Studies on the neutralization of herpes simplex virus. X. Demonstration of complement-requiring neutralizing (CRN) and slow-reacting CRN (s-CRN) antibodies in late IgG.

A sample of late IgG from a rabbit hyperimmunized with herpes simplex virus was analyzed for neutralizing (N) and complement-requiring neutralizing (CRN) antibodies. In a usual endpoint test, N and CRN titers were 1: 40 and 1: 160, respectively, but when virus-IgG mixtures were incubated at 0 C overnight before addition of complement (C), an endpoint of 1:1280 was obtained. Virus sensitized at 0 C overnight required more C for inactivation than did sensitized virus formed earlier. Sensitization kinetic curve experiments employing a proper initial virus concentration, which permitted differentiation of sensitized viruses requiring different amounts of C, indicated that formation of sensitized virus detectable only with a relatively large amount of C proceeded slowly at IgG dilutions where the ordinary CRN antibody requiring a smaller amount of C was negligible. The results strongly suggested that the IgG sample contained slow-reacting CRN (s-CRN) antibody in excess of the hitherto known CRN antibody. As to the mechanism of formation of s-CRN complexes, experiments failed to prove the occurrence of complexes initially insensitive to C, and it appears more likely that s-CRN antibody has a comparatively low avidity for virus.     

Studies on the neutralization of herpes simplex virus. XI. Differences between slow-reacting complement-requiring neutralizing (s-CRN) antibodies in IgG and IgM

Late IgG and IgM from a rabbit immunized with herpes virus were tested for ordinary neutralizing (N) antibody, complement-requiring neutralizing (CRN) antibody and in addition CRN antibody detectable by overnight sensitization at 0 C (s-CRN antibody). Heat stability tests showed that IgG s-CRN antibody was slightly less resistant to heating at 70 C than were N and CRN antibodies, whereas all three activities of IgM were quickly degraded at this temperature. Dose-response curves with varying amounts of complement (C) or anti-antibody revealed a marked difference between IgG s-CRN and IgM s-CRN antibodies. While 1-hr sensitization at 37 C was insufficient to detect IgG s-CRN antibody, it had the same effect as overnight sensitization at 0 C for IgM s-CRN antibody. When sensitization at 0 C was prolonged to 3 days, unexpectedly high endpoints exceeding 1:10,000 were obtained even with IgM. consequently, enhancement by C was several hundred-fold with IgM in contrast to 5- to 10-fold enhancement of IgG s-CRN antibody, which was similar to that after overnight sensitization. Also IgM obviously required more C than did IgG. These results suggest that IgM of late serum is slower reacting and more C-dependent than IgG s-CRN antibody. Tests with early rabbit sera indicated that the s-CRN antibody detectable by 3-day sensitization reaches a high level before the appearance of N antibody.     

Studies on the neutralization of herpes simplex virus. VIII. Significance of viral sensitization for inactivation by complement.

Early and late IgG of rabbits immunized with herpes virus showed, respectively, 8-fold and 2-fold enhancement of neutralization endpoint in the presence of complement (C). Kinetic curve experiments employing an appropriate amount of virus revealed that both neutralization and sensitization followed first-order reaction, and each IgG possessed a certain range of concentration where neutralization was negligible while sensitization was marked. Dose responses of neutralization and sensitization velocities demonstrated that the C enhancement of late IgG was about 7-fold and that of early IgG more than 20-fold. These facts suggested that the IgGs contained two different entities of complement-requiring (CRN) and non-requiring neutralizing (N) antibodies at different proportions, only the former being responsible for sensitization. The different CRN: N ratios obtained by the endpoint and kinetic methods may mean either that the two antibodies differ in avidity for the virus or that the number of critical sites per virion for CRN antibody is greater than that for N antibody. In this interpretation, sensitization by CRN antibody as well as neutralization by N antibody is thought to result from attachment of a single antibody molecule to the viral critical site. Alternative explanations, ascribing the mechanism of neutralization to steric hindrance of critical sites or to multiple hit of those sites by antibody, were denied by analyses of the present data.     

Complement requirement of neutralizing antibodies in different classes of immunoglobulin appearing in rabbits and guinea pigs after primary and booster immunizations with herpes simplex virus.

