METHODOLOGIC ASPECTS OF ROUTINE PROCEDURES FOR IDENTIFICATION OF NEISSERIA GONORRHOEAE BY IMMUNOFLUORESCENCE Inga Lind The Neisseria Department Statens Seruminstitut Copenhagen, Denmark
In Denmark, the laboratory diagnosis of gonococcal infections is centralized at Statens Seruminstitut. TABLE 1 outlines the annual number of specimens received for cultivation at The Neisseria Department since 1957. In 1970, a direct immunofluorescence (IF) test was established as a routine procedure for the identification of gonococci; the present communication summarizes the experience gained with this technique during the past 4 years. During this period, the incidence of gonorrhoea in the Danish population has been high; consequently, the annual number of specimens received also attained a high level (about 300,000 per year). In 1970, the percentage of positive cultures began t o decrease, whereas the annual number of registered patients with gonorrhea did not decline until 1973. Our routine procedure is, in principle, based o n the combined use of culture on selective and nonselective media' and identification of gonococci by IF. Smears for the IF test are made only from gonococcal-like colonies that give a positive reaction with oxidase reagent. The technical details of the IF test used have been published p r e v i ~ u s l y . ~ ~ ~ TABLE 1 IDENTIFICATION OF N. gonorrhoem BY CULTURE AT THE NEISSERIA DEPARTMENT. STATENS SERUMINSTITUT
1957 1958 1959 1960 1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973
Number of Cultures 76,418 79,541 88,713 102,463 109,309 109,863 106,612 109,976 117,125 128,525 145,173 16 1,809 183,301 225,115 266,785 314,311 311,946
Positive Cultures Number % 7055 9.2 7289 9.2 8823 9.9 11,287 11.0 12,349 11.3 11,026 10.1 9833 9.2 9638 8.8 10,322 8.8 10,383 8.1 12,204 8.4 15,755 9.7 18,668 10.1 20,682 9.2 20,489 7.7 22,554 7.2 20,746 6.7
400
Lind: Identification of N. gonorrhoeae
40 1
T h e methodologic aspects that have been subjected t o continuous assessment are given in TABLE 2 . Points A and C will not be discussed in this context. However, point A is included in the Table t o emphasize that the actual yield of potential positive specimens is determined primarily by two factors: conditions for the transPoint . ~ C is included t o recall that portation of specimens and for the c ~ l t u r e . ~ the quality of the microscope is particularly important when working with the direct IF method at high magnifications. From the very beginning, the optimal reagent for use in the I F test was determined empirically, and t h e choice was a heavily labeled, crude preparation of rabbit antigonococcal g l o b ~ l i n . The ~ ~ ~increasing demand for physicochemical characterization and standardization of immunologic reagents initiated experiments in which this reagent was compared with more well-defined products (point B). First, the suitability of antisera obtained by different immunization procedures was studied. The influence of the following factors was examined: preparation of antigen for immunization: suspension of formalin-killed washed cells versus heat-killed ( 5 6 O C , 20 min) cells (pool of 20-30 freshly isolated strains); route of injection: repeated intravenous injections versus a single or two doses given subcutaneously in Freund's complete adjuvant (in some cases followed by intravenous booster injections); stage of immune response a t which the blood samples were drawn (during primary or secondary responses). An optimal reagent, namely, a rabbit antigonococcal globulin, which after labeling with FlTC exhibited only minor variations in reactivity with different gonococcal strains, was only obtained from rabbits undergoing a secondary immune response to formalin-treated washed suspensions of bacteria administered intravenously. This result is consistent with the findings of Mohr6 who demonstrated that the staining of gonococci in the IF test is based on a reaction with FITC-labeled IgG antibodies. Next, three different globulin preparations were made from rabbit antigonococcal serum, and their content of various serum proteins was determined b y crossed immunoelectrophoresis.7 The results are shown in FIGURE 1. The main effect of ammonium sulfate precipitation (FIGURE l,B and C compared to A) is the removal of albumin and a relative increase in the content of immunoglobulin. Precipitation with ammonium sulfate was performed either once at 50% saturation (FIGURE 1,B)or three times at 35% saturation (FIGURE 1,C) as recommended by Hebert et aL8 The third globulin preparation was purified by ammonium sulfate precipitation and subsequent chromatography on DEAE-Sephadex. Portions of the three globulin
