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Research Note: Avidity of Chicken Yolk Antibodies to Enterotoxigenic Escherichia coli Fimbriae
Research Note: Avidity of Chicken Yolk Antibodies to Enterotoxigenic Escherichia coll Fimbriae YUTAKA IKEMORI,1 ROBERT C. PERALTA, MASAHIKO KUROKI, HIDEAKI YOKOYAMA, and YOSHIKATSU KODAMA Immunology Research Institute in Gifu, 839-1, Sana, Gifu City, 501-11, Japan ABSTRACT The yolk antibodies from chickens and the serum and colostrum antibodies from cows were obtained after immunization of these animals with inactivated bacterin or purified K99 fimbriae from enterotoxigenic Escherichia coli (ETEC). The avidity of anti-K99 fimbriae antibodies produced from either chickens or cows was measured by competitive binding assay of ELISA. The yolk antibodies competed strongly with the serum and colostrum antibodies from immunized cows and inhibited 40 to 80% of the binding of these antibodies. Results demonstrate that the avidity of antibodies obtained from immunized chickens compares with that obtained from immunized cows. Thus, the yolk antibody from immunized chickens, aside from its use for prophylaxis against some infectious diseases, may also serve as effective ligand for purification of biologically active substances such as fimbrial antigens by affinity chromatographic procedures. (Key words: antibody avidity, yolk antibody, K99 fimbriae, enzyme-linked immunosorbent assay, competitive binding assay) 1993 Poultry Science 72:2361-2365
The practical value of utilizing antibodies in therapy lies in the specific binding Passive antibody immunization, in ad- capabilities of antibodies to harmful antidition to innovative vaccine technology, gens. This property of an antibody to react may have a far-reaching importance in specifically with an antigen or the strength animal health maintenance and control of of combination between an antibody and infectious diseases of animals. Passive an antigen may be a desirable indicator of antibodies derived from serum, colostrum, the protective capacity of an antibody. So and yolks of immunized chickens have far, there has been little research on the been used for prophylaxis and treatment. avidity of chicken yolk antibodies against In passive immunization experiments, a particular antigen. The objective of this yolk antibodies were effective in the study was to evaluate the avidity of prevention and control of intestinal infec- chicken yolk antibodies against ETEC K99 tions due to rotavirus (Bartz et al., 1980) fimbriae in comparison with the antibodand enterotoxigenic Escherichia coli (ETEC) ies of serum and colostrum from im(Yokoyama et al, 1992). The feasibility of munized cows. cost-effective, large-scale production of antibodies has favored the use of yolk MATERIALS AND METHODS antibodies. INTRODUCTION
Immunization of Chickens Received for publication May 18, 1993. Accepted for publication September 2, 1993. x To whom correspondence should be addressed. ^Charles City, IA 50616.
Enterotoxigenic Escherichia coli strain 431 (0101:K30;K99;F41.NM,ST+) was obtained from Salsbury Laboratories Inc.2 The bacteria were grown in Minca broth (Guinee et
2361
2362
KEMORI ET AL.
