Further standardization of the agglutinin-absorption test in the serology of leptospires*

Bull. Org. mond. San ) 1970, 42, 733-738 Bull. Wid Hith, Org. Further Standardization of the Agglutinin-absorption Test in the Serology of Leptospires* EMIL KMETY,' MILDRED M. GALTON 2 & CATHERINE R. SULZER3 Four factors, suspected ofinfluencing the final results ofagglutinin-absorption tests used in the diagnosis and classification of leptospires, were investigated in comparative studies: (1) the time required for adequate absorption, (2) the quantitative relationship between antibody titres and amounts ofantigen neededfor absorption, (3) the possible effect of the Danysz phenomenon, and (4) the absorptive potency of live andformol-treated antigen. It was found that a 90-minute absorption time was adequate and that with increasing amounts of antigen, titres were continuously reduced, indicating a certain degree of non- specific absorption. The Danysz phenomenon was found to occur in leptospiral serology and the addition ofantigen to serum in 3 equalparts at 10-minute intervals is recommended. The titres of sera absorbed with formol-treated antigen were often found to be lower than titres ofsera absorbed with the same amounts of live antigen and some damaging effect offormol on antibody is suspected. The absorption test of Castellani was first applied to the diagnosis of leptospirosis by Ruys & Schuiffner (1934). Subsequently, Borg-Petersen (1938, 1944) proved, in pioneer studies of some strains of the Icterohaemorrhagiae and Hebdomadis serogroups of Leptospira, the possibility of revealing by this test serological differences in strains not sufficiently distinguishable in simple cross-agglutination pro- cedures. The importance of the absorption test for diagnosis, epidemiology and classification of lepto- spires led more laboratories throughout the world to adopt this technique and to develop it in modifica- tions differing more or less from the original pro- cedure. For example, Wolff (1954) performed the absorption test on immune sera diluted to a titre of 1: 3000, absorbing them with formol-killed, * From the Institute of Epidemiology, Medical Faculty, Komensky University, Bratislava, Czechoslovakia, and the Epidemiological Program, National Communicable Disease Center, Health Services and Mental Health Administration, Public Health Service, Department of Health, Education, and Welfare, Atlanta, Ga., USA. This study was supported in part by the World Health Organization. 1 Director, Institute of Epidemiology, Medical Faculty, Komensky University. 2 Deceased March 1968; formerly Chief, Veterinary Public Health Laboratory, National Communicable Disease Center. 3 Chief, Leptospirosis Unit, Epidemiologic Services Laboratory Section, National Communicable Disease Center. concentrated antigens. Babudieri & Mateew (1961) used a technique differing mainly in the way serum and antigen were mixed. Alexander et al. (1955) reported on repeated absorptions and 4-fold serum dilutions. At the National Communicable Disease Center, Atlanta, Ga., USA, a slightly modified ver- sion of Wolff's (1959) technique, recommended by Alexander et al. (1955), was adopted, whereas at the Institute of Epidemiology, Komensky Univer- sity, Bratislava, Czechoslovakia, a technique was developed that was based on the use of pooled, undiluted immune sera with titres of 1: 12 800 or 1: 25 600 absorbed with live concentrated antigens (Galton et al., 1962; Kmety, 1967). None of the above-mentioned methods determined with any greater precision the exact amounts of antigen which should be used for absorption. Some discrepancies in the typing of strains encountered in recent years explain the growing