Indirect Complement Fixation Research Articles

Indirect Complement Fixation in Foot-and-Mouth Disease

Indirect Complement Fixation in Foot-and-Mouth Disease

Indirect Complement Fixation in Foot-and-Mouth Disease

Indirect Complement Fixation in Foot-and-Mouth Disease

Indirect Complement Fixation in Foot-and-Mouth Disease

Zusammenfassung Indirekte Komplementbindung bei Maul- und Klauenseuche II. Überprüfung von Rinderseren Bei der Suche nach empfindlichen Rindern wurden die Seren mittels der indirekten Komplementbindung (IKB) und des zum Vergleich ausgeführten Neutralisationstestes in Gewebekulturen überprüft. Die Ergebnisse beider Teste stimmten einigermaßen gut überein. Die IKB war als einfache und schnelle Prüfungsmethode geeignet; sie spart Zeit, Material und Arbeit. Beide Methoden ergaben als Folge unspezifischer Inhibitoren gelegentlich zweifelhafte Resultate. Der IKB-Inhibitor war bei 12 (= 8%) von 148 Seren, die beiden Tests unterzogen worden waren, in störenden Konzentrationen vorhanden und schien von dem Neutralisations-Inhibitor verschieden zu sein. Keiner der Inhibitoren machte die Serumspender gegenüber einer Infektion mit dem MKS-Virus resistent. Im Gegensatz zu spezifischen Antikörpern war die Wirkung des IKB-Inhibitors nicht typspezifisch. Zur Bestimmung des Inhibitors wurden die Seren neben den Antigenen A und O auch mit SAT 1 überprüft, da SAT 1-Antikörper bei der jüngeren Rinderpopulation im Iran nicht mehr zu erwarten sind. Vorläufige Versuche, den IKB-Inhibitor durch einfache Maßnahmen auszuschalten, ohne daß der parallel überprüfte Antikörpertiter von Rinder-Immunseren sich vermindert, hatten keinen Erfolg. Zwei Hypothesen über die mögliche Natur des IKB-Inhibitors werden diskutiert. Résumé Fixation indirecte du complément lors de fièvre aphteuse II. Examens de sérums bovins Lors d'une recherche d'animaux positifs, on examine les sérums à l'aide de la fixation indirecte du complément (FIC) et, à titre comparatif, à l'aide du test de neutralisation sur cultures de tissus. Les résultats des 2 tests concordent assez bien. La FIC est indiquée pour un examen simple et rapide; elle épargne temps, matériel et traival. Les 2 méthodes donnent à l'occasion des résultats dounteux, à cause de la présence d'inhibiteurs non spécifiques. On trouve l'inhibiteur de la FIC à des concentrations gênantes dans 12 sur 148 sérums (8 %) soumis aux 2 tests. II semble se différencier de l'inhibiteur de neutralisation. Aucun des inhibiteurs ne parvient à rendre les donneurs de sérums résistants à une infection par le virus de la fièvre aphteuse. A l'encontre des anticorps spécifiques, l'action de l'inhibiteur de la FIC n'est pas spécifique du type. Pour déterminer l'inhibiteur, on teste les sérums avec les antigènes A et O, puis également avec SAT 1, car on ne s'attend guère à trouver des anticorps SAT 1 dans les populations de jeunes bovins de l'Iran. Les expériences préliminaires pour éliminer l'inhibiteur de la FIC par des méthodes simples, sans our autant diminuer le titre des anticorps des immunsérums bovins examinés parallèlement, demeurent jusqu'ici sans succès. On émet deux hypothèses sur la nature probable de l'inhibiteur de la FIC. Resumen Fijacién indirecta del complemento en la glosopeda II. Control de los sueros sanguíneos bovinos Iniciando la búsqueda de bovinos sensibles, se controlaron los sueros sanguíneus en cultivos hísticos mediante la fijacién indirecta del complemento (FIC) y la prueba de neutralizacién, efectuada en calidad de comparacién. Los resultados de ambas pruebas coincidían bastante bien. La FIC resultaba aprobpiada como método simple v rápido de control: ahorra tiempo, material y trabajo. A veces, ambas técnicas ofrecieron resultados dudosos como consecuencia de hallarse presentes inhibidores inespecíficos. El inhibidor de la FIC se detecté en concentraciones perturbadoras en 12 (8 %) de 148 sueros sanguíneos que se habían sometido a ambas pruebas y parecía ser distinto al inhibidor de la neutralizacién. Ninguno de los inhibidores tornaba resistente a los donadores del suero sanguíneo frente a la infeccién con el virus aftoso. En contraposicién a los anticuerpos específicos, la accién del inhibidor de la IC no era específica de tipo. Para diagnosticar el inhibidor, se controlaron los sueros sanguíneos además de con los antígenos A y O con el SAT 1, ya que en las populaciones bovinas jévenes del Irán no se suele contar más con los anticuerpos SAT 1. No tuvieron resultado feliz los ensayos provisionales para eliminar el inhibidor de la FEC mediante medidas simples, sin que disminuyese el título de anticuerpos de los sueros sanguíneus inmunes bovinos, controlado de forma paralela. Se discuten dos hypétesis sobre la naturaleza posible del inhibidor de la FIC.