al, 1977), were harvested by centrifugation, and the K99 fimbriae were purified by a combination of gel filtration and ion exchange chromatography (Isaacson, 1977). Purified fimbrial vaccine containing .5 mg of fimbrial antigen in emulsion oil mixed with 5% Arlacel 803 was injected i.m. in the breast muscle of 10 5-mo-old Hy-Line® W36 White Leghorn chickens. Six weeks after the initial injection, the chickens were boosted in the same manner, and the eggs were harvested from the 10 hens 3 to 4 wk after the second injection. Collection of Yolk Antibodies The yolks were individually and carefully separated from the egg whites and then mixed to homogeneity by an automated mixer. Yolks of eggs from each hen were pooled separately to yield 10 different samples. The yolks were subsequently diluted with 8 vol of distilled water. An aqueous dispersion of 5% (vol/vol) hydroxy propyl methyl cellulose phthalate (HPMCP)4 was added to the diluted yolk to make a final .5% (vol/vol) mixture, which was then centrifuged at 10,000 xg for 20 min at 4 C. The supernatant was collected as the Ig-rich fraction. Yolks from unvaccinated chickens were used as a source of native yolk Ig. Immunization of Cows
trum were collected from cows after parturition; serum and whey were separated. The purified fimbrial vaccine was immunized in two cows that were not pregnant using the same immunization schedule for chickens. Serum was collected 3 wk after the second vaccination. Blood and colostrum from unvaccinated cows were used as a source of native serum and whey. Binding and Competitive Binding Assay of Antibodies The procedures for binding assay by ELISA were performed as described previously with slight modification (Yokoyama et al., 1992). Microdilution plates were coated overnight at 4 C with 5 jtig/mL of K99 purified fimbrial antigen. The plates were blocked with bovine serum albumin and washed three times. The wells were filled with 100 pL of serial twofold dilutions of antibodies and the plates were incubated at 37 C for 1 h. Rabbit anti-chicken IgG or anti-bovine IgG conjugated with horseradish peroxidases diluted 1:12,800 and 1:6,400, respectively, were applied and incubated at 25 C for 30 min. These dilutions were determined by preliminary plotting of optical density (OD) values against each type of purified IgG6 and the desired dilution of conjugates correlated .1 OD value with 1 ng of antibodies. The ophenylenediamine dihydrochloride and hydrogen peroxide were added as the substrate and the color reaction was inhibited by addition of 3 N H2S04 and the OD values were obtained in a microdilution plate ELISA reader at 492 nm. The ELISA antibody titer was defined as the reciprocal of the highest dilution of antibodies with .1 OD value.
Two kinds of vaccines, a commercial inactivated bacterin5 and purified fimbrial vaccine, were used to immunize Holstein cows. The cows were 3 to 5 yr old and had no history of disease or vaccination against E. coli. The bacterin was immunized in cows according to the recommendation of the manufacturer. Eight cows were given a Competitive binding between chicken 2-mL dose of bacterin i.m. 6 wk before and bovine antibodies was assayed by anticipated calving and similar doses incubating simultaneously the competitor spaced 3 wk apart. Blood and early colos- and probe antibody dilutions. Two phases of binding assays were performed: the first utilized yolk antibodies as the probe antibody and the bovine sera (from cows 3 Maine Biological Laboratories, Waterville, ME vaccinated with bacterin or purified fim04901. briae) and whey (from cow vaccinated with ^Shinetsu Chemical Co., Tokyo, 100, Japan. 5 Norden Laboratories Inc., Lincoln, NE 68501. bacterin only) were used as competitor ^appel, Organon Teknika Co., West Chester, PA antibodies; the second phase utilized three 19380. kinds of bovine antibodies as the probe and 7 MR650 Microplate Reader, Dynatech Laboratories yolk antibodies as the competitor. The Inc., Chantilly, VA 22021.
RESEARCH NOTE
2363
concentration of the competitor antibodies was adjusted to 1:1,024 of ELISA titer and concentrations of probe antibody were from ELISA titers of 1:1 to 128 in the binding assay. A duplicate assay of each antibody was done on different occasions. Results were expressed as percentage competition, calculated using the formula 100[1 - (B/A)], where A and B are the OD values in the absence and in the presence of competitor antibody, respectively. The different combinations of yolk antibodies with serum or whey antibodies accounted for 180 observations in the assays. Eighty (serum or whey from cows immunized with bacterin) and 20 (serum from cows immunized with K99 fimbriae) readings were represented by the mean.