Rabbits and guinea pigs were immunized with herpes simplex virus and bled periodically. The sera were fractionated into slow IgG, fast IgG and IgM by DEAE-cellulose column chromatography, and complement-requiring (CRN) and nonrequiring neutralizing (N) antibody activities were estimated. In early sera of rabbits, the two IgG and IgM fractions possessed about equal CRN activities, although some animals showed a slightly lower activity in fast IgG. In guinea pigs, the early CRN activity resided mainly in slow IgS (7 S gamma2). The early IgG antibody of guinea pigs differed from that of rabbits in that it resembled IgM in resistances to heating at 70 C and to 2-merceptoethanol. The level of CRN IgM antibody in rabbits declined following a peak reached in 2 to 3 weeks, whereas such a decline was never observed in guinea pigs. N IgG antibody was developed a few weeks after the first immunization in rabbits and much retarded in guinea pigs. In both species, booster immunization quickly evoked N antibody in the two IgG fractions and also CRN IgM antibody, but in the case of rabbits the IgM antibody disappeared soon. It is concluded that IgG plays an important role in humoral immunity from the initial stage of the immunization course.     

Complement Requirement of Neutralizing Antibodies in Different Classes of Immunoglobulin Appearing in Rabbits and Guinea Pigs after Primary and Booster Immunizations with Herpes Simplex Virus

Rabbits and guinea pigs were immunized with herpes simplex virus and bled periodically. The sera were fractionated into slow IgG, fast IgG and IgM by DEAE-cellulose column chromatography, and complement-requiring (CRN) and nonrequiring neutralizing (N) antibody activities were estimated. In early sera of rabbits, the two IgG and IgM fractions possessed about equal CRN activities, although some animals showed a slightly lower activity in fast IgG. In guinea pigs, the early CRN activity resided mainly in slow IgG (7 S γ₂). The early IgG antibody of guinea pigs differed from that of rabbits in that it resembled IgM in resistances to heating at 70 C and to 2-mercaptoethanol. The level of CRN IgM antibody in rabbits declined following a peak reached in 2 to 3 weeks, whereas such a decline was never observed in guinea pigs. N IgG antibody was developed a few weeks after the first immunization in rabbits and much retarded in guinea pigs. In both species, booster immunization quickly evoked N antibody in the two IgG fractions and also CRN IgM antibody, but in the case of rabbits the IgM antibody disappeared soon. It is concluded that IgG plays an important role in humoral immunity from the initial stage of the immunization course.     

A differential effect of IgM and IgG antibodies on the blastogenic response of lymphocytes to rubella virus

Peripheral blood lymphocytes of rabbits immunized with live rubella vaccine respond to rubella virus antigens in tissue culture with increased DNA synthesis as measured by incorporation of ³H-thymidine. This reaction can be inhibited by rubella antibody. A dose dependent effect was observed when antibodies in whole serum were mixed with virus prior to addition to lymphocyte cultures. When antisera were fractionated and their individual immunoglobulins tested, a paradoxical effect was obtained. Immune IgG although it was highly effective in neutralizing the virus was incapable of inhibiting the lymphocyte response and at times caused an increased response. In contrast, immune IgM which was less efficient in neutralizing virus caused significant suppression of the blastogenic reaction. By themselves these results might have signified that IgG and IgM antibodies have different specificities or different binding properties with respect to viral surface antigens. However, immune complexes consisting of virus and IgM reduced response of both rubella immune and normal rabbit lymphocytes to PHA. This nonspecific inhibitory action required a specific step of antigen and IgM antibody interaction and normal IgM-virus mixtures or mixtures of anti-rubella IgM and poliovirus or influenza virus did not suppress lymphocyte response to PHA. Anti-rubella IgG complexed with rubella virus did not suppress the PHA response. The IgG function was apparently limited to neutralization of the infectivity of rubella virus whereas the major role of IgM was manifested through its suppressive effect on lymphocyte reactions.     