Indirect Complement Fixation in Foot-and-Mouth Disease

Indirect Complement Fixation in Foot-and-Mouth Disease

A case of anti-p200 pemphigoid: evidence for a different pathway in neutrophil recruitment compared with bullous pemphigoid

Conflicts of interest: none declared. Sir, Anti‐p200 pemphigoid was first described in 1996 by Zillikens et al. as a subepidermal blistering disease that involves autoantibodies directed against a 200‐kDa component (p200) of the basement membrane zone (BMZ).1 Although p200 is known to be an acidic noncollagenous N‐linked glycoprotein, which localizes to the lower lamina lucida of the BMZ, the nature of this molecule had not been elucidated for a long time.1 Currently, it is reported that patient sera react with laminin γ1, which is a component of the lamina densa, suggesting that p200 may be laminin γ1.2 In patients with anti‐p200 pemphigoid, histopathological examination reveals subepidermal blisters and infiltration predominantly by neutrophils, but occasionally also by eosinophils.3 Interleukin (IL)‐8 is a major chemotactic cytokine for neutrophils and has been implicated in the inflammatory process of bullous pemphigoid (BP).4 A 75‐year‐old Japanese man presented with bullae and pustules on his palms, which had arisen suddenly, then spread over the whole body (Fig. 1a). Mucous membranes, including the oral mucosa and conjunctiva, were also affected. Both flaccid and tense blisters were found. Nikolsky’s sign was negative. Milia arose after blister healing in some cases. Blood tests showed a white blood count of 11·85 × 109 L−1 (normal 3·3–9·2 × 109) and a neutrophil ratio of 81·2% (normal 45·3–73·2%). Complement C3 and C4 and immunoglobulin (including IgG and IgA) levels were within the normal range. Histopathological examination of a skin biopsy specimen from the neck revealed the presence of subepidermal bullae containing many neutrophils and some eosinophils (Fig. 1b). Direct immunofluorescence (DIF) revealed linear deposits of IgG and C3 at the BMZ. Indirect immunofluorescence (IIF) using 1 mol L−1 salt‐split skin showed that IgG antibody bound on the dermal side (Fig. 1c). Immunoblotting with dermal extracts showed that the patient’s IgG autoantibodies reacted with a 200‐kDa protein (Fig. 1d). The titre of autoantibody determined by IIF was 1 : 160. IgG subclass of autoantibodies against the BMZ was determined by DIF and IIF. The results of DIF showed linear deposits of IgG2, but not IgG1, IgG3 and IgG4 (Fig. 1e). The deposition of IgG2 autoantibody, but not IgG1, IgG3 and IgG4, was detected on the basement membrane by IIF (Fig. 1e). An indirect complement fixation test with patient serum revealed weak C3 deposits along the basement membrane (Fig. 1f). Enzyme‐linked immunosorbent assay (ELISA) revealed that the concentrations of antibodies against BP180, BP230 and desmoglein 1 and 3 were all within the normal range. The patient was diagnosed as having anti‐p200 pemphigoid and was initially treated with oral prednisolone 10 mg daily, but serum levels of autoantibodies, as determined by IIF, continued to increase, and the clinical symptoms worsened. In response to a treatment regimen of prednisolone 30 mg daily, dapsone 50 mg daily and azathioprine 100 mg daily, the blisters gradually disappeared. As the clinical symptoms resolved, the autoantibody titre against p200, as determined by IIF, also decreased gradually. At 7 months after onset, prednisolone therapy was tapered to 20 mg daily, but at this time the patient died due to sepsis, which was probably caused by aspiration pneumonia.