demand for the absorption technique to be stan- dardized as far as possible in order to ensure repro- ducibility of results in different laboratories. The absorption test is now the basic and essential pro- cedure for the definitive identification of leptospiral serotypes and is thus the basis on which the present classification system of leptospires is founded. This study attempted to resolve the problem by com- 2512 -733 E. KMETY, M. M. GALTON & C. R. SULZER parative investigations of the four following factors which may significantly influence the final results of the test: (1) the time required to obtain sufficient absorption; (2) the quantitative relationships be- tween antibody titre and amount of absorbing antigen; (3) the possible influence of the Danysz phenomenon; and (4) the absorptive potency of live and formol-treated antigen. MATERIALS AND METHODS Pools of rabbits' sera (3 animals) preserved with 0.5% phenol were used in most of the tests while single-rabbit sera preserved with equal amounts of glycerol were used in the remaining tests. Reference strains of the Bataviae and Autumnalis serogroups, and in one case the Icterohaemorrhagiae serogroup, were used (Bull. Wld Hlth Org., 1965). In studies of the Bataviae serogroup, a new and recently described serotype, L. argentiniensis, was included (Szyfres et al., 1967). The majority of the tests on the Bataviae group were performed at the National Communicable Disease Center with anti- gens grown in Stuart's or Ellinghausen's media. The antigens were nephelometrically measured before use in agglutination tests and, if necessary, adjusted to a standard of 25-30 nephelos according to Roessler & Brewer (1967). For tests carried out in Bratislava, strains grown in modified Korthof's medium and selected both by eye and by microscopic examination were used (Kmety, 1957). For all agglutination tests, equal volumes of living antigen suspensions and diluted serum were used, beginning with a serum dilution of 1 : 100 and proceeding in a 2-fold series. After the mixture had been left to stand for about 2 hours at room tem- perature (20°C-25°C), the result was read according to the 50% agglutination criterion; readings were made at first by two persons independently. How- ever, when differences in the end-points as determined exceeded 1 dilution on few occasions only, the double readings were considered to be unnecessary and were discontinued. Nevertheless, for consistency, the results obtained by only one of these observers are quoted. Antigens for absorption tests were cultured in 200-ml or 500-ml prescription bottles. Well-grown cultures, mostly between 5 and 10 days old, were spun at the equivalent of about 5000 g for 25-30 minutes in a high-speed, angle-headed centrifuge and concentrated about 40-80 times. The killed antigen was prepared by the addition of a 10% formol-buffered saline solution to a well-grown culture to a final concentration of 0.25% of formol. The formolized culture was left to stand for 3 hours at room temperature (20°C-25°C) before centrifuga- tion. To ensure that the same amounts of antigen were used in comparative studies, the technique was modified so that the concentrated living antigen was at first adjusted to McFarland Standard No. 10 (see Helper, 1953) by the addition of saline (McFar- land, 1907) and the antigen was then divided into 2 equal parts, one of which was treated with formol as previously described. The concentrated and adjusted antigen, living or killed, was added to the undiluted serum in 2 or 3 equal parts at 10-minute intervals, usually in a ratio of 24 (i.e., 3 x 8) parts of antigen to 1 part of serum. After each addition of antigen, the mixture was shaken and