Complement-requiring neutralizing antibody in guinea pigs with primary and recurrent genital herpes

Guinea pigs inoculated intravaginally with herpes simplex virus type 2 (HSV-2) strain 1868 produced a serum complement-requiring neutralizing (CRN) antibody during primary acute infection, i.e., 10 days postinoculation. The CRN antibody titers in the guinea pig sera decreased to less than 1:10 after heating at 56 degrees C for 30 min. It was found that 32 units of complement were necessary to obtain a satisfactory HSV-2 neutralizing antibody titer. Nonheated sera significantly reduced virus infectivity titers when mixed with 3.5 log10 PFU of HSV-2 and incubated at 37 degrees C for 20 to 60 min (P less than 0.001), whereas the same sera after heating at 56 degrees C for 30 min showed no inhibitory effect. Only 27.3% of infected guinea pigs had low serum non-CRN antibody titers ranging from 1:20 to 1:40. In addition, no evidence of increase in CRN antibody titers was noted during spontaneous recurrent genital herpes infection.     

Effect of selective complement deficiency on the rate of neutralization of enveloped viruses by human sera.

The capacity of human sera genetically deficient in selective complement (C) components to enhance neutralization of enveloped viruses was examined by kinetic plaque reduction assays. Vaccinia virus, a DNA virus, and vesicular stomatitis virus (VSV), an RNA virus, were studied. Exogenous rabbit: or human antibody to vaccinia virus, and guinea pig or human antibody to VSV were provided in limiting, C-dependent concentrations. IgG antibodies predominated in most of the antisera employed. C5-deficient and C6-deficient human sera consistently supported normal rates of neutralization of either virus; this effect was heat-labile. C4-deficient human serum did hot exceed heat-inactivated serum in any neutralization assay. C1r-deficient serum displayed slight heat-labile neutralizing capacity against vaccinia but none against VSV. C2- and C3-deficient sera consistently exhibited measurable but clearly subnormal rates of neutralization. Two fresh agammaglobulinemic sera failed to inactivate either virus in the absence of added antibody. These results confirm and extend earlier evidence, based on neutralization of herpes simplex and Newcastle disease viruses in the presence of early (IgM) antibody and functionally pure guinea pig C components or C-deficient animal sera, that the late-acting components C5-C9 are not required for C-dependent neutralization. Data on four enveloped viruses now agree that this function is mediated by C1-C3, although C1 plus C4 appear to have some neutralizing capacity. This requirement for C1-C3 is overcome, however, in the presence of higher antibody cohcentrations, suggesting that the contribution of the C system to viral neutralization in vivo may be chiefly in the early phase of infection when antibody is limited.     

Defense mechanisms against primary influenza virus infection in mice. I. The roles of interferon and neutralizing antibodies and thymus dependence of interferon and antibody production.

To investigate the defensive roles and production of interferon and antibodies, C3H/He mice were subjected to various immunosuppressive treatments and infected with influenza virus. In infected normal control mice the pattern of pulmonary viral growth can be divided into three phases. The first phase is characterized by an exponential increase of virus titer, the second by a rapid decrease, and the third by a moderate decrease. At the time of transition from the first phase to the second in pulmonary virus growth, interferon could be detected in the tracheobronchial washings of infected mice, but neutralizing antibodies could not. In infected B cell-deprived mice and infected anti-mu-treated mice, the transition from the first phase to the second occurred without any detectable antibody production, and interferon could be induced in the early stage of infection. However, the pulmonary virus in these mice increased again exponentially until the death of the mice. In infected T cell-deprived mice which could not induce interferon, but produced IgM-neutralizing antibodies, the second phase was not observed after the first phase, but a transient plateau phase could be demonstrated, and then the pulmonary virus increased again exponentially until the death of the mice. In anti-gamma-treated infected mice, pulmonary virus growth and production of interferon and neutralizing antibody were almost similar to those of infected normal control mice except for the absence of IgG neutralizing antibody production. Although anti-alpha-treated infected mice produced interferon and no IgA antibody, the transition from the first exponential increase of pulmonary virus to the second rapid decrease was seen, but then the virus increased exponentially again until the death of the mice. These results suggest that interferon plays an important role in the transition from the first phase to the second, and that T cells are required for interferon induction in mice infected with influenza virus. These data also suggest that IgA antibodies play an important role in the inhibition of virus propagation in the lungs after the disappearance of interferon. Moreover, infected T cell-deprived mice could produce only IgM neutralizing antibodies, but not IgG and IgA antibodies. Therefore, T cells are required for the production of IgG and IgA antibodies and even     

Virus-neutralizing antibodies of immunoglobulin G (IgG) but not of IgM or IgA isotypes can cure influenza virus pneumonia in SCID mice.