antibody titer in the serum was moderately lower than that in the whey. The avidities of anti-K99 fimbriae antibodies were measured by competitive binding assays. The yolk antibodies strongly competed with antibodies of serum and whey from cows immunized with inactivated bacterin and inhibited 40 to 80% of the binding of these antibodies (Figure 1, A and B). On the other hand, the antibodies obtained from cows competed less strongly with the yolk antibodies, and inhibited 0 to 30% of the binding of the latter. Difference in antibody avidity of serum and whey of immunized cows could be recognized, and the antibodies of whey inhibited more strongly the binding of the yolk antibodies than serum competitor antibodies. Almost similar results were obtained Statistical Analysis from experiments with the antibodies of Results of competitive binding assays cows immunized with purified fimbrial antigen. In spite of almost similar antiwere analyzed using the Student's t test to body titers obtained, the yolk antibodies determine whether differences between inhibited 40 to 90% of the binding of those mean percentage competition of yolk anti- antibodies. The serum antibodies of cows body and three kinds of bovine antibodies raised against purified fimbrial antigen used as the competitor were significant. inhibited 0 to 30% of binding of the yolk Significance was set at P < .001. antibodies and inhibited a bit stronger the binding of the yolk antibodies than antibodies from cows immunized with inactiRESULTS AND DISCUSSION vated bacterin (Figure 1, C). The differThe mean ELISA antibody titers were 1: ences recognized between the mean 97,420 in the yolks and 1:115,852 in the percentage competition of the yolk antiserum from cows immunized with puri- bodies and the cow antibodies regardless fied K99 fimbriae (Table 1). In the cows of immunized antigens were significant (P immunized with the inactivated bacterin, < .001). mean ELISA titers were 1:137,772 in the All control antibodies from chickens whey and 1:13,280 in the serum; the and cows using the same competitive
TABLE 1. ELISA antibody titers of antibodies from immunized chickens and cows against enterotoxigenic Escherichia coli K99 fimbriae
Antibody
Animal
Number of samples tested
Yolk
Chicken
10
K99 fimbriae
21657 ± .75
Serum
Cow
2
K99 fimbriae
216.82 ± .5
Serum
Cow
8
Bacterin
213.70 ± .48
Whey
Cow
8
Bacterin
217.07 ± 1.09
Immunized antigen
Antibody titer1 (40,960 to 163,840) (81,920 to 163,840) (10,240 to 20,480) (81,920 to 327,680)
a
Data expressed as X ± SD and (minimum to maximum).
2364
KEMORI ET AL.
dilutions did not inhibit the binding of probe antibodies in the assays (data not shown), indicating that the environmental or buffer component had insignificant influence in bringing about the observed binding assay results. These results demonstrate that the avidity of the antibodies obtained from chicken immunized with antigen, at least to fimbrial antigen, compares with or is higher than that obtained from mammalian species (bovine) immunized with the same antigen. The strong avidity of chicken yolk antibodies to fimbrial antigens may explain their efficacy against infectious intestinal diseases in animals (Bartz et al, 1980; Yokoyama et al, 1992). Purification of antibodies for assay was not carried out because it might result in a selective loss of certain antibody subpopulations. The samples of serum, whey, and yolk included many classes or subclasses of antibodies. Immunoglobulin G accounted for about 90% of antibodies in these samples. These samples were diluted from 100 times to 100,000 times to test competitive binding assay, so IgM and IgA were not detected in the high dilution samples in the ELISA, suggesting that IgG played an important role in the competition of antibody binding abilities. The development of B cell memory and affinity maturation is well known to be by selection and expansion of clones bearing receptors with high affinity for the immunizing antigen and by accumulation of
somatic mutations in the rearranged V region genes of B cell (Klinman and Linton, 1990). The difference in the structures of Ig and the mechanism of the rearrangement of the Ig genes between chickens and mammals was also defined. However, there is little data on how to stimulate B cells with high affinity for an antigen. The selection of a high affinity subpopulation of antibody-forming cells is thought to occur more rapidly with lower doses of antigen during the initial stages of immune response (Eisen and Siskind, 1964; Werblin et al, 1973). In this study, chickens and cows were immunized with the same quantity of the fimbrial antigen and the quantity of antigen per body weight was remarkably lower in cows than in chickens. Nevertheless, chickens produced a high avidity of antibodies. There are reports that helper T cell activity is required for the production of a high avidity response (Gershon and Poul, 1971; Francus et al, 1991; Rizzo et al, 1992). Thus, sufficient antigen might be required for the preferential selection of high affinity B lymphocytes to stimulate helper T cells. The reason for the high avidity of chicken antibody is not yet clear. Oral administration of antibodies derived from serum and colostrum has been successful in preventing diseases. However, it is expensive to obtain the large amounts of antibodies by these means. Antibodies are actively transported to the yolks from serum of immunized hens and
,»-. ^*... =-*J rn
•^-»-_.
