Production Of Complement-fixing Antibodies Research Articles

Complement regulatory protein Crry deficiency contributes to the antigen specific recall response in experimental autoimmune myasthenia gravis

BackgroundMyasthenia gravis (MG) and animal model of experimental autoimmune myasthenia gravis (EAMG) is the most common autoimmune disorder of neuromuscular transmission. The disease is caused by the breakdown of the acetylcholine receptor (AChR) which is largely due to complement activation at the neuromuscular junction (NMJ). Limited knowledge exists to the extent that complement receptor 1-related gene/protein y deficiency (Crry −/−) modulates the adaptive immune response and EAMG outcome.MethodsMouse EAMG was induced by s.c. administrations of purified acetylcholine receptor (AChR) to Crry −/− and age- matched WT (C57BL/6) mice. Disease severity was assessed by clinical score assessment and muscle grip strength measurements. Serum complement activity was determined by hemolytic assay. ELISA was used to detect the level of AChR specific antibodies. Splenic cells were analyzed for T and B cells subsets distribution, release of cytokines and AChR specific recall responses. Deposition of complement components at the NMJ was assessed by immunofluorescence staining.ResultsIn comparison to WT EAMG, Crry −/− EAMG mice showed signs of augmented muscle weakness but differences, except for one time point, were not statistically significant. Serum complement activity was reduced in Crry −/− EAMG mice and no substantial changes in deposition of C3, C3b/iC3b and C5b-9 (MAC) at the NMJ between WT EAMG and Crry −/− EAMG mice were detected. Lack of Crry affected adaptive immune response. Crry −/− EAMG mice showed increases in the number of AChR specific splenic T-cells secreting IFN-γ and IL-4. Production of complement fixing antibodies (IgG2b, IgG2c) was also augmented. More Th1, Th2 and Th17 cytokines were released into the bloodstream of Crry −/− EAMG mice.ConclusionsData suggest that Crry deficiency modulates the adaptive immune response in EAMG, but its effect on disease outcome is limited. This was due to the generally lower serum complement level caused by increased C3 turnover. Modulation of complement activity with soluble or membrane bound regulators of complement activity represents a potentially effective approach to modify autoimmune processes in MG and EAMG.

Immunological and therapeutic strategies against salmonid cryptobiosis.

Salmonid cryptobiosis is caused by the haemoflagellate, Cryptobia salmositica. Clinical signs of the disease in salmon (Oncorhynchus spp.) include exophthalmia, general oedema, abdominal distension with ascites, anaemia, and anorexia. The disease-causing factor is a metalloprotease and the monoclonal antibody (mAb-001) against it is therapeutic. MAb-001 does not fix complement but agglutinates the parasite. Some brook charr, Salvelinus fontinalis cannot be infected (Cryptobia-resistant); this resistance is controlled by a dominant Mendelian locus and is inherited. In Cryptobia-resistant charr the pathogen is lysed via the Alternative Pathway of Complement Activation. However, some charr can be infected and they have high parasitaemias with no disease (Cryptobia-tolerant). In infected Cryptobia-tolerant charr the metalloprotease is neutralized by a natural antiprotease, α2 macroglobulin. Two vaccines have been developed. A single dose of the attenuated vaccine protects 100% of salmonids (juveniles and adults) for at least 24 months. Complement fixing antibody production and cell-mediated response in vaccinated fish rise significantly after challenge. Fish injected with the DNA vaccine initially have slight anaemias but they recover and have agglutinating antibodies. On challenge, DNA-vaccinated fish have lower parasitaemias, delayed peak parasitaemias and faster recoveries. Isometamidium chloride is therapeutic against the pathogen and its effectiveness is increased after conjugation to antibodies.

Cryptobia (Trypanoplasma) salmositica and salmonid cryptobiosis.