allowed to stand at room temperature (20°-25°C) for 90 min- utes, unless it is stated otherwise. The antigen was then removed by centrifugation and the supernatant (absorbed serum) was stored at -20°C, if it was not to be tested immediately. In assays of the Danysz phenomenon, the serum was first diluted with saline (1 part of whole serum or 2 parts of glycerolated serum being added to 25 parts or 24 parts of saline, respectively. The diluted serum was then added in 3 approximately equal parts (9 + 9 + 8) at 10-minute intervals to 24 parts of antigen if it is not stated otherwise (Fig. 3, p). Again, the antigen-serum mixture was centrifuged and the supernatant was stored at -20°C if it was not to be tested at once. RESULTS Time relationship in absorption In the first series of absorption tests, a comparative study was performed to show whether overnight storage for 18 hours of the serum-antigen mixture, as practised in most laboratories, results in better absorption than when the serum is exposed to antigen for a shorter period. Results of parallel tests were compared; in 5 tests with live antigens and in 2 with killed antigens, the serum being exposed to antigen for 18 hours and for 90 minutes, respectively. In 3 additional tests with live antigen, the time of absorption was shortened to 60 minutes. The results of these tests (Fig. 1) revealed clearly that there was no significant difference in titre in any of the 10 investigated pairs of sera, whether absorbed with live or formol-treated antigen. It may be assumed, therefore, that a period of 90 minutes, and apparently also of 60 minutes, is sufficient to ensure 734 FIG. I RESULTS OF COMPARATIVE ABSORPTION TESTS PERFORMED AFTER SPECIFIED PERiODS OF ABSORPTION WITH LIVE AND KILLED LEPTOSPIRAL ANTIGENS 12,600- 6400- 3200- 1600- 800- 400- 200- '00- 0- 90 MINUTEs IS HOURS S. IMMUNE SERUM PAIDJAN WITH CLAYTONI LIVE HOURS 6400- 3200- 600- 800- 400- 200- 100- 0- 1 van Tiesen 2 Paidjan 3 djotui, NS 26 4 cloyleni, LT 515 50 MINUTES 15 HOURS 90 MINUTES IS HOURS 90 MINUTES IS HOURS 90 MINUTES IS HOURS 5 kobh,CZ 320 6 balboo,LT 761 7 b..IIIeae., 776 8 drgenIImismSs, LT 019 f. IMMUNE SERUM AKIYAMI ABSORBED WITH RACHMAT LlVE LIVE 60 MINUTES IS HOURS 12500- 6400- 3200- 1600- *00- 400- 200- 100- h. Rschmel 4 S.ngkinong 2 Erineceus spites 670 5 moons, Moses 3 eelemnelle,.Akiyanii A 6 rms Bragg 7 Nlkuleevo 10 8.1w-b. B wemlruno. SspuIette 9 jet sneusove WHO 00406 FIG. 2 RESULTS OF COMPARATIVE ABSORPTION TESTS PERFORMED WITH VARYING AMOUNTS OF LIVE OR KILLED ANTIGENS IMMUNE SERUM VAN TIENEN ABSORBED WITH CLAYTONI KILLED, ISh 1+30 I +60 b. t IS IMMUNE SERUM VAN TIENEN WITH ARGENTINIENSIS KILLED, 90 mitt 1+25 + 35 5, IMMUNE SERUM ARGENIlNIENSIS WITH VAN TIENEN LIVE, 90mm 1+15 1+25 1+35 IMMUNE ABSORBED I + IS SERUM ARGENTINIENSIS WITH VAN TIENEN, KILLED,SOmin 1+25 1+35 1 + 48 I +74 IMMUNE SERUM VAN TIENEN 6. WITH ARGENTINIENSIS; LIVE, 90mm I + IS 1+25 1+35 I. I +98 I.IMMUNE SERUM DJATZI WITH PAIDJAN L1VE, 90mm I + 24 I + 34 1 + 44 IMMUNE SERUM PAIDJAN WITH DJATZI LIVE, 90mm + 74 IMMUNE SERUM DJATZI WITH PAIDJAN KILLED, 90mm I + 24 I + 34 + 44 600 - 600- 400 - 200- 100- IMMUNE SERUM VAN TIENEN WITH PAIDJAN LIVE, 90mm 1+14 1+20 I + 26 I + 32 1 +40 k. 1+14 U. IMMUNE SERUM VAN TIENEN WITH CLAYTONI LIVE, 90mm 1+20 1 + 26 1+32 IMMUNE SERUM DJATZI WITH PA1DJAN flOG- 6400- 3200- 1 600 - 600- 400- 200- too - 0- I + 14 1+20 LIVE, 90mm I + 26 I + 32 N. IMMUNE SERUM DJATZI ABSORBED WITH CLAYTONI LIVE. 90mm 1+14 1+20 1+26 1+32 1+40 12600 - 6 400- 3 200- 12 600- 6400- 3200 - I 600 - 500 - 400- 200 - 100- 0- 1 *n Tienen 2 Paidbon 3 djatzi,H526 4 clayloni, LT BIB LIVE, 18 hours + 32 9 5 kobbe, CZ 320k 6 balboa, LT 761 7 brosilienslS, 776 & orpntiniensis, LT 1019 p IMMUNE SERUM BANGKINANG LIVE, 90mm 1+14 1+20 WITH SAPULETTE + 26 1+23 I. 25600- 12500- 6400- 3200- 1600- 600 - 400 - 200- 100- C- II. C I. 12100- 6400- 3 200- * 600- 600- 400- 200- 100- 0- h. I. 6400- 3200- 1600 - 600- 400 - 200- 100- 0- I. I + 40 I + 40 1+14 1 + 20 LIVE, 90mm + 26 1 ±32 1 +40 + 32 WHO 00407 FIG. 3 RESULTS OF COMPARATIVE TESTS ON THE OCCURRNCE OF THE DANYSZ PHENOMENON IN LEPTOSPIRAL ABSORPTION TESTS IMMUNE -SERUM VAN TIENEN ABSORBED WITH CLAYTONI ANTIt;EN ANTIEN WSEU b ER --_ANTIGEN ANTIGEN---+SERUM .LIVE, lll ; :. .,r. " __.II- . ,- Lg vt IMMUNE SERUM VAN TIENEN WITH ARGENTNIENSIS 0. SERtUM _-- ANTIGEN ANTIGEN -_ SERUMA. 6 400- 1.2 00- Bo- 400- 200- 100- O - SERUM _-- I C a * 0 IMMUNE SERUM ARGENINIENSIS WITH VAN TIENENd. 12smt- AO - 3200- Iw10- So- 400- ZOO- 100- O - .h.a 12,00 - 320- IW- No- 400- '200- *100- O- IMMUNE SERUM VAN TIENEN WITH DJ.ATZI SERUM -+ATIGEN ANTIGEN-+SRUM LIVE I iT-''I MMUNE SERUM BALBOA ABSORBED WITH- ARGENTINENSIS PAIDJAN .L I.S-a A. A - S SERtJM--+ATGE ANTIGN-4SERUM k. SERUM-ANTON ANTIGEN-+ SERU UI L 1 L4.".l..". .,.,.oL., 2 4 2 34 8 -5476 1 2 -0 56T'? 6 2 3 4 G 7@0 Z 3 4 567 A CLAYTONI_IMMUNE SERUIV WI VAN TIENEN " S ---- A A--- 4 8 LIVE L I V E L I V E .SERUM -+ANtIltE SERUM -+ANTIGEN ANTIGEN --+SERUM I Van Tienen 2 Paidion 3 djatzi, HS 26 4 c'Iytoni, LT 818 5 kobbe, C Z 320 k 6 balboa, LT 761 7 brasiliensis, 776 8 argentiniensis, LT 1019 12 800 - $.400- 3 200- I 00o- 800O- 400 - 200 - 100- O- go IMMUNqE SERlUM AKIYAMI ABSORBEDE WITH BANGKINANG L IYVE 1 Rochmnot 6 Fort BraggSERUM --+AicNTIE A-NTI6EN _- SERUM 2 Erinoceus ouritus 670 7 Nikoloevo3 200- 1 600- 800- 400- - ZOO- 100- O-' 3 outumnolis, Akiyami A 4 Bangkinang 1 5 mooris, Moores 8 sumotmand, Sapulettes 9 Jez Bratislava 10 Butembo 12800- 6 400- J 260 - I $W- 800o - 400- 200- 100 - O - I RGA 2 M-20 ~~~~~~~~~~FIG.4 RESULTS OF COMPARATIVE ABSORPTION TESTS WITH LIVE AND KILLED LEPTOSPIRAL ANTIGEN h. IMMUNE SERUM PAIDJAN WITH CLAYTONI LIVE KILLED a. IMMUNE SERUM BALBOA AB'SOR8ED WITK BRASILIENSIS . IMMUNE SERUM DJATZI WITH VAN TIENEN LIVE KILLED LIVE KILLED25600- 1 2li0- 6400 - 3200 - t 600- Boo0- 400 - 200- 100 - O- f. b. IMMUNE SERUM CLAYTONI WITH ARGENTINIENSIS LIVE KILLED --. I 11 13 IMMUNE SERUM DJATZI WITH ARGENIINIENSIS LIVE KIXLLED 12800- 6400 - 3200- 1 600- 800- 400- 200- 100- O- L I 2 3 4 5 6 7 8 L 2 3 4 5 6 7 0 |@ IMMUNE SERUM AROENTINIENSIS WITH VAN TIENEN LIVE KILLED IMMUNE SERUM DJATZI WITH CLAYTONI LIVE KILLED I Van Tionen 2 Poidjon 3 djatzi, HS 26 4 cboytoni, LT 818 5 kobbe,CZ 32Ok 6 boiboo,LT6,m 7 tbmsiliensis, 776 8, agentin;enos,sLT 1019 WHO .00409 12 B00- 6 400- 3200- 1 600- Boo - 400- 200- 100- O - I MMUNE SERUM BALBO>A WITH ARGENTINIENSIS f.SERUM ---ANTIGEN ANTIGEN-- SERUM DSERUM-+ ANTIGEN ANTIGEN ---SERM L I VIE -KILLED 45 ?1 2 34 S6 7 0 1 2 3 4 5 6 7 s ES =1 . EEi . .RT 5 SY . Y| W 0@080W, 2 s 4 1 2 s 4 : M. IMMUNLE SERUM PAIDJAN WITH DJATZI I n. IMMUNE SERUM DJATZI WITH PAIDJAN 1 2 34 5 6 7 83 p. IMMUNE SERUM M-2-0 WITH RGA LlIVE K.SRUM DROMD b. SERUM AND ANTI- C. ANTIGEN DROPPED d ANTIGEN DROPPED SLOWLY INTO GEN MIXED INTO SERUM IN * INTO SERUM IN ANTIGEN AT ONE TWO PARTS THREE., PARTS 12 ~~~~12 I 2 1 2 d. IMMUNE SERUM.BALBOA WITH ARGENTNIENS;IS LIVE KILLED 4 5 7 8' WIHVN TIEN 0 0 0 t.. ] O.