- Volume 64 , Number 3
- Page: 316–9
70th anniversary of the Rubino reaction
Seventy years ago, the Uruguayan scientist Miguel C. Rubino,1 while working with complement fixation reactions for the diagnosis of syphilis using formolized sheep red blood cells, noted that among approximately 800 human sera tested there was one that induced rapid cell sedimentation and supernatant clarification. Since he had not observed this phenomenon previously, he determined the origin of the serum and identified it as a sample from a leprosy patient.
Rubino then devoted his time to the study of the nature of the reaction, to the standardization of the conditions under which it occurs, and to its application to a large number of human sera of different origins.2-6 He reproduced the phenomenon in 84% of 38 other sera from leprosy patients, observing red cell sedimentation within less than an hour.1 Only rarely did sera from nonleprosy subjects cause sedimentation of formolized sheep red cells so rapidly and these sera, in contrast to those from leprosy patients, also caused the sedimentation of natural, nonformolized red cells. He thus concluded that the reactivity of sera from leprosy patients was mainly characterized by a different behavior in the presence of formolized and natural red blood cells.3-5-6 These first observations by Rubino demonstrated the specificity of the reaction, which was positive only for leprosy patients; a single exception among 300 control sera tested was serum from a patient with filariasis who, however, was suspected to be also infected with Mycobacterium leprae 4
The pioneering work of Rubino motivated the study of the reaction by several investigators. Marchoux and Caro,7 while testing 10 sera from leprosy patients, observed 50% positivity without the occurtected a 44% rate of positive reactions among leprosy patients with the absence of nonspecific reactions. Bier and Arnold9 obtained only one positive reaction among 954 sera from individuals without leprosy. In 1929 Rubino3 reported that he had tested more than 2000 sera from nonleprosy individuals without observing any positive reactions. Evidence for the absolute specificity of the reaction accumulated during the following years.10-24 The few reports which questioned the specificity of the test by other investigators can be attributed to the fact that no countertest with natural red blood cells was performed.25-29.
The nature of the Rubino reaction was studied extensively by Marchoux and Caro,7 who attributed it to the presence of a specific factor in the serum of leprosy patients which was selectively adsorbed to formolized red cells. They observed that the adsorption of this specific factor required the participation of a second factor also present in the serum of normal individuals, and that formolized sheep red blood cells could completely deplete leprosy serum of the active substance. They reported an unsuccessful attempt to isolate the specific factor in leprosy serum by solubilizing the factor adsorbed to the surface of formolized sheep red blood cells.
Rubino5 considered the reaction described by him to correspond to agglutinosedimentation of formolized red cells of the same nature as microbial agglutinations and serum flocculations. Ambrogio13 attributed this reactivity to a principle present in positive serum that possibly acted synergistically with electrolytes causing a change in potential, increased repulsion between globules and consequent sedimentation.
Benetazzo30 considered the reaction to be related to colloidal plasma changes caused by the absolute increase of globulins. Despite the facts and conjectures about the origin of the Rubino reaction, in 1936 Bier22 referred to the factor responsible for the Rubino reaction as an "enigmatic factor" not related to the immunologic factors known at the time.
All of the studies carried out invariably indicated that the Rubino reaction was absolutely specific, although its sensitivity was low and variable and its positivity occurred only when the diagnosis of leprosy was already implicit. These characteristics limited its diagnostic applicability, a fact probably justifying the hiatus of almost three decades in the study of the Rubino reaction.
In 1962, Almeida31 demonstrated the absence of correlation between positivity of the Rubino reaction and anti-complementary activity of sera from leprosy patients. During the same year, Curban32 published a comprehensive review of the literature in which he emphasized the importance of the test not as a diagnostic tool but rather as an unexplored field that might contribute to the understanding of the immune response in leprosy.