Salmonid cryptobiosis is caused by Cryptobia (Trypanoplasma) salmositica. The haemoflagellate has been reported from all species of Pacific Oncorhynchus spp. on the west coast of North America. It is normally transmitted by the freshwater leech, Piscicola salmositica, in streams and rivers, and sculpins, Cottus spp., are considered important reservoir hosts. The pathogen can also survive on the body surface of fish because it has a contractile vacuole to osmoregulate when the fish is in fresh water. This allows for direct transmission between fish, especially in aquaculture facilities. The parasite divides rapidly by binary fission in the blood to cause disease, the severity of which is directly related to parasitaemia. Cryptobia salmositica has a mitochondrium and it normally undergoes aerobic respiration; however, if its mitochondrium is damaged it will switch to glycolysis. Its glycolytic enzymes and catalase are contained in glycosomes. Cysteine protease is a metabolic enzyme, and its neutralization inhibits oxygen consumption and multiplication of the parasite. An important virulent factor in cryptobiosis is a secretory metalloprotease. The protective mechanism involves production of complement fixing antibodies, phagocytosis by macrophages, and cell-mediated cytotoxicity. Recovered fish are protected, probably for life as the immunity is non-sterile. Clinical signs of the disease include anaemia, anorexia, splenomegaly, general oedema and abdominal distension with ascites. The metabolism and swimming performance of infected fish are significantly reduced and the bioenergetic cost of the disease is very considerable. Fish are susceptible to hypoxia and their immune system is depressed during acute cryptobiosis. Severity of the disease and mortality rates vary significantly between species and stocks of salmon. Protective strategies include selective breeding of Cryptobia-resistant fish. This is innate resistance to infection and it is controlled by a dominant Mendelian locus. In these fish the parasite is lysed via the alternative pathway of complement activation. In Cryptobia-tolerant fish (infected with the pathogen but which do not suffer from disease) the metalloprotease secreted by the parasite is neutralized by alpha2 macroglobulin. Hence, the production of a transgenic Cryptobia-tolerant salmon is an option. This strategy has the advantage in that human intervention (e.g. vaccination, chemotherapy) is not required once the transgenic fish is produced. Acquired immunity is another option; a single dose of the attenuated live vaccine protects fish for at least 2 years. The protective mechanism in vaccinated fish is similar to that in recovered fish. The trypanocidal drug, isometamidium chloride, is an effective therapeutic and prophylactic agent. It accumulates in the mitochondrium of the parasite and significantly disrupts aerobic respiration by causing lesions in the organelle. Efficacy of the drug is significantly increased after its conjugation to antibodies. This immuno-chemotherapeutic strategy has the advantage in that it will lower the drug dosage and hence side-effects of chemotherapy. It will probably reduce the accumulation of the drug in fish, an important consideration in food fish.

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Impaired humoral responses to subgenus D adenovirusenovirus infections in HIV-positive patients

HIV-positive patients are at increased risk of developing adenovirus infection, particularly of the gastrointestinal tract and with unusual subgenus D strains. To investigate humoral immunity to these strains of adenoviruses, the humoral immune response was examined in longitudinal samples of serum against isolates collected from a prospective study of HIV-positive patients with subgenus D adenovirus infection. Of 10 HIV-positive patients developing adenovirus infection, 3 had chronic infection (8->27 months) with one serotype, 3 had chronic infection (>/=10 months) with changing serotypes and 4 had acute and self-limiting adenovirus infection (<1 month). Fifty-one sera were tested, and samples collected before adenovirus infection were available in 8 patients. Neutralising assays were performed against the patient's own isolate (adenoviruses 9, 17, 19, 19/23, 19/37, 23, 26, 23/26, 43 and 46) and common circulating strains of adenovirus 1-5. Indirect immunofluorescence tests were carried out against the autologous isolate and complement-fixation tests were undertaken using a standard assay. Immunofluorescence test antibodies were detected (titre >/=160) in all patients, and present to high titre (>/=320) in 8/10 patients. Complement-fixing antibodies were not detected in significant titre. Of particular note, there was no significant neutralising antibody response to the patient's own isolate after acute infection. Neutralising antibody to adenovirus 3 (titre 20) was transiently detected in two patients. In the remaining patients neutralising antibody directed against adenoviruses 1-5 was not detected. Persistent carriage of subgenus D adenoviruses in HIV-positive patients is probably the result of failure of cell-mediated immune responses to clear primary infection. Nevertheless, there is marked impairment of B cell responses resulting in poor neutralising and complement-fixing antibody production even though immunofluorescence test determined antibodies are produced in high titre. These possibly reflect impairment of effective B cell priming mechanisms within the germinal centres of lymph nodes, or the polyclonal activation of B cells driven by HIV infection.