-O O.O1 2 3 4 S 7'8- T 2 3 4 5 6 7 8 k. IMMUNE SERUM BALBYONI WITH PAN IDJNE LIVE KILLED IMMUNE SERUM BALBOA WITH ARGENTINIENSIS *. [iSERUM _-- ANTIGEN] LIVE KILLED 2 3 4 5 6 7 6 STANDARDIZATION OF THE AGGLUTININ-ABSORPTION TEST FOR LEPTOSPIRES the expected binding of antibodies to the correspond- ing sites of antigen. Thus, the time to be allowed for absorption procedures requires no standardization. From a practical point of view, however, an absorp- tion time of 90 minutes is suggested. Quantitative relationships between serum titre and amount of antigen The amount of antigen necessary for the complete absorption of antibodies detectable by the agglutina- tion test is a matter of great importance because antigen used in too small amounts is not able to remove all corresponding antibodies, and in too large amounts it is believed to cause non-specific absorp- tion (Kmety, 1967). To obtain a better insight into the quantitative relationship between antibody, as expressed by the titre, and the amounts of antigen required for absorption, 17 tests (including 4 with killed antigen) were performed, using increasing amounts of antigen. The smallest proportion was 1 part of serum to 14 parts of antigen; the largest was 1 part of serum to 98 parts of antigen. The proportions used in the tests are shown in Fig. 2. The results of these investigations show with considerable consistency that increasing the amounts of antigen caused a continuous decrease in titre. From the following tabulation it can be seen that if the titres in tests performed with the smallest and the largest amounts of antigens are compared, the titre was lower in more than 4 out of 5 of the latter tests, and that there was no change in titre in fewer than 1 out of 5. Titre * Same 1 lower 2 lower 3 lower No. ofreactions 17 53 22 77 (82%) 2J Total 94 * Of serum absorbed with large amounts of antigen, in comparison with the titre of serum absorbed with small amounts of antigen. It was noted that even 4-fold amounts of antigen (Fig. 2, f and g) caused, at the most, a difference of 2 titre steps. However, even if the sera absorbed with large amounts of antigen did not show a sub- stantial decrease in titre, the agglutination was much weaker in the highest dilution; in fact, the reaction was sometimes on the borderline of positivity. The results presented in Fig. 2 also allow an estimation to be made of the amounts of antigen required to achieve adequate absorption. In most cases, the ratio of 1 part of serum to 24 parts of concentrated antigen of a density corresponding to McFarland Standard No. 10 is sufficient to give adequate absorption of the antibodies present"in a titre of 1: 12 800. If the titre of antibodies to be absorbed is lower than 1: 12 800 (for instance, about 1: 3 200), satisfactory absorption can be obtained with smaller amounts of antigen as indicated in Fig. 2, tests b, c, d, j, 1, n, o and p. It must be admitted that the evaluation of the antigen density by eye, in comparison with an adjustment to the McFarland Standard, may intro- duce a considerable error. However, the above- mentioned results show that even a 2-fold increase in the amount of antigen does not greatly change the residual titres. Therefore, an exact nephelometric measurement of antigen density for absorption procedures does not seem to be absolutely necessary. In antigen-antibody relationships, there seems to be a certain difference between antibodies corre- sponding to antigens in the absorbing strain (anti- bodies to be absorbed), and antibodies not corre- sponding to antigens in the absorbing strain (anti- bodies which should remain in the absorbed serum), as is clearly shown in the results of tests b, e, j, k, m, n, p and q in Fig. 2. These tests indicate that even slightly increased (less than double) amounts of antigen are able to reduce the residual titre against the absorbing strain by 1 step, whereas considerably larger amounts of antigen are usually necessary to cause a titre reduction of the non-corresponding antibodies. The quantitative antigen-antibody relationships are apparently more critical as far as antibodies