium. The growth promoting activity of these fractions was parallel to their total carbohydrate content. On the other hand, bovine colostrum M-1 glycoprotein had a relatively low growth promoting activity, whereas human serum orosomucoid had none.     

Effect of aeration on the production of carotenoid pigments by Rhodotorula rubra-lactobacillus casei subsp. casei co-cultures in whey ultrafiltrate

Under intensive aeration (1.3 l/l min) the associated growth of Rhodotorula rubra GED2 and Lactobacillus casei subsp. casei in cheese whey ultrafiltrate (55 g lactose/l) proceeded effectively for both cultures with production of maximum carotenoids (12.4 mg/l culture fluid). For maximum amount of carotenoids synthesized in the cell, the yeast required more intensive aeration than the aeration needed for synthesis of maximum concentration of dry cells. Maximum concentration of carotenoids in the cell (0.49 mg/g dry cells) was registered with air flow rate at 1.3 l/l min, and of dry cells (27.0 g/l) at 1.0 l/l min. An important characteristic of carotenogenesis by Rhodotorula rubra GED2 + Lactobacillus casei subsp. casei was established--the intensive aeration (above 1.0 l/l min) stimulated beta-carotene synthesis (60% of total carotenoids).     

Studies on Immunoglobulins and Trypsin Inhibitor in Colostrum and Milk from Sows and in Serum of Their Piglets

The concentrations of IgG, IgM, IgA and the specific sow colostrum trypsin inhibitor (SCTI) were measured by radial immunodiffusion in colostrum and milk samples from sows and in serum samples from their offspring during the suckling period. A clear time dependence was found for all the measured variates in both whey and serum. Statistically significant positive correlations were found between, on the one hand, concentrations of IgG and IgA, but not IgM, in sera from 39 suckling piglets 1 and 3 days old, and, on the other hand, concentrations of the same immunoglobulins and of the trypsin inhibitor in maternal colostrum (n = 7). Multiple regression analyses showed that at day 1 and day 3 the levels of both IgG and IgA in serum samples from the suckling piglets were positively influenced by both the SCTI and the IgG or IgA contents in maternal colostrum.     

Enhancement effects of BSA and linoleic acid on hybridoma cell growth and antibody production

The effects of linoleic acid and bovine serum albumin on hybridoma cell growth and antibody production were investigated. In dish cultivation, linoleic acid on its own promoted cell growth when used at concentrations below 50 mg L^–1, but strongly inhibited growth at a concentration of 100 mg L^–1 on more. However, linoleic acid bound to bovine serum albumin did not inhibit cell growth, even at a concentration as high as 100 mg L^–1. Also, linoleic acid did not affect the specific antibody production rate, with or without bovine serum albumin. In order to elucidate the enhancement of antibody production by bovine serum albumin, fractions were prepared by ultrafiltration (98% molecular weight cut-offs, 50,000 and 17,000) and the effects of the fractionation on antibody production were studied in batch cultivation. The high-molecular-weight fraction (50,000) promoted antibody production whereas the low-molecular-weight fraction (17,000) inhibited it. In continuous cultivation, the high-molecular-weight fraction was also found to enhance antibody production.