In vitro and in vivo effects of rabbit anti-thymocyte serum on circulating leucocytes and production of complement fixing antibodies in thymectomized Oncorhynchus mykiss (Walbaum) infected with Cryptobia salmositica Katz 1951.

Rabbit anti-thymocyte serum (RATS) against thymocytes of rainbow trout was toxic to leucocytes from intact and thymectomized rainbow trout at 10 °C under in vitro conditions. The total number of leucocytes decreased significantly in 24 h after RATS was injected intraperitoneally into intact rainbow trout, but the number returned to pre-injection level within 1 week. RATS destroyed a lower percentage of leucocytes in thymectomized fish than in intact fish under both in vitro and in vivo conditions and the recovery in the number of leucocytes was slower in thymectomized fish. The parasitaemia, packed cell volume and production of complement fixing antibody in thymectomized and intact fish (injected with RATS before Cryptobia salmositica infection) were not significantly different from control fish (not injected with RATS), and they both acquired protective immunity against cryptobiosis on recovery. This indicates that RATS is not cytotoxic to B-like cells in the lymphoid tissue which produce complement fixing antibody against C. salmositica and that the protective antigen in C. salmositica seems to be thymus-independent.

Vaccination of rainbow trout, Oncorhynchus mykiss (Walbaum), against cryptobiosis: efficacy of the vaccine in fresh and sea water

Adult rainbow trout, Oncorhynchus mykiss (Walbaum), maintained in either fresh or sea water were vaccinated with a live Cryptobia salmositica vaccine. All vaccinated fish were protected 4 weeks later against the cryptobiosis, while unvaccinated rainbow trout developed the disease (e.g. high parasitaemia and severe anaemia) after challenge with virulent C. salmositica. There was also no disease in vaccinated fish when they were transferred from fresh to sea water immediately after vaccination. Complement fixing antibodies (CFAbs) were detected in vaccinated fish and the CFAbs lysed parasites under in vitro conditions. The antibody titres increased rapidly at one week post‐challenge in vaccinated fish in fresh water and vaccinated fish transferred from fresh water to sea water after vaccination. However, the production of CFAbs was delayed by one week in vaccinated fish in sea water and the antibody titre was significantly lower than that in fish maintained in fresh water.

Studies on the induction of anterior chamber-associated immune deviation (ACAID). II. Eye-derived cells participate in generating blood-borne signals that induce ACAID.

Despite an ever increasing body of information concerning general in vivo immune reactivity to exogenous Ag, relatively little is known concerning regional tissue influences on these responses. One expression of regionally distinct immune regulation is the deviant form of systemic immunity induced after the introduction of Ag, such as BSA, into the anterior chamber of the eye--anterior chamber-associated immune deviation (ACAID). ACAID is characterized by a selective, systemic immune deficiency in which both Ag-specific delayed hypersensitivity (DH) and complement-fixing antibody production are impaired by populations of Ts lymphocytes, although other immune effector modalities are preserved. Recent evidence demonstrates that the blood of mice that receive an anterior chamber injection of BSA 48 h earlier contains leukocytes capable of inducing cell-mediated Ag-specific efferent suppression of DH similar to that seen in mice with ACAID. Additional analysis of these bloodborne suppression-inducing leukocytes revealed them to express the mature macrophage/monocyte marker F4/80. On the basis of these findings, we wished to determine the source of the ACAID-inducing cells present in the peripheral blood of mice after anterior chamber inoculation of BSA. The data reveal that cells harvested from the normal eye are capable of selective induction of DH suppressive activity similar to that seen in ACAID. Interestingly, this property is unique to F4/80 expressing cells of the iris and ciliary body (I/CB). F4/80- I/CB cells do not possess this property. Although the F4/80 expressing cells that are present in extraocular sites do not constitutively possess ACAID-inducing properties, exposure of these cells to the anterior chamber microenvironment endows them with this ability. Finally, I/CB cells exposed to Ag in situ, via direct anterior chamber Ag inoculation, induce suppressed Ag-specific DH reactivity when infused into naive syngeneic recipients--a form of systemic immune deviation similar to that seen in mice with ACAID. The results presented here represent the first in vivo description of APC, altered by factors in a local microenvironment, that can initiate a systemic immune response characterized by Ag-specific immune suppression.