tui Antibody titers
FIGURE 1. Competition between the antibodies of yolks and cows immunized with enterotoxigenic Escherichia coli antigens by competitive binding assay. Competitors used were yolk antibodies (•) and A) serum of cows immunized with inactivated bacterin; B) whey of cows immunized with inactivated bacterin; C) serum of cows immunized with K99 fimbriae (A). Probes were subsequently diluted twofold. Mean percentage competition of yolk and cow antibodies were different (P < .001).
RESEARCH NOTE
provide a convenient and suitable material for extraction of large amount of antibodies. The results of this study indicate that yolk antibodies have high avidity to antigen comparable to antibodies of cows. Thus, yolk antibodies derived from chickens immunized strongly against a protective antigen may serve as effective prophylaxis for some infectious diseases and because of their high avidity may be utilized for purification by affinity chromatography to isolate objective substances as ligand.
REFERENCES Bartz, C. R, R H. Conklin, C. B. Tunstall, and J. H. Steele, 1980. Prevention of murine rotavirus infection with chicken egg yolk immunoglobulins. J. Infect. Dis. 142:439-441. Eisen, H. N., and G. W. Siskind, 1964. Variation in affinities of antibodies during the immune response. Biochemistry 3:996-1008. Francus, X, Y. Francus, and G. W. Siskind, 1991. Memory T cells enhance the expression of highavidity native B cells. Cell. Immunol. 134: 520-527.
2365
Gershon, R K., and W. E. Poul, 1971. Effect of thymus-derived lymphocytes on amount and affinity of anti-hapten antibody. J. Immunol. 106:872-874. Guinee, P.A.M., J. Veldkamp, and W. H. Jansen, 1977. Improved minca medium for detection of K99 antigen in calf enterotoxigenic strains of Escherichia coli. Infect. Immun. 15:676-678. Isaacson, R E., 1977. K99 surface antigen of Escherichia coli: Purification and partial characterization. Infect. Immun. 15:272-279. Klinman, N. R., and P. J. Linton, 1990. The generation of B
The practical value of utilizing antibodies in therapy lies in the specific binding Passive antibody immunization, in ad- capabilities of antibodies to harmful antidition to innovative vaccine technology, gens. This property of an antibody to react may have a far-reaching importance in specifically with an antigen or the strength animal health maintenance and control of of combination between an antibody and infectious diseases of animals. Passive an antigen may be a desirable indicator of antibodies derived from serum, colostrum, the protective capacity of an antibody. So and yolks of immunized chickens have far, there has been little research on the been used for prophylaxis and treatment. avidity of chicken yolk antibodies against In passive immunization experiments, a particular antigen. The objective of this yolk antibodies were effective in the study was to evaluate the avidity of prevention and control of intestinal infec- chicken yolk antibodies against ETEC K99 tions due to rotavirus (Bartz et al., 1980) fimbriae in comparison with the antibodand enterotoxigenic Escherichia coli (ETEC) ies of serum and colostrum from im(Yokoyama et al, 1992). The feasibility of munized cows. cost-effective, large-scale production of antibodies has favored the use of yolk MATERIALS AND METHODS antibodies. INTRODUCTION
Immunization of Chickens Received for publication May 18, 1993. Accepted for publication September 2, 1993. x To whom correspondence should be addressed. ^Charles City, IA 50616.