In 1970, Almeida,33 in a review of theserology of leprosy, referred to the factor responsible for the Rubino reaction as a gamma-globulin, and stated that there wasno correspondence between the factor and the complement-fixing antibodies that recognize tubercle antigen preparations. Thesame author later demonstrated that the Rubino reaction was inhibited when carried out in the presence of antigen preparations of M. leprae or some, but not all, mycobacteria.34,35
A new stage in the investigation of the Rubino reaction became possible after standardization of the criteria for the classification of the clinical forms of leprosy.36
Silva, et al 37-39 studied 60 patients with the lepromatous clinical form and 16 with the tuberculoid form and observed that the Rubino reaction was invariably negative among patients with the tuberculoid form and positive in 50% of the patients with the lepromatous form. In a study of a group of 178 leprosy patients, Opromolla, et al. 40 confirmed the negativity of the reaction among tuberculoid patients and reported 9% positivity among borderline patients.For the group of lepromatous patients, they obtained 55% positive reactions for those with active disease and 6% for those considered to be clinically cured. These observations were confirmed when these investigators expanded their series, a fact that led them to propose the existence of an inverse relationship between positivity of the Rubino reaction and host resistance to M. leprae. 41 As these studies were being conducted, a new application of the Rubino test was suggested, i.e., as a criteria for the cure of the disease.40 However, this application became unfeasible when positive reactions were detected among 57% of clinically cured patients.42
Research interest then started to converge on the relationship between the positivity of the Rubino reaction and the impaired specific immune response in leprosy (The Figure). Subsequent studies were implicitly based on the search for a correspondence between the Rubino factor and specific components of the serum of lepromatous patients. Hypergammaglobulinemia wasobserved in lepromatous patients and in-creased IgM levels were observed only inRubino-positive sera.37,38 Rheumatoid factor was detected in lepromatous patients butits presence was not associated with positivity of the Rubino reaction. Anti-thyroglobulin antibodies were detected only inthe serum of Rubino-positive patients.39 A subsequent study showed that the positivity of the reaction did not correlate withchanges in serum IgG or IgM levels, an ob-servation interpreted to indicate the inde-pendence between the Rubino reaction andthese immunoglobulin isotypes.44
The figure. Correlation between positivity of the Rubino reaction and the clinical forms of leprosy. LL = lepromatous leprosy; BL = borderline lepromatous leprosy; BB = borderline leprosy; BT = borderline tuberculoid leprosy; TT = tuberculoid leprosy. AFB = acid-fast bacilli. Adapted from Bloom and Jacobs,1989.43
The specific factor responsible for theRubino reaction (Rubino factor) was recently identified as an immunoglobulin of isotype M.45 The Rubino factor/IgM wasisolated from a pool of sera from 20 patients with lepromatous leprosy whose IgM levels were higher than those of normal individuals but similar to those of 20 othersera from Rubino-negative patients withlepromatous leprosy. Anti-IgM antibodies block the Rubino activity of positive serumor of the purified factor. The availability of purified Rubino factor/IgM permitted the unequivocal demonstration of the requirement of a cofactor present in normal serumfor the Rubino reaction.46
Recent interest in the mechanisms which underly the Rubino reaction is based on the expectation that they may contribute to the understanding of the cell-mediated immune unresponsiveness typical of lepromatous leprosy. However, the Rubino reactioncompletes 70 years as an enigma still to be understood.
- Ademilson Panunto-Castelo, M.Sc.
Maria-Cristina Roque-Barreira, M.D., Ph.D.
Department of Parasitology, Microbiology and Immunology
Faculty of Medicine of Ribeirão Preto
University of São Paulo
14049-900 Ribeirão Preto, SP, Brazil
1. Rubino, M. C. Nueva reaction sorológica en la lepra. Rev. Med. Uruguay 29(1926)143-155.
2. Rubino, M. C. Nuevas reacciones sorológicas en la lepra. Rev. Soc. Arg. Biol. 2(1926)407-416.
3. Rubino, M. C. Nouvelle reaction serologique dans la lepre. Rev. Med. Uruguay 32(1929)85-116.
9. Bier, O. and Arnold, K. Estudos sobre a sorologia da lepra. I. Sobre a especificidade e a sensibilidade da reação de Rubino. Pesquisas sobre o mecanismo da reação. Folia Clin 7(1935)1-7.
10. Monacelli, M. SuIla reazione di Rubino nella lepra. G. Ital. Derm. Sif. 69(1928)1472-1476.
11. Figueiredo, A. P. O diagnóstico sorológico da lepra pela reação de Rubino , doctoral thesis, Faculty of Medicine of Rio de Janeiro, Rio de Janeiro, Brazil,1931.
12. Adant, M. Au sujet de la réaction de Rubino. C. R.0. Soc. Biol. 110(1932)119-120.
13. Ambroggio, A. La reazione di Rubino nella lepra.Pathologica 24(1932)258-268.
14. Furtado, D. and heite, S. A reacão de Rubino e o diagnóstico precoce da lepra. Lisboa Medica 9(1932)1019-1027.
15. Hombria, M. Contribución al estudio sorologicade Ia lepra. Acta Dermo.-Sifiliogr. 25(1932)193-2(8).
16. Lepine, P., Markianos, J. and Papayoannou, A.Valour pratique de la reaction de Rubina pour le serodiagnostic de la lepre. Bull Soc. Path. Exot. 25(1932)543-546.
17. Travassos, J. Reação de Rubino. Arch. Riogrand. Med. 7(1932)7-11.
18. Zevallos, C. A. La reacción de Ruhino en el diagnóstico de la lepra. Arch. de Lepra 4(1932)22-64.
19. Silveira, G. F. and Mesquita, M. P. Contribuicáo ao estudo da reacão de Rubino. Rev. Assoc. Paul. Med.3(1933)21-31.
20. Roca-de-Viñals, R. Contnbucian al estudio de serodiagnastico de la lepra. La reaccion de Rubino.Cron. Méd.-Quir. Habana 60(1934)27-30.