corresponding to the absorbing strain are con- cerned. Both effects must be taken into account in order to increase the reliability of the absorption test. The Danyszphenomenon Danysz (1902) described the observation that mixtures of the same amounts of toxin and antitoxin resulted in different toxicities, depending on the way in which the two components were mixed. If toxin was added drop by drop to antitoxin, the final mixture remained toxic. In contrast, if antitoxin was dropped into toxin, free, unbound antitoxin was found in the final mixture. This phenomenon was later explained by Heidelberger & Kendall (1929) as the result of the different proportions in which antigens and antibodies are able to combine. It was thought that the Danysz phenomenon might occur in the absorption procedures with 735 E. KMETY, M. M. GALTON & C. R. SULZER leptospires. In order to study this possibility, 16 comparative tests, including 4 tests with killed antigens, were performed. The results presented in Fig. 3 (mainly in tests a, c, d, e and o) show clearly that the same amounts of antigen, when dropped into serum, give better absorptive effects than when serum is added to antigen. The difference in titre is equal to 1 or 2 steps. An excellent example is Fig. 3, p, which shows that the addition of antigen to serum in 2 or 3 equal parts at 10-minute intervals improves the final absorption, reducing the residual titre against the absorbing strain by 2 or 3 steps, respectively. In some tests (Fig. 3, f, g, j, k and n), where apparently the amounts of antigen used for absorp- tion were too large, the titre of the residual anti- bodies also was lower in those mixtures in which antigen was added to serum. This must be explained as a consequence of non-specific absorption in tests that quantitatively were not well balanced, as discussed in the previous section. Evidently, the Danysz phenomenon applies also to the serology of leptospires. Thus the technique of agglutinin-absorption by the addition of antigen to serum in 2 or 3 equal parts at intervals of about 10 minutes seems to be appropriate. Absorption with live and formol-treated antigens At the present time, absorption, whether per- formed with formol-treated or with live, concen- trated antigen, seems to give similar results; however, comparative tests giving exact results have not yet been reported. To evaluate both procedures, a series of 14 comparative tests were performed. Exactly the same amounts of antigen for absorption and the same live cultures for titration of the absorbed sera were used. No substantial differences were seen in the results obtained in these tests (Fig. 4 and Fig. 2, h and i). However, sera absorbed with killed antigen showed a lower titre in nearly 2 out of every 3 tests. The differences in titre in the comparative absorp- tion tests are summarized in the following tabulation: Titre * 1 higher Same 1 lower 2 lower 3 lower Total No. of tests run 2 19 18 1 31(60%) 2 52 * Of serum absorbed with killed antigen, in comparison with the titre of serum absorbed with live antigen. In 52 cases, corresponding residual titres could be compared; in 19 the titres did not differ, but in 33 they differed by 1 or 2, or occasionally 3, titre steps. However, while the residual titre was higher in the serum absorbed with formol-treated antigen in only 2 cases, it was higher in the serum absorbed with live antigen in 31. Since the same amounts of formol-treated antigen and live antigen were used, and since the assumption of a higher absorbing potency of formol-treated antigen is considered to be unreasonable, some damaging effect of formol on the antibodies must be taken into account. In order to approach this