Epstein-Barr Virus Infection and Antibody Synthesis in Patients With Multiple Sclerosis

We studied infectious and immune mechanisms in demyelinating disease. The clinical diagnosis in this study of 313 consecutive cases of multiple sclerosis (MS) was based on the clinical conclusions of two or more neurologists and definite abnormalities in CSF IgG. Measurement of antibodies to six microbial agents was compared in 313 patients with MS and 406 controls in the same age range. Using a standardized immunofluorescent antibody (IFA) technique, we found a significantly higher prevalence of Epstein-Barr virus (EBV) infection and a higher level of serum viral capsid antigen IgG antibody titer in the MS population than in the controls. The MS population had a lower cytomegalovirus (CMV) infection rate and lower CMV complement fixing antibody production than controls. Except for the higher measles infection rate and antibody titer in patients with MS, data on the other viruses did not differ from controls.

Immunoregulatory Responses in Experimental Disseminated Histoplasmosis: Lymphoid Organ Histopathology and Serological Studies

To study immunoregulatory mechanisms in systemic histoplasmosis, a highly reproducible model of infection was established in C3H/Anf mice. Intravenous inoculation of 6- to 8-week-old mice with from 5 x 10(5) to 10 x 10(5) cells of yeast-phase Histoplasma capsulatum strain G-217B produced disseminated infection that resolved over an 8-week period without therapeutic intervention. Serological studies demonstrated complement-fixing antibody production to yeast- and mycelial-phase antigens of H. capsulatum. Complement-fixing antibody to the former was detected at week 1, and it peaked at week 3 and declined thereafter. Complement-fixing antibody to mycelial-phase antigen(s) appeared later (week 3) and did not peak until week 18. Grossly, the spleens of infected mice were enlarged from three to four times normal size during peak infection, whereas the thymuses were markedly involuted. Conversely, at week 8 the average spleen size was considerably smaller and the thymic mass was increased relative to the mass at week 3. Histopathologically, the paracortical regions of lymph nodes and the white pulp (periarteriolar lymphocyte sheaths) and marginal zones of the spleen were heavily infiltrated by granulomata at week 1. By week 8, the infiltrates in these areas had largely resolved. Thymocytes were severely depleted from the cortical lobules of the thymus at week 1; however, thymic cellularity was restored by week 8. These reciprocal changes in cellularity of the thymus and spleen during active infection may be of importance with reference to the disturbances of immunoregulation that we have observed in Histoplasma-infected mice.

Immunological relationship between delta herpesvirus of patas monkeys and varicells-zoster virus of humans

The Delta herpesvirus (DHV) which produced outbreaks of exanthematous disease in patas monkeys was shown to have a close immunological relationship to varicella-zoster (V-Z) virus of man. Immunization of rhesus monkeys with DHV or V-Z virus resulted in the development of neutralizing antibodies to both viruses and also in the production of complement-fixing antibodies to V-Z virus. Immunoglobulin M neutralizing antibody to V-Z virus was demonstrated in the serum of a rhesus monkey immunized with DHV, suggesting a primary antibody response rather than recall of antibody to a related virus. Convalescent-phase sera from human zoster cases had comparable levels of neutralizing antibody for both DHV and V-Z virus. Patas monkeys involved in an outbreak of DHV infection showed seroconversion to both DHV and V-Z virus by neutralization tests and to V-Z virus by complement fixation tests. The demonstration of the close antigneic relationship between DHV and V-Z virus suggests that DHV may be useful in an animal model system for studies on the latency and reactivation of V-Z virus.

DEVELOPMENT OF A VACCINE AGAINST MENTAL RETARDATION CAUSED BY CYTOMEGALOVIRUS INFECTION IN UTERO

Abstract Cytomegalovirus infection in utero is an important cause of mental retardation. A live tissue-culture-adapted strain of the virus was tested in volunteers. The subcutaneous route of inoculation was successful in stimulating neutralising and complement-fixing antibody production without important side-effects. It is suggested that the use of such a vaccine in adolescent girls would reduce the incidence of primary cytomegalovirus infection in pregnancy and thus eliminate fetal brain damage due to this cause.

Inapparent congenital cytomegalovirus infection with elevated cord IgM levels. Casual relation with auditory and mental deficiency.