Enterotoxigenic Escherichia coli strain 431 (0101:K30;K99;F41.NM,ST+) was obtained from Salsbury Laboratories Inc.2 The bacteria were grown in Minca broth (Guinee et
2361
2362
KEMORI ET AL.
al, 1977), were harvested by centrifugation, and the K99 fimbriae were purified by a combination of gel filtration and ion exchange chromatography (Isaacson, 1977). Purified fimbrial vaccine containing .5 mg of fimbrial antigen in emulsion oil mixed with 5% Arlacel 803 was injected i.m. in the breast muscle of 10 5-mo-old Hy-Line® W36 White Leghorn chickens. Six weeks after the initial injection, the chickens were boosted in the same manner, and the eggs were harvested from the 10 hens 3 to 4 wk after the second injection. Collection of Yolk Antibodies The yolks were individually and carefully separated from the egg whites and then mixed to homogeneity by an automated mixer. Yolks of eggs from each hen were pooled separately to yield 10 different samples. The yolks were subsequently diluted with 8 vol of distilled water. An aqueous dispersion of 5% (vol/vol) hydroxy propyl methyl cellulose phthalate (HPMCP)4 was added to the diluted yolk to make a final .5% (vol/vol) mixture, which was then centrifuged at 10,000 xg for 20 min at 4 C. The supernatant was collected as the Ig-rich fraction. Yolks from unvaccinated chickens were used as a source of native yolk Ig. Immunization of Cows
trum were collected from cows after parturition; serum and whey were separated. The purified fimbrial vaccine was immunized in two cows that were not pregnant using the same immunization schedule for chickens. Serum was collected 3 wk after the second vaccination. Blood and colostrum from unvaccinated cows were used as a source of native serum and whey. Binding and Competitive Binding Assay of Antibodies The procedures for binding assay by ELISA were performed as described previously with slight modification (Yokoyama et al., 1992). Microdilution plates were coated overnight at 4 C with 5 jtig/mL of K99 purified fimbrial antigen. The plates were blocked with bovine serum albumin and washed three times. The wells were filled with 100 pL of serial twofold dilutions of antibodies and the plates were incubated at 37 C for 1 h. Rabbit anti-chicken IgG or anti-bovine IgG conjugated with horseradish peroxidases diluted 1:12,800 and 1:6,400, respectively, were applied and incubated at 25 C for 30 min. These dilutions were determined by preliminary plotting of optical density (OD) values against each type of purified IgG6 and the desired dilution of conjugates correlated .1 OD value with 1 ng of antibodies. The ophenylenediamine dihydrochloride and hydrogen peroxide were added as the substrate and the color reaction was inhibited by addition of 3 N H2S04 and the OD values were obtained in a microdilution plate ELISA reader at 492 nm. The ELISA antibody titer was defined as the reciprocal of the highest dilution of antibodies with .1 OD value.
Two kinds of vaccines, a commercial inactivated bacterin5 and purified fimbrial vaccine, were used to immunize Holstein cows. The cows were 3 to 5 yr old and had no history of disease or vaccination against E. coli. The bacterin was immunized in cows according to the recommendation of the manufacturer. Eight cows were given a Competitive binding between chicken 2-mL dose of bacterin i.m. 6 wk before and bovine antibodies was assayed by anticipated calving and similar doses incubating simultaneously the competitor spaced 3 wk apart. Blood and early colos- and probe antibody dilutions. Two phases of binding assays were performed: the first utilized yolk antibodies as the probe antibody and the bovine sera (from cows 3 Maine Biological Laboratories, Waterville, ME vaccinated with bacterin or purified fim04901. briae) and whey (from cow vaccinated with ^Shinetsu Chemical Co., Tokyo, 100, Japan. 5 Norden Laboratories Inc., Lincoln, NE 68501. bacterin only) were used as competitor ^appel, Organon Teknika Co., West Chester, PA antibodies; the second phase utilized three 19380. kinds of bovine antibodies as the probe and 7 MR650 Microplate Reader, Dynatech Laboratories yolk antibodies as the competitor. The Inc., Chantilly, VA 22021.