Indirect Complement Fixation in Foot and Mouth Disease. II. Screening of Cattle Serums

Indirect Complement Fixation in Foot and Mouth Disease. II. Screening of Cattle Serums

Serological tests for the detection of antibodies to Mycoplasma gallisepticum in chickens and turkeys

SUMMARYA comparison was undertaken of several serological tests in determining the response of chickens and turkeys experimentally infected with the A 514 strain of Mycoplasma gallisepticum.After a single intratracheal inoculation of chickens with a culture of the organism, the highest titres were obtained by the indirect complement fixation (ICF) test, followed by the tube agglutination (TA), haemagglutination inhibition (HI), slide agglutination (SA) and metabolic inhibition (MI) tests. By all these tests positive titres were observed within the first week and peak titres between the first and second weeks. At 5 months there was no positive reaction by the ICF test but most chickens gave positive readings by the TA, HI and SA tests for at least 14 months after infection, but turkey sera became negative by all tests after 3 months.A disadvantage of the ICF test was that sera up to a dilution of 1/8 and 1/16 for chicken and turkey respectively were anticomplementary, and in turkeys this masked the ICF titre, which presumably was low following one intratracheal inoculation. Titres in turkeys with the TA, HI and SA tests followed the pattern seen with chickens and were generally lower than those found by other workers probably because of the avirulent nature of the inoculum used.The WB test was the least sensitive of the agglutination tests but is useful as a flock test which can be undertaken on the farm.The MI test gave the lowest titres of all and antibodies could be detected for only 4 months following one intratracheal inoculation. Even with serum prepared by multiple inoculations in chickens the titre was never higher than 1/32 compared with 1/1024 for serum similarly prepared in rabbits.Precipitins were detected by the agar gel method in the sera of chickens and turkeys after two intratracheal inoculations but in only some of the chickens and none of the turkeys after one inoculation.By all tests higher titres were observed with chicken than turkey sera and antibodies persisted for a longer time.Re-infection of chickens when antibodies to the initial infection had become low, and of turkeys when antibodies were no longer detectable, gave rise to an anamnestic response with titres which were higher than before.Antiserum to M. gallisepticum prepared in chickens is comparable with that prepared in rabbits except for low titres by the MI test.

Characteristics of Direct and Indirect Complement-Fixing Antibodies Obtained from Turkeys Infected with Ornithosis Agent