The ability of monoclonal antibodies (MAbs) to passively cure an influenza virus pneumonia in the absence of endogenous T- and B-cell responses was investigated by treating C.B-17 mice, homozygous for the severe combined immunodeficiency (SCID) mutation, with individual monoclonal antiviral antibodies 1 day after pulmonary infection with influenza virus PR8 [A/PR/8/34 (H1N1)]. Less than 10% of untreated SCID mice survived the infection. By contrast, 100% of infected SCID mice that had been treated with a single intraperitoneal inoculation of at least 175 micrograms of a pool of virus-neutralizing (VN+) antihemagglutinin (anti-HA) MAbs survived, even if antibody treatment was delayed up to 7 days after infection. The use of individual MAbs showed that recovery could be achieved by VN+ anti-HA MAbs of the immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgG3 isotypes but not by VN+ anti-HA MAbs of the IgA and IgM isotypes, even if the latter were used in a chronic treatment protocol to compensate for their shorter half-lives in vivo. Both IgA and IgM, although ineffective therapeutically, protected against infection when given prophylactically, i.e., before exposure to virus. An Fc gamma-specific effector mechanism was not an absolute requirement for antibody-mediated recovery, as F(ab')2 preparations of IgGs could cure the disease, although with lesser efficacy, than intact IgG. An anti-M2 MAb of the IgG1 isotype, which was VN- but bound well to infected cells and inhibited virus growth in vitro, failed to cure. These observations are consistent with the idea that MAbs of the IgG isotype cure the disease by neutralizing all progeny virus until all productively infected host cells have died. VN+ MAbs of the IgA and IgM isotypes may be ineffective therapeutically because they do not have sufficient access to all tissue sites in which virus is produced during influenza virus pneumonia.     

Immunoregulatory role of Ig isotypes. I. Induction of contrasuppressor T cells for contact sensitivity responses by antibodies of the IgM, IgG1, and IgG3 isotypes

A profound state of specific tolerance for the contact sensitivity reaction can be produced by i.v. exposure to hapten on the surface of syngeneic macrophages. When the same haptenated cells are incubated with specific antibody to form cell-bound Ag-antibody complexes, i.v. injection induces immunity rather than tolerance. We observe that such cell-bound Ag-antibody complexes induce not only effector cells for contact sensitivity but also hapten-specific contrasuppressor T (Tcs) cells, which are capable of rendering effector cells resistant to the inhibitory effects of Ts cells. Whereas the induction of the effector cells of contact sensitivity by cell-bound complexes required I region compatibility between the injected cells and the recipient, the induction of Tcs cells showed no genetic restriction. On the other hand, induction of contrasuppression required intact Fc on the complexed antibody, inasmuch as F(ab')2 fragments of specific antibody did not induce immunity. In addition, Tcs cells could also be induced by Ag-antibody complexes on opsonized TNP-mouse RBC treated with anti-TNP antibody. Immunity induced by cell-bound Ag-antibody complexes was observed only when antibodies of the IgM, IgG3, or IgG1 isotypes are used to generate the complexes. Further studies demonstrated that the Tcs cells induced in this way displayed the phenotype of Tcs cells described in other systems (Lyt-1+,2- I-J+, Vicia villosa lectin-adherent) and released a hapten-specific contrasuppressor factor. These studies indicate that Tcs cells can be induced independently of other T cells (such as the effector cells of contact sensitivity) and are likely to be responsible for some of the immunoregulatory effects of cell-bound Ag-antibody complexes. The role of antibody isotype in the induction of Tcs cells is discussed.     

Influence of Complement on the Neutralization of Murine Cytomegalovirus by Rabbit Antibody

Antiserum against murine cytomegalovirus produced in the rabbit contained complement (C')-requiring neutralizing (CRN) antibody. The proportion of CRN was extremely high (up to 98%) during the early portion of an immunization procedure, whereas the antisera produced late had a much lower proportion that required C'. The antiserum produced was specific for MCMV with or without C'.     