hypothesis, the results of agglutination tests performed with 13 sera of the Bataviae serogroup treated with formol in the same concentration as that used in the absorption proce- dure were compared with the results of agglutination tests performed with untreated sera. In 77 tests no difference in titre was observed and in 16 tests the titre of the formolized serum was lower by 1-2 steps; but in 11 tests a higher titre was found. It must be admitted, however, that the formol generally acted on the unabsorbed sera for a much shorter time than it did on the absorbed sera. The absorbed sera had, in most cases, been stored at -20°C for several weeks before being tested, and the results may have been influenced also by the effects of this storage. The question why sera absorbed with formol- treated antigen gave mostly lower titres remains open, but some damage to antibodies by the formol is suspected. DISCUSSION The agglutinin-absorption test is a fundamental procedure in the current classification of leptospires which is based mainly on serological properties of the strains. There is a general need to establish a standard technique in order to ensure the repro- ducibility of results and to avoid discrepancies between the results of different laboratories. The present study shows that certain quantitative relationships between antibodies and antigens must be carefully considered and that the ability of anti- bodies to combine with antigens in different ratios, depending on the way in which the antibodies and antigens are mixed, must be respected. The use of formol-treated antigens is not recommended because the treatment may result in damage to the antibodies. All these factors may cause some variability in the results. However, even in well-balanced absorption tests, there remain some other factors, such as the immune serum, the agglutinability of strains, tech- 736 STANDARDIZATION OF THE AGGLUTININ-ABSORPTION TEST FOR LEPTOSPIRES nical precision, etc., which may possibly affect the perfect conformity of results. These other possible causes of variability probably cannot be eliminated completely. The criterion for differentiating serotypes contains the statement that " adequate amounts " of antigen should be used in absorption tests, but there is no specification of how much antigen should be con- sidered adequate (Bull. Wld Hlth Org., 1965). Wolff & Broom (1954) considered complete absorp- tion to be achieved when no agglutinin against the absorbing strain could be demonstrated in a serum dilution of 1: 30 which had had a homologous titre of 1: 3000 before absorption. The possibility of non- specific absorption caused by excessive amounts of absorbing antigen was not considered. Nevertheless, the results reported in the present paper indicate that the use of absorbing antigens in excessive amounts does cause some degree of non-specific absorption. The problem of " adequacy " is appar- ently a question of the minimum amount of antigen required to achieve appropriate absorption of the corresponding antibodies. The use of antigen of an adjusted density in suitable proportions makes a substantial contribu- tion to the standardization of the test but the proof that the absorption test was not performed with an excess of antigen is given only when the first dilution of the absorbed serum still reacts with the absorbing strain. This requirement is very strongly recom- mended in cases where the required 10% post- absorption titre limit is just reached, and in cases where the differences between two strains are below the 10% limit. In such cases, precision is of particu- lar importance because a difference of only 1 titre step is decisive for the recognition of a