Summary The antibody response of turkeys experimentally infected with ornithosis was measured by three different methods: indirect complement-fixation (ICF), direct complement-fixation utilizing a detergent-extracted soluble antigen (DCF-det), and a DCF method in which a small amount of fresh normal fowl serum was added (DCF-Brum). Possible explanations for the discrepancies between the titers obtained by these three assay methods were tested by studying the physical properties of the antibodies. Early acute-phase antisera (9–14 days) contained 7 S γ2 antibody, as determined by all three tests. These early antisera also contained anti-ornithosis activity detectable only by ICF, which sedimented in sucrose gradients intermediate between the 7 S and 19 S fractions. The ICF antibody migrated electrophoretically in the γ1- and γ2-globulin regions. Primary anti-BSA turkey sera contained three distinct populations of hemagglutinins (HA): 19 S γ1, 19 S γ2 and 7 S γ2. In contrast, only 7S anti-BSA antibodies were detected in primary antisera by an antigen-binding technique. The chromatographic properties of 7 S anti-ornithosis antibody and 7 S anti-BSA HA differed; the anti-ornithosis antibodies were eluted with a lower salt concentration than were the more strongly anionic-binding 7 S anti-BSA HA. Reasons for the absence of 19 S anti-ornithosis antibody are discussed.

Studies on the Biophysical Characteristics of Antibodies Produced in Birds and Mammals in Response to Experimental Chlamydial Infection (Psittacosis)

Summary Circulating complement-fixing and agglutinating antibodies—produced in turkeys, pigeons, sheep, rabbits and a calf at early and late stages of experimental infection with agents of the genus Chlamydia (psittacosis group)—were analyzed by sucrose gradient centrifugation and electrophoretic methods. All species, except pigeons, produced antibodies of the fast sedimenting type early in the course of experimental infection and slow sedimenting type in the later stages. Only slow sedimenting antibodies were found in pigeons. Both molecular types were readily detectable in turkey serums using the indirect complement fixation (ICF) method and in the other species using the direct complement fixation method. Agglutinating antibodies detected by the capillary tube method of Mason were principally of the fast sedimenting type. Electrophoretic patterns of whole serums and gradient separated fractions confirmed that the antibodies were of the γ-globulin type. Inactivating serums at 56°C or 65°C caused a marked shift in the electrophoretic mobility of serum albumin, and 65°C inactivation reduced the γ2-globulin content. The serologic activity of fast sedimenting antibodies produced in the calf was eliminated by 65°C heating (as was previously shown by others). However, heating the serums or gradient fractions thereof from turkeys or sheep not only inactivated the fast sedimenting antibodies but also reduced the titers of slow sedimenting antibodies.

Modification Including the Use of Rabbit Serum as Indicator in the Indirect Complement Fixation Procedure with Avian Serums for Diagnosis of Ornithosis

In the course of conducting complement fixation tests with avian serums for the diagnosis of ornithosis, it was observed that some batches of phenolized yolk sac ornithosis antigen that reacted with anti-ornithosis chicken or turkey serum in the Karrer et al. indirect complement fixation (ICF) procedure were inactive in the Brumfield's direct complement fixation (DCF) technique (6). In such instances testing must be done by ICF, which is not only cumbersome but requires human or p,igeon anti-ornithosis indicator serum which may not be available. In view of these difficulties, the use of rabbit immune serum in the ICF procedure in place of human or pigeon serum was attempted. This report describes the use of rabbit immune serum as an indicator serum together with a modification of the ICF procedure as a means for detecting ornithosis antibodies in suspected turkey or chicken serums.

Serologic Differences between the Beaudette and Massachusetts Strains of Infectious Bronchitis Virus

It has been generally accepted that the embryo lethal strain of avian infectious bronchitis virus (IBV), commonly referred to as the Beaudette strain, is nonimmunogenic, (1,3,4). This strain has been widely used to test avian serum for IBV neutralizing antibodies by the virus neutralization (VN) test performed in chicken embryos (2). This test, using the Beaudette strain as the antigen, has been useful in that it detected antibodies to the Massachusetts strain and thereby aided in the discovery of strains antigenically different from the Beaudette and Massachusetts strains of IBV virus (3,4). In the development of an indirect complement-fixation test for IBV, antibodies were produced for the Beaudette strain in adult chickens (5). This communication reports the production of antibodies to the Beaudette strain and their relation to the Massachusetts strain.