RESEARCH NOTE
2363
concentration of the competitor antibodies was adjusted to 1:1,024 of ELISA titer and concentrations of probe antibody were from ELISA titers of 1:1 to 128 in the binding assay. A duplicate assay of each antibody was done on different occasions. Results were expressed as percentage competition, calculated using the formula 100[1 - (B/A)], where A and B are the OD values in the absence and in the presence of competitor antibody, respectively. The different combinations of yolk antibodies with serum or whey antibodies accounted for 180 observations in the assays. Eighty (serum or whey from cows immunized with bacterin) and 20 (serum from cows immunized with K99 fimbriae) readings were represented by the mean.
antibody titer in the serum was moderately lower than that in the whey. The avidities of anti-K99 fimbriae antibodies were measured by competitive binding assays. The yolk antibodies strongly competed with antibodies of serum and whey from cows immunized with inactivated bacterin and inhibited 40 to 80% of the binding of these antibodies (Figure 1, A and B). On the other hand, the antibodies obtained from cows competed less strongly with the yolk antibodies, and inhibited 0 to 30% of the binding of the latter. Difference in antibody avidity of serum and whey of immunized cows could be recognized, and the antibodies of whey inhibited more strongly the binding of the yolk antibodies than serum competitor antibodies. Almost similar results were obtained Statistical Analysis from experiments with the antibodies of Results of competitive binding assays cows immunized with purified fimbrial antigen. In spite of almost similar antiwere analyzed using the Student's t test to body titers obtained, the yolk antibodies determine whether differences between inhibited 40 to 90% of the binding of those mean percentage competition of yolk anti- antibodies. The serum antibodies of cows body and three kinds of bovine antibodies raised against purified fimbrial antigen used as the competitor were significant. inhibited 0 to 30% of binding of the yolk Significance was set at P < .001. antibodies and inhibited a bit stronger the binding of the yolk antibodies than antibodies from cows immunized with inactiRESULTS AND DISCUSSION vated bacterin (Figure 1, C). The differThe mean ELISA antibody titers were 1: ences recognized between the mean 97,420 in the yolks and 1:115,852 in the percentage competition of the yolk antiserum from cows immunized with puri- bodies and the cow antibodies regardless fied K99 fimbriae (Table 1). In the cows of immunized antigens were significant (P immunized with the inactivated bacterin, < .001). mean ELISA titers were 1:137,772 in the All control antibodies from chickens whey and 1:13,280 in the serum; the and cows using the same competitive
TABLE 1. ELISA antibody titers of antibodies from immunized chickens and cows against enterotoxigenic Escherichia coli K99 fimbriae
Antibody
Animal
Number of samples tested
Yolk
Chicken
10
K99 fimbriae
21657 ± .75
Serum
Cow
2
K99 fimbriae
216.82 ± .5
Serum
Cow
8
Bacterin
213.70 ± .48
Whey
Cow
8
Bacterin
217.07 ± 1.09
Immunized antigen
Antibody titer1 (40,960 to 163,840) (81,920 to 163,840) (10,240 to 20,480) (81,920 to 327,680)
a
Data expressed as X ± SD and (minimum to maximum).