TABLE 2 METHODOLOGIC ASPECTS OF ROUTINE PROCEDURES FOR IDENTIFICATION OF N. gonorrhoeae BY IMMUNOFLUORESCENCE A. Conditions for transportation and culture of specimens B. Preparation of FITC-labeled rabbit antigonococcal globulin Immunization schedule Preparation of globulin solution Labeling procedure Performance of the test (one-step inhibition test) C. Ootical eauioment
402
Annals New York Academy of Sciences
FIGURE 1. Characterization of rabbit antigonococcal serum (A) and different 1% globulin preparations from the same serum (B-D) by crossed immunoelectrophoresis.7 The crude globulin preparations were made by precipitation with ammonium sulfate either once at 50% saturation (B) or three times at 35% saturation (C); the latter procedure removed the major part of the albumin. A globulin preparation that was purified by DEAE chromatography both before and after labeling with FITC contained only traces of other serum proteins (D).The analyses were performed by means of swine antiserum to rabbit plasma proteins (Nordic).
preparations were labeled with FlTC2 t o a different degree; some of the conjugates were finally subjected to DEAE chromatography.s For all conjugates, the OD ratios ( E ~ 9 s / E ~ , 0 )were 9 ~ 1measured, 0 although these values are of limited value for characterization when mixed protein solutions are used for labeling (cf. FIGURE l,B and C). The conjugates were examined for the ability t o stain gonococci and for the degree of background staining in the IF test, which was performed both with and without the admixture of unlabeled rabbit antistaphylococcal serum.’ The unsurpassed reagent for identification of gonococci in smears from cultures was the heavily labeled (OD ratio 1.2-1.4) crude globulin preparation l9
Lind: Identification of N. gonorrhocae
403
( F I G U R E 1 ,B) mixed with a surplus of unlabeled rabbit antistaphylococcal serum; we therefore still use this for our routine diagnostic work. For special purposes, for example, identification of gonococci in smears of secretion, an FITC-labeled IgG purified by DEAE chromatography (OD ratio 0 . 8 - 0 . 9 ) mixed with unlabeled rabbit antistaphylococcal serum gave a fairly brilliant staining of gonococci without any background staining. As mentioned before, the yield of positive cultures is determined primarily by the conditions for transportation and culture of the specimens. The motive for introducing the IF test for the identification of gonococci was t o save time and costs. An attempt to survey these factors has been made based on the results obtained for specimens received in January-February 1974 (TABLE 3). For practical reasons, only urogenital and rectal specimens received on weekdays, except Fridays, were included. Specimens from the fauces (and rare sites) are not examined by the IF test but are subjected t o bacteriologic identification. Among 34,847 cultures, 235 1 contained gonococcal-like colonies that reacted positively with oxidase reagent. TABLE 3 IDENTIFICATION OF N . gonorrhoem B Y IMMUNOFLUORESCENCE (IF TEST)* Pa ti en t s (No.)
Males Females Total
6413 11,832 18,245
Specimens (No.)
Cultures Examined by IF Test (No.)
8998 25,849 34,847
976 1375 235 1
* Specimens received on weekdays, except Fridays, January-February
1974.
The findings for specimens received from male patients and examined by the IF test are summarized in TABLE 4. The plates were inspected for visible gonococcal-like colonies for the first time after 18-24 hr of incubation; negative plates were reinspected after an additional 24 hr of incubation. The majority, 86% (820/950), was found and identified within 2 4 hr after receipt of the specimen. For female patients (TABLE 5), the diagnosis could be confirmed within 2 4 hr in 76% of cases (977/1293). For these patients, the results were then reported,
TABLE 4 IDENTIFICATION OF N . gonorrhoem BY IMMUNOFLUORESCENCE (IF TEST)* IF Test Incubation (hr)
Primary Culture
Positive Subculture
Total
Negative
Total Number Examined
18-24 48
820 84 904
8 38 46
828 122 950
14 12 26
842 134 976
Total
* Smears from gonococcal-like colonies that gave a positive reaction with oxidase reagent: 976 cultures from 954 male patients.