Abstract A longitudinal clinical, virologic and immunologic study found 18 patients with inapparent congenital cytomegalovirus infection among 267 neonates with elevated umbilical-cord IgM levels. Virus excretion persisted in most patients through the third year of life, and the concomitant antigenic stimulation resulted in an accelerated development of immunoglobulins M and G and in continued complement-fixing antibody production in 14. Some degree of sensorineural hearing loss occurred in nine of 16 patients tested as compared with two of 12 controls, and, in four, an auditory handicap was either proved or considered likely. A trend toward subnormal intelligence was observed in the infected children, and two manifested definite mental and social disability. Cytomegalovirus infection probably has an important causal role in mild to moderate auditory and mental dysfunction in childhood. (N Engl J Med 290:291–296, 1974)

DEVELOPMENT OF A VACCINE AGAINST MENTAL RETARDATION CAUSED BY CYTOMEGALOVIRUS INFECTION IN UTERO

Cytomegalovirus infection in utero is an important cause of mental retardation. A live tissue-culture-adapted strain of the virus was tested in volunteers. The subcutaneous route of inoculation was successful in stimulating neutralising and complement-fixing antibody production without important side-effects. It is suggested that the use of such a vaccine in adolescent girls would reduce the incidence of primary cytomegalovirus infection in pregnancy and thus eliminate fetal brain damage due to this cause.

Complement-fixing antibodies produced in guinea pigs immunized with purified N and H antigens of poliovirus.

Highly purified poliovirus possessing N reactivity (N antigen) and H antigen which was prepared by heating the former, were used to pursue the production of complement-fixing antibodies in guinea pigs. By four successive intraperitoneal injections of approximately 1.0μg of viral protein of the N antigenicity, both N and H antibodies were produced in all guinea pigs tested. By the same procedure and the same amount of viral protein of the H antigenicity, the animals produced only H antibody. However, titers of H antibody were greatly varied from animal to animal in contrast with those observed in the animals injected with N antigen. Practical purpose to obtain mono-reactive N or H antisera was accomplished by using absorption technique. and

The association of murine lymphoma with reovirus type 3 infection.

SummaryThe development of a thymic lymphoma 4261/T in a mouse suffering from the chronic reovirus type 3 disease is reported. Transplantation of the tumor resulted in macroscopic lymphoma masses in the kidneys and/or jaw area. Low levels of reovirus antigen were found in the early transplantation passages. Antibody to reovirus was not detected in tumor-bearing mice from these passages. Mice from transplantation passages 12 and 13 showed evidence of reovirus type 3 infection with concomitant production of complement-fixing antibody to reovirus. Tests for reovirus in subsequent transplantation passages have proved negative. There has been no evidence to date for association of murine leukemia virus with lymphoma 4261/T.

Use of guinea pig eye in study of intraocular infections produced by mumps virus.

Conclusions(1) When a mixture of mumps virus with specific immune serum was injected into the eyes of guinea pigs, the serum completely prevented the corneal reaction and the production of complement fixing antibodies. Pooled sera collected from normal mumps susceptible children, when mixed with mumps virus and injected into the eyes of guinea pigs irregularly prevented the corneal reaction and the production of complement fixing antibodies. (2) Subsequent to the injection of mumps virus into the eye of the guinea pig, complement fixing antibodies appear in the convalescent sera between the 4th and 8th day, persist at significant levels until the 20th to 24th days, and then decline. (3) Mumps virus persists or grows in guinea pig eyes for at least 8 days after inoculation. (4) Chick embryo adapted strains of mumps virus recently recovered from parotid tissue, saliva and testicular fluid when injected into guinea pig eyes produce the corneal reaction, and complement fixing antibodies were demonstrated in t...