new serotype. When the homologous titre, i.e., the titre with the strain used for immunization, is not significantly reduced after absorption, and the 10% titre limit is evidently exceeded, a negative reaction with the absorbing strain in the first dilution of the ab- sorbed serum may also be considered to reflect a well-balanced test. The acceptance of this recommendation, as well as the fact that a variability of + 1 or -1 titre step is within the limitations of the technique itself, indicates that a serum with a high titre should be used. An immune serum with a titre of 1 : 12 800 or 1 : 25 600 is considered optimal for use in an agglutinin- absorption test for purposes of classification. ACKNOWLEDGEMENTS The authors wish to express their sincere appreciation to Dr A. D. Alexander, Dr C. Borg-Petersen and Dr L. H. Turner, for reviewing the manuscript and offering constructive suggestions. RItSUME NOUVELLE NORMALISATION DE L'EPREUVE D'ABSORPTION DES AGGLUTININES DANS LA SEROLOGIE DE LA LEPTOSPIROSE Par une s6rie d'essais comparatifs, on a recherch6 dans quelle mesure certains facteurs peuvent modifier les resultats des epreuves d'absorption des agglutinines ser- vant au diagnostic de la leptospirose et a la classification des souches de Leptospira. Ont ete etudies: a) le laps de temps requis pour obtenir une absorption suffisante; b) les relations quantitatives entre les titres d'anticorps et les quantites d'antigene necessaires a l'absorption; c) le r6le 6ventuel d'un ph6nomene de Danysz; d) la capacite d'absorption des antigenes vivants et des antigenes tues par le formol. D'epreuves effectuees avec des antigenes vivants ou tues, il ressort que l'absorption n'est pas sensiblement am6lioree par un contact de l'antigene et des anticorps prolonge pendant 18 heures. Du point de vue pratique, on peut admettre que la liaison antigene-anticorps est realis6e apres 90 minutes de contact. Si l'on utilise une trop grande quantite d'antigene, on constate reguliere- ment une diminution du titre des anticorps seriques, ce qui denote un certain degre d'absorption non speifique. On a mis en evidence l'existence d'un phenomene de Danysz: pour une meme quantit6 d'antigene, l'absorp- tion est plus effective lorsque l'antigene est incorpore au serum en petites quantites successives que lorsqu'on ajoute le serum a l'antigene. La meilleure technique, semble-t-il, consiste a ajouter l'antigene au serum en deux ou trois fractions aliquotes a intervalle d'environ 10 minu- tes. Des epreuves comparatives indiquent que les serums absorbes par un antigene formole presentent des titres inferieurs a ceux des serums absorbes par des antigenes non traites. Les implications de ces donnees en ce qui concerne la methodologie des epreuves d'absorption et les crit6res a utiliser pour la classification des leptospires sont examin6es. 737 738 E. KMETY, M. M. GALTON & C. R. SULZER REFERENCES Alexander, A. D., Wetmore, P. W., Evans, L. B., Jefferies, H. & Gleiser, C. A. (1955) Amer. J. trop. Med., 4, 492-506 Babudieri, B. & Mateew, D. (1961) R. C. Ist. sup. Sanita, 24, 614-622 Borg-Petersen, C. (1938) Leptospirenuntersuchungen in Danemark. In: Proceedings of the 3rd International Congresses on Tropical Medicine and Malaria, Amster- dam, Netherlands Society of Tropical Medicine, pp. 396-406 Borg-Petersen, C. (1944) Acta path. microbiol. Scand., 21, 165-179 Bull. Wld Hlth Org., 1965, 32, 881-891 Danysz, J. (1902) Ann. Inst. Pasteur, 16, 331-345 Galton, M. M., Menges, R. W., Shotts, E. B., Jr, Nahmias, A. J. & Heath, C. W., Jr (1962) Leptospirosis-epide- miology, clinical manifestations in man and animals, and methods in laboratory diagnosis. 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