Annals New York Academy of Sciences
404
TABLE 5 IDENTIFICATION OF N. gonorrhoeue BY IMMUNOFLUORESCENCE (IF TEST)* IF Test Positive
(hr)
Primary Culture
Subculture
Total
Negative
Total Number Examined
18-24 48 Total
971 223 1200
7 86 93
984 309 1293
36 46 82
1020 355 1375
Incubation
* Smears from gonococcal-like colonies that gave a positive reaction with oxidase reagent: 1375 cultures from 826 female patients. even though other specimens received simultaneously from the same patient were thereby not examined further. This line of procedure tends t o decrease the percentage of positive specimens without influencing the number of patients registered (cf. TABLE 1). Pharyngeal gonorrhea was rediscovered in Scandinavia about 3 years ago, and since that time, t h e number of specimens received from each patient, including controls for treatment, has increased. This rise also tends t o decrease the percentage of positive cultures. In conclusion, the IF test enabled identification of gonococci in the primary culture for 95% of the male patients and for 93% of the female patients (TABLE 6). This test therefore provides a considerable saving of media and working hours for a laboratory that handles a large number of specimens every day. TABLE 6 IDENTIFICATION OF N. gonorrhoeue BY IMMUNOFLUORESCENCE (IF TEST)* Cultures Examined After Incubation for
Male Patients
Female Patients
Total
18-24 hr 48 hr Total
86.3 8.8 95.1
75.6 17.2 92.8
80.1 13.7 93.8
* Percentage of definitive positive results obtained by examination of primary culturcs from 2243 specimens that contained gonococci. ACKNOWLEDGMENT DEAE chromatography of globulin preparations and conjugatcs was kindly performed by B. Mansa, Department of Biophysics, Statens Seruminstitut.
REFERENCES 1. REYN, A. 1969. Recent developments in the laboratory diagnosis of gonococcal infections. Bull. World Health Org. 40: 245-255.
Lind: Identification of N. gonorrhoeae
405
2. LIND, I. 1967. Identification of Neisseria gonorrhoeae by means of fluorescent antibody technique. Acta Pathol. Microbiol. Scand. 70: 61 3-629. 3. LIND, I. 1969. Combined use of fluorescent antibody technique and culture o n selective medium for t h e identification of Neisseriu gonorrhoeae. Acta Pathol. Microbiol. Scand. 76: 279-287. 4. DANIELSSON, D. & G . JOHANNISON. 1973. Culture diagnosis of gonorrhoeae. A comparison of the yield with selective and non-selective gonococcal culture media inoculated in the clinic and after transport of specimens. Acta Dermato-Venereol. 53: 75-80. 5. LIND, I . & J. M. RHODES. 1970. Specific and non-specific immunofluorescent reactions shown by different preparations of FITC-labelled rabbit antigonococcal globulin. Acta Pathol. Microbiol. Scand. B 78: 153-162. 6 . MOHR, J. 1972. Problems encountered in the preparation of bacterial conjugates. I. Communication: testing the suitability of bacterial antisera for labelling, and the influence of immunoglobulin isotypes. Zentr. Bakteriol. Parasitenk. Abt. I Orig. A 221: 106-115. 7. WEEKE, B. 1973. Crossed immunoelectrophoresis. In A Manual of Quantitive immunoelectrophoresis. Methods and Applications. N. H. Axelsen, J. Kr#ll & B. Weeke, Eds.: 47-56. Universitetsforlaget. Oslo, Norway. 8. HEBERT, G . A., P. L. PELHAM & B. PITTMAN. 1973. Determination of the optimal ammonium sulfate concentration for the fractionation of rabbit, sheep, horse and goat antisera. Appl. Microbiol. 25: 2 6 - 3 6 . 9. THC, T. H. & T. E. W . FELTKAMP. 1970. Conjugation of fluorescein isothiocyanate to antibodies. 1. Experiments o n the conditions of conjugation. Immunology 18: 865 873. 10. T H h , T. H. & T. E. W. FELTKAMP. 1970. Conjugation of fluorescein isothiocyanate to antibodies. 11. A reproducible method. Immunology 18: 875-881.