IMMUNITY IN MUMPS

Since the experimental data have already been discussed at some length, it is here necessary only to review those observations which appear to be of most significance. The manifestations of the disease we have produced in monkeys have in general been similar to those described by previous workers. It has been found that members of the species M. mulatta are regularly susceptible to infection with the virus of mumps, provided they have not been in contact with animals which have recently been infected. Such contact may be followed by the development of resistance to inoculation unattended by overt signs of infection. Following the intraparotid injection of virus into a susceptible animal, an antigen appears in the gland which reacts specifically in the complement fixation test with sera of monkeys which have recovered from an attack of mumps. This antigen has not been demonstrated in any other organ of the infected monkey which has been examined, nor does it persist or increase in amount when introduced into the brain or the testes. Attempts to propagate it in the tissues of other animal species including the developing hen's egg and tissue cultures have so far been uniformly unsuccessful. The antigen is relatively thermostabile, resisting a temperature of 65 degrees C. for at least 20 minutes at the optimal hydrogen ion concentration. It is unimpaired by a concentration of formalin of 0.3 per cent. Together with the virus it may be preserved indefinitely in the frozen state. The maximal quantity of antigen in the gland, which may be considerable, is attained on or about the 5th day following inoculation of the virus. An antibody which reacts with the antigen appears in the serum from 8 to 14 days after infection. This antibody has not been found in the serum of normal monkeys which have had no opportunity for contact with the virus. It has been demonstrated during convalescence in the serum of all animals which have been examined. The antibody which is present in large amounts soon after recovery, although decreasing in concentration with the lapse of time, may continue to be present for many months. At 62 degrees C. for 20 minutes its activity is not reduced and on storage at 4 degrees C. its titer has remained unaltered for over 2 years. A similar factor which appears in human beings convalescent from mumps is less stabile, since its activity is reduced appreciably under these conditions. In the CO(2) cabinet, however, both antibodies can be preserved for long periods of time. The definition of these reagents has afforded means whereby the simian infection with the virus of mumps, whether it is actively in progress or has occurred at some time in the past, can be recognized. Upon the establishment of these facts, the estimation of the quantity of antigen in the glands of vaccinated animals was employed as an index of their resistance in studies on the effect of the injection of formolized and alumprecipitated formolized suspensions of infected parotid gland. It has been shown that such vaccines, in which the virus has been rendered inactive, lead to the production of complement-fixing antibody. In about 60 per cent of the vaccinated animals, moreover, evidence of increased resistance was obtained as indicated by the partial or complete suppression of the formation of antigen in the inoculated gland. In certain instances no evidence of resistance was observed, yet antibody was found to be present. It is clear, therefore, that resistance is not determined solely by the presence of the complement-fixing antibody. The possibility, however, of its participation as a factor in immunity cannot be excluded. Employing the same criterion of resistance, the virus-neutralizing capacity of a pool of human mumps convalescent sera and of a globulin concentrate prepared from the pool has been studied. Neither of these materials was found to prevent entirely the formation of antigen, although each of them appeared to exert some inhibitory effect.

PRODUCTION IN MONKEYS OF COMPLEMENT-FIXING ANTIBODIES WITHOUT ACTIVE IMMUNITY BY INJECTION OF KILLED PLASMODIUM KNOWLESI

Injection into rhesus monkeys of Plasmodium knowlesi killed by heat, formalin, drying, or freezing and thawing stimulates the production of complement-fixing antibodies, but no demonstrable agglutinating or protective antibodies are formed. Possible differences in the immunity mechanisms concerned in active infection and in artificial immunization are discussed.

The Antigenic Properties of Globin, with a Note on the Independence of the Properties of Serum and Tissue Proteins, as Exemplified by the Absence of Antibody from the Globin of an Immunised Animal

Summary Globin can act as an antigen. In addition to two antisera for guinea-pig's globin, two antisera for ox globin have also been obtained from rabbits. With these antisera and a number of different specimens of the homologous globins powerful complement-fixation reactions have been obtained. The obtaining of the reaction depends on suitable quantitative relationships between antigen and antibody and also, as was shown previously, on a suitable hydrogen-ion concentration. Only certain rabbits apparently respond to injections of globin by the production of complement-fixing antibodies. The injections of globin caused no obvious toxic effects in rabbits. Hemoglobin seems to elicit antibody production more rarely. The reactions with antisera show, in certain cases, marked species-specificity of globin; thus the antiserum to guinea-pig's globin does not fix complement with ox globin. On the other hand, anti-ox globin fixes complement along with goat, duck and guinea-pig globin but not with rabbit globin; no explanation is offered of the contradictory behavior of ox and guinea-pig globins in the crossed experiments. But the results taken together seem to exclude bacterial contamination as the cause. The evidence points to the phenomena being due to a genuine antibody to globin and not to adventitious protein contamination. Further recorded facts (namely, that anti-guinea-pig globin does not contain hemolytic immune body for guineapig corpuscles and does not react with guinea-pig's hemoglobin, serum or acid serum-albumin, and that guinea-pig globin does not react with antihuman serum) also exclude the probable contaminations which might arise in the course of preparation of the globin solutions.