2364
KEMORI ET AL.
dilutions did not inhibit the binding of probe antibodies in the assays (data not shown), indicating that the environmental or buffer component had insignificant influence in bringing about the observed binding assay results. These results demonstrate that the avidity of the antibodies obtained from chicken immunized with antigen, at least to fimbrial antigen, compares with or is higher than that obtained from mammalian species (bovine) immunized with the same antigen. The strong avidity of chicken yolk antibodies to fimbrial antigens may explain their efficacy against infectious intestinal diseases in animals (Bartz et al, 1980; Yokoyama et al, 1992). Purification of antibodies for assay was not carried out because it might result in a selective loss of certain antibody subpopulations. The samples of serum, whey, and yolk included many classes or subclasses of antibodies. Immunoglobulin G accounted for about 90% of antibodies in these samples. These samples were diluted from 100 times to 100,000 times to test competitive binding assay, so IgM and IgA were not detected in the high dilution samples in the ELISA, suggesting that IgG played an important role in the competition of antibody binding abilities. The development of B cell memory and affinity maturation is well known to be by selection and expansion of clones bearing receptors with high affinity for the immunizing antigen and by accumulation of
somatic mutations in the rearranged V region genes of B cell (Klinman and Linton, 1990). The difference in the structures of Ig and the mechanism of the rearrangement of the Ig genes between chickens and mammals was also defined. However, there is little data on how to stimulate B cells with high affinity for an antigen. The selection of a high affinity subpopulation of antibody-forming cells is thought to occur more rapidly with lower doses of antigen during the initial stages of immune response (Eisen and Siskind, 1964; Werblin et al, 1973). In this study, chickens and cows were immunized with the same quantity of the fimbrial antigen and the quantity of antigen per body weight was remarkably lower in cows than in chickens. Nevertheless, chickens produced a high avidity of antibodies. There are reports that helper T cell activity is required for the production of a high avidity response (Gershon and Poul, 1971; Francus et al, 1991; Rizzo et al, 1992). Thus, sufficient antigen might be required for the preferential selection of high affinity B lymphocytes to stimulate helper T cells. The reason for the high avidity of chicken antibody is not yet clear. Oral administration of antibodies derived from serum and colostrum has been successful in preventing diseases. However, it is expensive to obtain the large amounts of antibodies by these means. Antibodies are actively transported to the yolks from serum of immunized hens and
,»-. ^*... =-*J rn
•^-»-_.
tui Antibody titers
FIGURE 1. Competition between the antibodies of yolks and cows immunized with enterotoxigenic Escherichia coli antigens by competitive binding assay. Competitors used were yolk antibodies (•) and A) serum of cows immunized with inactivated bacterin; B) whey of cows immunized with inactivated bacterin; C) serum of cows immunized with K99 fimbriae (A). Probes were subsequently diluted twofold. Mean percentage competition of yolk and cow antibodies were different (P < .001).
RESEARCH NOTE
provide a convenient and suitable material for extraction of large amount of antibodies. The results of this study indicate that yolk antibodies have high avidity to antigen comparable to antibodies of cows. Thus, yolk antibodies derived from chickens immunized strongly against a protective antigen may serve as effective prophylaxis for some infectious diseases and because of their high avidity may be utilized for purification by affinity chromatography to isolate objective substances as ligand.
REFERENCES Bartz, C. R, R H. Conklin, C. B. Tunstall, and J. H. Steele, 1980. Prevention of murine rotavirus infection with chicken egg yolk immunoglobulins. J. Infect. Dis. 142:439-441. Eisen, H. N., and G. W. Siskind, 1964. Variation in affinities of antibodies during the immune response. Biochemistry 3:996-1008. Francus, X, Y. Francus, and G. W. Siskind, 1991. Memory T cells enhance the expression of highavidity native B cells. Cell. Immunol. 134: 520-527.
2365
Gershon, R K., and W. E. Poul, 1971. Effect of thymus-derived lymphocytes on amount and affinity of anti-hapten antibody. J. Immunol. 106:872-874. Guinee, P.A.M., J. Veldkamp, and W. H. Jansen, 1977. Improved minca medium for detection of K99 antigen in calf enterotoxigenic strains of Escherichia coli. Infect. Immun. 15:676-678. Isaacson, R E., 1977. K99 surface antigen of Escherichia coli: Purification and partial characterization. Infect. Immun. 15:272-279. Klinman, N. R., and P. J. Linton, 1990. The generation of B