DISCUSSION DR. W. C. EVELAND: Using the Chairman’s prerogative, I want t o report on the work of one of our graduate students who has produced a highly specificG.C. antiserum by the inoculation of live cultures into the “golf ball” unit implanted into the skin of a rabbit. This antiserum has been used t o type strains of the organism and also t o show numerous serotypes of the so-called pili present on the virulent strains. The work will be published by Milton Takeguchi. DK. R. G. WHITE: What do you mean by the “golf ball” method of immunization of rabbits? DR. EVELAND: We use an ordinary (sterile) practice “golf ball” that is hollow and has several holes in it. This is embedded surgically in the scapular area of the rabbit. After 2-3 weeks, material can be injected directly through the skin into the chamber. I think the original description was by Arco. DR. W. HIJMANS: It would be useful t o have quantitative information o n the cost-benefit aspect of your approach t o obtain the appropriate support for research. DR. LIND: Unfortunately, I d o not have such data. However, it should be possible to get them. DR. E. V. HESS: Is there a difference in positive percentages with different sources of primary cultures, such as cervical, urethral, rectal, and oral?
406
Annals New York Academy of Sciences
DR. LIND: Yes, there are. For example, in female patients, we have found that approximately 90% are positive for the cervical specimen, about 75% for the urethral specimens (7% positive from urethra alone), and approximately 35% for the rectal specimen (3% positive from rectum alone). DR. L. J. LOOMIS: What was the rate of false positivity in your test with the relatively crude antigen? DR. LIND: The rate of false positive results is very low, because the material examined by t h e IF test is highly selected (gonococcal-like colonies that give a positive oxidase reaction obtained from urogenital and rectal specimens). DR. LOOMIS: Have you tried a more specific antigen, such as pili isolated from the surface of types I and I1 N. gonorrhoeae? DR. LIND: So far, we have not tried to use antisera t o gonococcal pili. However, the antiserum used stains all four colony types brilliantly.
In Denmark, the laboratory diagnosis of gonococcal infections is centralized at Statens Seruminstitut. TABLE 1 outlines the annual number of specimens received for cultivation at The Neisseria Department since 1957. In 1970, a direct immunofluorescence (IF) test was established as a routine procedure for the identification of gonococci; the present communication summarizes the experience gained with this technique during the past 4 years. During this period, the incidence of gonorrhoea in the Danish population has been high; consequently, the annual number of specimens received also attained a high level (about 300,000 per year). In 1970, the percentage of positive cultures began t o decrease, whereas the annual number of registered patients with gonorrhea did not decline until 1973. Our routine procedure is, in principle, based o n the combined use of culture on selective and nonselective media' and identification of gonococci by IF. Smears for the IF test are made only from gonococcal-like colonies that give a positive reaction with oxidase reagent. The technical details of the IF test used have been published p r e v i ~ u s l y . ~ ~ ~ TABLE 1 IDENTIFICATION OF N. gonorrhoem BY CULTURE AT THE NEISSERIA DEPARTMENT. STATENS SERUMINSTITUT
1957 1958 1959 1960 1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973
Number of Cultures 76,418 79,541 88,713 102,463 109,309 109,863 106,612 109,976 117,125 128,525 145,173 16 1,809 183,301 225,115 266,785 314,311 311,946
Positive Cultures Number % 7055 9.2 7289 9.2 8823 9.9 11,287 11.0 12,349 11.3 11,026 10.1 9833 9.2 9638 8.8 10,322 8.8 10,383 8.1 12,204 8.4 15,755 9.7 18,668 10.1 20,682 9.2 20,489 7.7 22,554 7.2 20,746 6.7