A micro-neutralization system for detection of s-CRN antibody to herpes simplex virus

It was learned that the ordinary micro-neutralization system with herpes simplex virus (HSV) gave a composite result of the initial neutralization and the effect of antibody on subsequent growth of unneutralized virus. In the case of slow-reacting complement-requiring neutralizing (s-CRN) antibody, which was detected by incubating virus-serum mixtures at 4 C for 3 days before addition of complement, the titer obtained was lower than expected from the result of the plaque reduction test. This was thought ascribable to its low ability to prevent viral breakthrough caused by growth of unneutralized virus. This was overcome by adding an appropriate amount of hyperimmune antibody at 3 hr after addition of cells. The endpoint of s-CRN antibody so determined was but slightly lower than that obtained by the plaque reduction test. Early (1-week) rabbit sera, which were negative in the ordinary micro-neutralization test, titered 1:2,560 to 1:5,120 when tested by this method. When the 3-day sensitization in the cold was substituted by 5-hr incubation at 37 C, the titer obtained was 2 to 4-fold lower; in this case, however, the whole process could be finished within 3 days. Also, s-CRN antibody reactive with type 2 HSV in homologous and heterologous sera could be detected by the same method using type 1 hyperimmune serum as the additional antibody.     

A new microplate neutralization test for typing of herpes simplex virus.

A microplate serum neutralization test for estimation of complement-requiring neutralizing (CRN) antibody was established as the first step for simplification of typing of herpes simplex virus (HSV). When guinea pigs were immunized with type 2 HSV, the late sera could mostly differentiate the types of HSV better than hyperimmune rabbit sera, the CRN titer against the heterologous type 1 HSV being much lower than the homologous titer. Sera of guinea pigs immunized with type 1 HSV showed about the same level of cross reaction against type 2 HSV as did rabbit antisera. Guinea pig sera having minimal levels of cross reaction were selected, and their high dilution (1:160) and complement were added to serial 10-fold dilutions of virus in the microplate titration of virus infectivity. Selective reduction of virus titer by either antiserum could determine the type of HSV. No equivocal intermediate case was found among a number of stock strains including many fresh isolates. The typing result coincided with that determined by a modification of Yang et al's method based on virus titers obtained with Vero and primary chick embryo cells. The typing based on plaquing in chick embryo cells sometimes failed to identify type 1 HSV.     

Studies on the Neutralizing Antibody to Herpes Simplex Virus

The type-specific antibody responses in rabbits immunized with both types of herpes simplex virus (HSV), measured by complement-requiring neutralizing (CRN) and slow-reacting CRN (s-CRN) antibody assays were compared. Titers of type-specific antibody measured by s-CRN antibody assay were always markedly higher than those measured by CRN antibody assay. The s-CRN antibody assay was so sensitive that even an undetectable level of CRN antibody could be readily detected by this method. The detection of type-specific antibody by s-CRN antibody assay may be useful when attempting to analyze human HSV infection.     

Development and characterization of monoclonal antibodies to foot and mouth disease virus type 'C'

Five fusion experiments were conducted with spleen cells from Balb/c mice immunized with purified 146S antigen of foot and mouth disease virus type 'C' (vaccine strain). Monoclones (31) thus developed were isotyped as IgM (3), IgG1 (6), IgG2a (5), IgG2b (3) and IgG3 (14). Eleven clones isotyped as IgM, IgG2a and IgG2b showed neutralizing activity in virus neutralization and plaque reduction tests. Six of the neutralizing clones precipitated 146S virus in Ouchterlony reaction. On the basis of location of MAb reactive epitopes in relation to intact virus (146S), 12S particles and VP1 in ELISA test, the clones were classified as Class II (6), Class III (11) and Class IV (14). These clones may be useful for purposes of antigen detection from field isolates and for estimation of antibody titres in vaccinated animals.     

Neutrophil receptors for IgG and complement: their roles in the attachment and ingestion phases of phagocytosis.