ニューカッスル病ウイルスの血清学的研究

It is well known that interest informations on the antigenic structure of viruses have been obtained with complement fixation. The serological properties of Newcastle disease virus, however, have been mainly studied by neutralization and hemagglutination and available references on its complement fixing properties are limited. For complement fixation many kinds of animals may be able to donate antibodies, but the natural host should be considered to be standard as source of antibodies. As chicken serum, which is of the most susceptible natural host for the virus, fails to fix complement when inactivated by heating at 56°C for 30 minutes, indirect complement fixation has been recommended as an available technique. Recently the possibility of direct complement fixation with chicken serum was suggested by Nitzschke (1956) and Brumfield (1957). The author tried to establish the direct complement fixation in the Newcastle disease virus-chicken serum system and to examine complement fixing properties of the virus and antigenic difference among several strains with the technique.The Sato, Miyadera and W strains of Newcastle disease virus, different in origin and pathogenicity, were chiefly employed. The Sato strain was isolated from an affected bird in the outbreak of the acute type disease in 1930 and the Miyadera strain, of the mild type disease in 1951. The W strain, originating from American living vaccine, is an avirulent one in chickens.Results are summarized as follows.1. Direct complement fixation was successfully performed in the Newcastle disease virus-chicken serum system, using sera in the active phase and the incubation at 37°C for 60 minutes in a water bath.2. Complement was specifically fixed with infected embryonic tissues under the presence of immune serum. The highest fixation was shown in the test with chorioallantoic membranes and no fixation was detected in the test with allantoic fluid.3. Two kinds of antigens, soluble and viral, existed in infected embryonic tissues. The former was prepared by the complete elimination of hemagglutinin from the suspension of chorioallantoic membranes, using centrifugation and absorption with chicken red cells, and the latter, by centrifugal concentration of hemagglutinin from allantoic fluid.Although hemagglutinability was approximately the same in titer, the amount of soluble antigen in chorioallantoic membranes seemed to be different among the strains.4. Antibodies against soluble and viral antigens were produced in chickens hyperimmunized with inactivated virus or infected with active virus. Antibody against viral antigen was usually from 2 to 8 times higher than that against soluble antigen in titer.5. Partial antigenic differences among the strains were revealed by cross hemagglutination inhibition with sera absorbed with heterologous antigens, particularly the Sato strain appeared to be different from the others. Cross complement fixation failed to detect the partial differences among them, even when viral antigen was used. The Miyadera and W antigens, both soluble and viral, showed stronger reaction with the Sato immune serum than that with the homologous. The Sato strain seemed to be more effective to produce antibodies in chickens.

A Modified Direct Complement Fixation Test for the Detection of Antibodies in the Serum of Cattle Previously Infected with Vesicular Stomatitis Virus

Summary A modified direct complement fixation method for the detection of antibodies in heat-inactivated sera of cattle infected with vesicular stomatitis virus is described. The addition of fresh unheated bovine serum to the diluted guinea pig complement constitutes the only modification. In its absence no fixation of complement is observed. The normal serum supplying the apparently essential heat-labile factor, is pretested for absence of nonspecific reactivity with test antigen. Sera of animals around 6 months of age show less nonspecific reactivity than sera of adult cattle. This method when applied in testing serial bleedings from cattle infected with either the New Jersey or Indiana type of vesicular stomatitis virus, detected the antibodies 9 to 10 days after intralingual infection. Antibodies were demonstrable at a diagnostic level for at least 90 days afterward. The test was shown to be sufficiently specific to indicate the type of virus in epithelial tongue tissue collected at the acute stage of infection and to reveal the type-specificity of the antibodies in serum of convalescent cattle. In indirect complement fixation tests, the inhibition titers of early convalescent sera were much lower than those recorded in the modified direct complement fixation test. The difference was negligible with hyperimmune sera.