400
Lind: Identification of N. gonorrhoeae
40 1
T h e methodologic aspects that have been subjected t o continuous assessment are given in TABLE 2 . Points A and C will not be discussed in this context. However, point A is included in the Table t o emphasize that the actual yield of potential positive specimens is determined primarily by two factors: conditions for the transPoint . ~ C is included t o recall that portation of specimens and for the c ~ l t u r e . ~ the quality of the microscope is particularly important when working with the direct IF method at high magnifications. From the very beginning, the optimal reagent for use in the I F test was determined empirically, and t h e choice was a heavily labeled, crude preparation of rabbit antigonococcal g l o b ~ l i n . The ~ ~ ~increasing demand for physicochemical characterization and standardization of immunologic reagents initiated experiments in which this reagent was compared with more well-defined products (point B). First, the suitability of antisera obtained by different immunization procedures was studied. The influence of the following factors was examined: preparation of antigen for immunization: suspension of formalin-killed washed cells versus heat-killed ( 5 6 O C , 20 min) cells (pool of 20-30 freshly isolated strains); route of injection: repeated intravenous injections versus a single or two doses given subcutaneously in Freund's complete adjuvant (in some cases followed by intravenous booster injections); stage of immune response a t which the blood samples were drawn (during primary or secondary responses). An optimal reagent, namely, a rabbit antigonococcal globulin, which after labeling with FlTC exhibited only minor variations in reactivity with different gonococcal strains, was only obtained from rabbits undergoing a secondary immune response to formalin-treated washed suspensions of bacteria administered intravenously. This result is consistent with the findings of Mohr6 who demonstrated that the staining of gonococci in the IF test is based on a reaction with FITC-labeled IgG antibodies. Next, three different globulin preparations were made from rabbit antigonococcal serum, and their content of various serum proteins was determined b y crossed immunoelectrophoresis.7 The results are shown in FIGURE 1. The main effect of ammonium sulfate precipitation (FIGURE l,B and C compared to A) is the removal of albumin and a relative increase in the content of immunoglobulin. Precipitation with ammonium sulfate was performed either once at 50% saturation (FIGURE 1,B)or three times at 35% saturation (FIGURE 1,C) as recommended by Hebert et aL8 The third globulin preparation was purified by ammonium sulfate precipitation and subsequent chromatography on DEAE-Sephadex. Portions of the three globulin
TABLE 2 METHODOLOGIC ASPECTS OF ROUTINE PROCEDURES FOR IDENTIFICATION OF N. gonorrhoeae BY IMMUNOFLUORESCENCE A. Conditions for transportation and culture of specimens B. Preparation of FITC-labeled rabbit antigonococcal globulin Immunization schedule Preparation of globulin solution Labeling procedure Performance of the test (one-step inhibition test) C. Ootical eauioment
402
Annals New York Academy of Sciences
FIGURE 1. Characterization of rabbit antigonococcal serum (A) and different 1% globulin preparations from the same serum (B-D) by crossed immunoelectrophoresis.7 The crude globulin preparations were made by precipitation with ammonium sulfate either once at 50% saturation (B) or three times at 35% saturation (C); the latter procedure removed the major part of the albumin. A globulin preparation that was purified by DEAE chromatography both before and after labeling with FITC contained only traces of other serum proteins (D).The analyses were performed by means of swine antiserum to rabbit plasma proteins (Nordic).