21. Imbert, M. F. P. La reaccion de Rubino en la lepra.Puerto Rico J. Publ. Hlth. 12(1936)246-256.
22. Bier, 0. Sorologia da lepra. Rev. Bras. Leprol. 4(1936)221-222.
26. Castro-Paullier, V. and Errecart, L. Nuevos casos de reacción de Rubino en sujetos no leprosos. Manera de obtenela experimentalmente en leprosos y no
30. Benetazzo, G. Rapporti tra reazioni di Rubino,velocity di sedimentazione e reazione sierologiche nella lebbra. Dermosif. 8(1933)241-255.
31. Almeida, J. 0. Serological studies on leprosy; a comparision of complement-fixation tests using antigens prepared from tubercle bacilli and beaf-heart lipids with others serological reactions. Bull. Who 26(1962)233-240.
32. Curban, G. V. Contribuicao para o estudo da reação de Rubino. Rev. Bras. Leprol. 30(1962)179-216.
33. Almeida, J. 0. Serology in leprosy. Bull. Who 42(1970)673-702.
34. Almeida, J. 0. and Kwapinski, J. B. Reatividade de antígenos de actinomicetos corn soros de lepra, avaliada por imunofluorescencia em suporte de acetato de celulose. Publcões Cent. Estud. Lepr. 14(1974)73-89.
35. Almeida, J. 0. Inhibition of Rubino factor as a test for detecting antigens common to leprosy bacilli. (Letter) Int. J. Lepr. 46(1978)436.
36. Ridley, I). S. and Jopling, W. H. Classification ofleprosy according to immunity; a five group system. Int. J. Lepr. 34(1966)255-273.
37. Silva, O. P. Ferri, R. G., Moraes, N. and Marques, A. L. V. Estudos imunoquímicos na lepra. I - Eletro e imunoeletroforese das proteínas séricas. Tentativa de associação com a reação de Rubino. Hansen. Int. 1(1976)33-41.
38. Silva, O. R, Ferri, R. G, Moraes, N. and Marques, A. L. V. Estudos imunoquímicos na lepra. II - Quantificação de imunoglobulinas séricas. Tentativa de associação com a reação de Rubino. Hansen. Int. 1(1976)43-51.
39. Silva, O. R, Ferri, R. G, Moraes, N. and Marques, A. L. V. Estudos imunoquímicos na lepra. III - Pesquisa de auto-anticorpos séricos. Tentativa de associação com a reação de Rubino. Hansen. Int. 1(1976)120-124.
40. Opromolla, D. V. A., Arruda, M. S., Ura, S., Pernambuco, J. C, Bastazini, I., Fleury, R. N., Tolentino, M. M., Tonelo, C. L. and Arruda, O. S. Aspectos evolutivos da reação de Rubino. Med. Cutan. Ibero Lat. Am. 10(1982)9-14.
41. Arruda, M. S. P, Arruda, O. S., Astolfi, C. S., Nogueira, M. E. S., Bastazini, I., Opromolla, D. V. and Ura, S. Reação de Rubino. Critério dc branqueamento para pacientes virchowianos. Med. Cur. Ibero Lat. Am. 11(1983)423-430.
42. Arruda, M. S. P., Arruda, O. S., Souza, L. C. D., Opromolla, D. V. A. and Ara, S. Aspectos da imunidade humoral de pacientes leprosos clinicamente curados. Salusvita 7(1988)22-30.
43. Bloom, B. R. and Jacobs, W. R. New strategies for leprosy and tuberculosis and for development of bacillus Calmette-Guénn into a multivaccine vehicle. Ann. N. Y. Acad. Sci. 569(1989)155-173.
44. Arruda, M. S., Costa, h. C, Souza, L. C. D. and Nobre, L. A. S. Avaliação das immunoglobulinas séricas em pacientes com hanseniase virchoviana. Salusvita 6(1987)96-101.
45. Panunto-Castelo, A. and Roque-Barreira, M. C. Identification of IgM as the leprosy patient serum factor responsible for rapid sedimentation of formalized sheep erythrocytes. Int. J. Lepr. 63(1995)231-240.
46. Panunto-Castelo, A., Almeida, I. C. and Roque-Barreira, M. C. Participação de um co-fator na reatividade Rubino induzida por IGM específica do soro de hansenianos. Annals of the XX Congress of the Brazilian Society of Immunology, abstract 313. Angra dos Reis, Rio de Janeiro, Brazil (1995).
Reprint requests to Dra. M.-C. Roque-Barreira at the above address or FAX =55-16-6336631; email = mcrbarre@ fmrp.usp.br.