A Capillary Tube Agglutination Test for Detecting Antibodies against Ornithosis in Turkey Serum

Summary and Conclusions A capillary tube agglutination test using a Giemsa-stained, buffered suspension of elementary bodies prepared from a toxigenic turkey strain of ornithosis virus propagated in the allantoic cavity of chicken embryos was compared with the indirect complement fixation test, and the results were promising. The CTA test seemed to be more sensitive in the first week or two after infection, and thereafter the two tests seemed equally sensitive. Both were a little more sensitive than the direct complement fixation test when the test material was turkey serum. In the CTA test false positive reactions were encountered, usually in low serum dilutions, but occasionally in dilutions as high as 1:32, when the serum was hemolyzed, contaminated or contained residual fibrin. Reactions at dilutions of 1:16 or higher constituted good presumptive evidence of infection or of some previous encounter with ornithosis agents; reactions at dilutions of less than 1:16 were less specific and were interpreted cautiously. Preliminary testing of serum from infected parrots and pigeons indicated that the CTA test can detect ornithosis antibodies in these species. The CTA test is economical and simple to perform. According to the results of these tests it could be a useful flock test, of value in epizootiologic surveys, and a rapid screening test of flocks before marketing in enzootic or epizootic areas. Flocks undergoing acute or subacute epizootics of ornithosis, the most dangerous from the standpoint of public health, could be detected easily.

The Measurement of Complement Fixation by Viruses

Publisher Summary This chapter discusses the measurement of complement fixation by viruses. The complement fixation test is an indirect way of detecting and measuring an antigen–antibody reaction that is not easily observed directly as an agglutination or as a precipitation; it is, therefore, particularly useful for the study of viruses that can rarely be obtained in the high concentrations and purity that will permit the application of direct methods. Complement fixation tests are usually employed for the study of virus-soluble antigens, as there are few other ways to detect these substances associated with the virus elementary body. Direct and indirect complement fixation tests certainly cover a very wide field both in the study of virus antigens and antibodies, and in the analysis of virus multiplication. The chapter mentions the three variables of a complement fixation test that are the three components of the primary system: the antigen, the antiserum, and the complement. The object of a quantitative complement fixation test is to measure the amount of complement fixed in a set of reaction mixtures in which the amounts of antigen and antiserum are independently varied over a selected range. The chapter also presents and discusses two methods of measuring the amount of complement fixed by a particular antigen-antibody complex.

Direct complement-fixation test for diagnosis of ornithosis in turkeys.

SummaryEmploying a soluble antigen, complement-fixation reactions were obtained with turkey antiornithosis sera. Antigens consisting of viable allantoic and killed yolk-sac virus suspensions did not show the same degree of activity. The direct complement-fixation reaction was as sensitive as the indirect complement-fixation test; therefore, the use of the direct test for the study and diagnosis of turkey ornithosis was suggested.

Detection of complement fixing antibodies against foot-and-mouth disease in cattle serum.

The complement fixation test has become of considerable value in the early diagnosis of foot-and-mouth disease (F and M) in cattle and in typing the virus from this disease. However, this method is less useful in later stages of the disease when infected tissue can no longer be obtained for antigen. Furthermore, it is difficult to detect complement fixing antibodies in serums from convalescent cattle when using conventional procedures. Therefore, many attempts have been made to improve the sensivitity of the test. These modifications of the complement fixation test have included the use of unheated serum (Fredericks, 1949) or performance of the entire test at higher salt concentration (Serra and Guarini, 1951). None of these have proved to be successful. An indirect method for demonstrating antibodies in late convalescent F and M serums has been suggested by Rice and Brooksby (1953). The indirect complement fixation test had earlier been applied by Rice (1948) in detecting antibodies in anti-pullorum serums, by Wolfe (1949) in anti-Newcastle disease serums, by Karrer et al (1950) and Hilleman et al (1951) in anti-ornithosispsittacosis, and by Downie et al (1950) in anti-variola serums from chickens. Rice and Boulanger (1953) have also used it to demonstrate antibodies against vesicular viruses in cattle, and Rice and McKercher (1954) in studies of vesicular stomatitis in horses, cattle and swine. The difficulty with the indirect test is its relative complexity, and it is time consuming. A simple modification of the F and M complement fixation test was described by Nordberg and Schjerning-Thiesen (1953) in a preliminary report. These workers were able to demonstrate antibodies against F and M in the serums of three hyperimmunized and seven convalescent cattle when a conventional test was employed but with temporary hypertonization of the serum component alone. The present report describes studies on a larger series of convalescent and vaccinated cows and presents the procedure in greater detail.