preparations were labeled with FlTC2 t o a different degree; some of the conjugates were finally subjected to DEAE chromatography.s For all conjugates, the OD ratios ( E ~ 9 s / E ~ , 0 )were 9 ~ 1measured, 0 although these values are of limited value for characterization when mixed protein solutions are used for labeling (cf. FIGURE l,B and C). The conjugates were examined for the ability t o stain gonococci and for the degree of background staining in the IF test, which was performed both with and without the admixture of unlabeled rabbit antistaphylococcal serum.’ The unsurpassed reagent for identification of gonococci in smears from cultures was the heavily labeled (OD ratio 1.2-1.4) crude globulin preparation l9
Lind: Identification of N. gonorrhocae
403
( F I G U R E 1 ,B) mixed with a surplus of unlabeled rabbit antistaphylococcal serum; we therefore still use this for our routine diagnostic work. For special purposes, for example, identification of gonococci in smears of secretion, an FITC-labeled IgG purified by DEAE chromatography (OD ratio 0 . 8 - 0 . 9 ) mixed with unlabeled rabbit antistaphylococcal serum gave a fairly brilliant staining of gonococci without any background staining. As mentioned before, the yield of positive cultures is determined primarily by the conditions for transportation and culture of the specimens. The motive for introducing the IF test for the identification of gonococci was t o save time and costs. An attempt to survey these factors has been made based on the results obtained for specimens received in January-February 1974 (TABLE 3). For practical reasons, only urogenital and rectal specimens received on weekdays, except Fridays, were included. Specimens from the fauces (and rare sites) are not examined by the IF test but are subjected t o bacteriologic identification. Among 34,847 cultures, 235 1 contained gonococcal-like colonies that reacted positively with oxidase reagent. TABLE 3 IDENTIFICATION OF N . gonorrhoem B Y IMMUNOFLUORESCENCE (IF TEST)* Pa ti en t s (No.)
Males Females Total
6413 11,832 18,245
Specimens (No.)
Cultures Examined by IF Test (No.)
8998 25,849 34,847
976 1375 235 1
* Specimens received on weekdays, except Fridays, January-February
1974.
The findings for specimens received from male patients and examined by the IF test are summarized in TABLE 4. The plates were inspected for visible gonococcal-like colonies for the first time after 18-24 hr of incubation; negative plates were reinspected after an additional 24 hr of incubation. The majority, 86% (820/950), was found and identified within 2 4 hr after receipt of the specimen. For female patients (TABLE 5), the diagnosis could be confirmed within 2 4 hr in 76% of cases (977/1293). For these patients, the results were then reported,
TABLE 4 IDENTIFICATION OF N . gonorrhoem BY IMMUNOFLUORESCENCE (IF TEST)* IF Test Incubation (hr)
Primary Culture
Positive Subculture
Total
Negative
Total Number Examined
18-24 48
820 84 904
8 38 46
828 122 950
14 12 26
842 134 976
Total
* Smears from gonococcal-like colonies that gave a positive reaction with oxidase reagent: 976 cultures from 954 male patients.
Annals New York Academy of Sciences
404
TABLE 5 IDENTIFICATION OF N. gonorrhoeue BY IMMUNOFLUORESCENCE (IF TEST)* IF Test Positive
(hr)
Primary Culture
Subculture
Total
Negative
Total Number Examined
18-24 48 Total
971 223 1200
7 86 93
984 309 1293
36 46 82
1020 355 1375
Incubation
* Smears from gonococcal-like colonies that gave a positive reaction with oxidase reagent: 1375 cultures from 826 female patients. even though other specimens received simultaneously from the same patient were thereby not examined further. This line of procedure tends t o decrease the percentage of positive specimens without influencing the number of patients registered (cf. TABLE 1). Pharyngeal gonorrhea was rediscovered in Scandinavia about 3 years ago, and since that time, t h e number of specimens received from each patient, including controls for treatment, has increased. This rise also tends t o decrease the percentage of positive cultures. In conclusion, the IF test enabled identification of gonococci in the primary culture for 95% of the male patients and for 93% of the female patients (TABLE 6). This test therefore provides a considerable saving of media and working hours for a laboratory that handles a large number of specimens every day. TABLE 6 IDENTIFICATION OF N. gonorrhoeue BY IMMUNOFLUORESCENCE (IF TEST)* Cultures Examined After Incubation for
Male Patients
Female Patients
Total
18-24 hr 48 hr Total
86.3 8.8 95.1
75.6 17.2 92.8
80.1 13.7 93.8
* Percentage of definitive positive results obtained by examination of primary culturcs from 2243 specimens that contained gonococci. ACKNOWLEDGMENT DEAE chromatography of globulin preparations and conjugatcs was kindly performed by B. Mansa, Department of Biophysics, Statens Seruminstitut.
REFERENCES 1. REYN, A. 1969. Recent developments in the laboratory diagnosis of gonococcal infections. Bull. World Health Org. 40: 245-255.
Lind: Identification of N. gonorrhoeae
405
2. LIND, I. 1967. Identification of Neisseria gonorrhoeae by means of fluorescent antibody technique. Acta Pathol. Microbiol. Scand. 70: 61 3-629. 3. LIND, I. 1969. Combined use of fluorescent antibody technique and culture o n selective medium for t h e identification of Neisseriu gonorrhoeae. Acta Pathol. Microbiol. Scand. 76: 279-287. 4. DANIELSSON, D. & G . JOHANNISON. 1973. Culture diagnosis of gonorrhoeae. A comparison of the yield with selective and non-selective gonococcal culture media inoculated in the clinic and after transport of specimens. Acta Dermato-Venereol. 53: 75-80. 5. LIND, I . & J. M. RHODES. 1970. Specific and non-specific immunofluorescent reactions shown by different preparations of FITC-labelled rabbit antigonococcal globulin. Acta Pathol. Microbiol. Scand. B 78: 153-162. 6 . MOHR, J. 1972. Problems encountered in the preparation of bacterial conjugates. I. Communication: testing the suitability of bacterial antisera for labelling, and the influence of immunoglobulin isotypes. Zentr. Bakteriol. Parasitenk. Abt. I Orig. A 221: 106-115. 7. WEEKE, B. 1973. Crossed immunoelectrophoresis. In A Manual of Quantitive immunoelectrophoresis. Methods and Applications. N. H. Axelsen, J. Kr#ll & B. Weeke, Eds.: 47-56. Universitetsforlaget. Oslo, Norway. 8. HEBERT, G . A., P. L. PELHAM & B. PITTMAN. 1973. Determination of the optimal ammonium sulfate concentration for the fractionation of rabbit, sheep, horse and goat antisera. Appl. Microbiol. 25: 2 6 - 3 6 . 9. THC, T. H. & T. E. W . FELTKAMP. 1970. Conjugation of fluorescein isothiocyanate to antibodies. 1. Experiments o n the conditions of conjugation. Immunology 18: 865 873. 10. T H h , T. H. & T. E. W. FELTKAMP. 1970. Conjugation of fluorescein isothiocyanate to antibodies. 11. A reproducible method. Immunology 18: 875-881.
DISCUSSION DR. W. C. EVELAND: Using the Chairman’s prerogative, I want t o report on the work of one of our graduate students who has produced a highly specificG.C. antiserum by the inoculation of live cultures into the “golf ball” unit implanted into the skin of a rabbit. This antiserum has been used t o type strains of the organism and also t o show numerous serotypes of the so-called pili present on the virulent strains. The work will be published by Milton Takeguchi. DK. R. G. WHITE: What do you mean by the “golf ball” method of immunization of rabbits? DR. EVELAND: We use an ordinary (sterile) practice “golf ball” that is hollow and has several holes in it. This is embedded surgically in the scapular area of the rabbit. After 2-3 weeks, material can be injected directly through the skin into the chamber. I think the original description was by Arco. DR. W. HIJMANS: It would be useful t o have quantitative information o n the cost-benefit aspect of your approach t o obtain the appropriate support for research. DR. LIND: Unfortunately, I d o not have such data. However, it should be possible to get them. DR. E. V. HESS: Is there a difference in positive percentages with different sources of primary cultures, such as cervical, urethral, rectal, and oral?
406
Annals New York Academy of Sciences
DR. LIND: Yes, there are. For example, in female patients, we have found that approximately 90% are positive for the cervical specimen, about 75% for the urethral specimens (7% positive from urethra alone), and approximately 35% for the rectal specimen (3% positive from rectum alone). DR. L. J. LOOMIS: What was the rate of false positivity in your test with the relatively crude antigen? DR. LIND: The rate of false positive results is very low, because the material examined by t h e IF test is highly selected (gonococcal-like colonies that give a positive oxidase reaction obtained from urogenital and rectal specimens). DR. LOOMIS: Have you tried a more specific antigen, such as pili isolated from the surface of types I and I1 N. gonorrhoeae? DR. LIND: So far, we have not tried to use antisera t o gonococcal pili. However, the antiserum used stains all four colony types brilliantly.