Structure of the influenza virus; the relation between biological activity and chemical structure of virus fractions.

Influenza virus elementary bodies can be disintegrated by treatment with ether with the liberation of two types of smaller particle, soluble antigen and red cell agglutinin.Soluble antigen derived from the elementary body is identical in serological and chemical behaviour with soluble antigen recovered from infected tissues. Its chemical properties are those of a ribonucleoprotein.The agglutinin is an enzyme, with a protein part carrying the enzymic activity and a combining group with affinity for red cells. The agglutinin does not react in complement-fixation tests by the short fixation technique, but by the use of prolonged fixation or by indirect complement fixation it can be shown to contain a strain specific antigen and also a non-specific antigen. The specific antigen is identical with the specific complement-fixing antigen demonstrable in the intact elementary body, and has combining affinity for red blood cells. The non-specific antigen is probably similar to the protein part of the soluble antigen. No evidence has been found that the agglutinin contains either carbohydrate or nucleic acid.From the ether used to disintegrate the elementary body a serologically active lipid can be recovered which has properties suggesting that it is derived from the host cell.The author is greatly indebted to Dr L. Dmochowski for assistance in experiments involving high-speed centrifugation, to Sir Macfarlane Burnet and Dr G. L. Ada for supplies of receptor-destroying enzyme, to Dr E. S. Duthie for crystalline trypsin and chymotrypsin, and to Dr R. R. Porter for a sample of crystalline ribonuclease.

The Indirect Complement Fixation, Hemagglutination and Conglutinating Complement Absorption Tests for Viruses of the Psittacosis-Lymphogranuloma Venereum Group

Summary Three new tests for demonstrating antibody against viruses of the psittacosis-lymphogranuloma venereum group are described. These are the indirect complement fixation test, which detects inhibitory antibody for these viruses in the sera of chickens, the hemagglutination inhibition technique for testing human and chicken sera and the conglutinating complement absorption test for use with sera from patients. The possible application of the indirect complement fixation and agglutination inhibition tests to the study of infection with these viruses in avian populations is discussed. The relative merits and demerits of the three tests as compared with the ordinary complement fixation test are also reviewed. Theoretical considerations regarding the mechanism of the indirect complement fixation test, the nature of the meningopneumonitis hemagglutinin and the inhibition of hemagglutination by calcium ion are discussed.

The conglutination phenomenon; further experiments on the importance of the choice of complement when examining antisera for the presence of complement-fixing or complement-absorbing antibodies.

1. Anti-mallein sera produced in thirteen species of mammals (including man) and one species of bird have been examined by the haemolytic complement fixation test using guinea-pig complement, and by the conglutinating complement absorption test using pig, horse and cat complements. Two methods of fixation have been used and compared, namely, fixation for half an hour at room temperature and fixation overnight at 4° C.2. The complement of the horse proved to be the most sensitive in nearly every case in demonstrating the specific antibodies in the different sera; in the few instances where it was not markedly the most sensitive it was for practical purposes the equal of any other complement used, independent of the method of fixation. This is clearly indicated in the text figures.3. The effects of overnight fixation on the titres obtained with the different complements are discussed.4. The sera of six fowls inoculated with mallein failed to show fixation of any of the four complements in the presence of the homologous antigen. The presence of immune bodies was demonstrated however by the Indirect Complement Fixation technique of Rice (1948b).