The functional roles of IgG and C3b in phagocytosis by human peripheral neutrophils were investigated. Phagocytosis of Staphylococcus aureus in the presence of human serum was severely depressed by heat inactivation of serum at 56 degrees C for 30 min. Experiments with varying particle: leukocyte ratios in the presence of complement-inactivated sera showed that particle-bound C3b can mediate a 10-fold enhancement of the overall phagocytic rate. When sheep erythrocytes were sensitized with either IgG or IgM, only the former were bound to and readily internalized by neutrophils. Erythrocytes sensitized with both IgM and C3b were bound but not internalized. Furthermore, the presence of Fc fragments during incubation of S. aureus or latex beads with neutrophils in the presence of IgG or fresh serum affected a total inhibition of internalization but did not significantly alter adherence. Quantitative data regarding IgG sensitization indicated that bound C3b results in at least a 3-fold decrease in the amount of sensitizing IgG required for 50% maximal phagocytic response by neutrophils. On the basis of the above results, it is argued that particle-bound C3b functions primarily in the adherence phase and that bound IgG serves as a trigger for the internalization phase of phagocytosis.     

The temperature-dependent stoichiometry of mixed cryoimmunoglobulins

The interaction of three monoclonal rheumatoid factor IgM molecules with IgG antigens has been studied utilizing immunoglobulins isolated from three mixed cryoglobulins. Static light scattering measurements show that the stoichiometry of these immune complexes changes in a temperature-dependent manner from IgM(IgG)0-2 at temperatures greater than 37 degrees C to IgM(IgG)5 complexes at temperatures below 15 degrees C. These results were confirmed by the analysis of the composition of polyethyleneglycol-precipitated complexes. For one mixed cryoglobulin (Glo), temperature-dependent changes in stoichiometry were also verified by chemical cross-linking studies. Binding constants were determined by Scatchard analysis of light scattering data and by fluorescence polarization measurements. Values on the order of 10(5) M-1 were obtained for three monoclonal rheumatoid factor IgM molecules. Glo was further investigated by dynamic light scattering and partial specific volume measurements. Both dynamic light scattering and partial specific volume measurements provided evidence for surprising shape changes of the IgM X IgG complex as a function of temperature and IgG stoichiometry. Collectively, the data support the simple hypothesis that cryoprecipitation of mixed cryoglobulins occurs as a consequence of increases in the size (stoichiometry) of the complexes that are formed at low temperatures.     

Rapid production of neutralizing antibody leads to transient hepadnavirus infection

Hepatitis B virus (HBV) frequently causes transient infections in adults but chronic infections in infants. The basis of these age-related outcomes is not known. Infection of ducks with duck hepatitis B virus (DHBV) displays a similar dependence of outcome on the age of the host at the time of infection. In this study we compared the infection of ducks at 3 days and 3 weeks of age. We found that the efficiency of infection of hepatocytes by virus in the inoculum was similar between the two age groups but that spread of the infection throughout the liver was severely inhibited in the 3-week-old-old ducks, while a rapid spread of the infection was observed in 3-day-old ducklings. Inhibition of virus spread was accompanied by the appearance in the serum of virus neutralizing activity, as assayed by blocking of infection of primary hepatocyte cultures. Neutralizing activity appeared as early as 1 or 2 days postinfection and increased during the next 2 weeks. Depletion of immunoglobulins from serum eliminated the neutralizing activity. The specific depletion of IgM indicated that IgM appeared as the dominant fraction of neutralizing antibody in the first 2 days postinfection, but declined from day 3 on while IgG antibody rose. We conclude that excess neutralizing antibody arising rapidly in birds inoculated at 3 weeks of age but not in newly hatched ducks prevented secondary cycles of infection, resulting in a limited infection in the liver and contributing to the eventual transient outcome of the infection.     

A calcium-dependent cryoglobulin IgM kappa/polyclonal IgG.

The mechanisms responsible for cold-induced precipitation of mixed cryoglobulins are not well understood. A mixed cryoglobulin IgM kappa/IgG (type II) of a patient with Sj?gren's syndrome was studied because of its unique properties. This cryoglobulin precipitated in serum but not in serum containing 10 mM EDTA. The cryoprecipitation was shown to require calcium (Ca) and was optimal at 1 mM of free Ca. Cryoprecipitation was also induced by Ba, Mn, and Sr, but not by Mg and Co. Purified IgM kappa/IgG complexes precipitated in the presence of Ca, but not IgM kappa alone. There was no significant binding of 45Ca to the purified IgM kappa, IgM kappa/IgG complexes formed with purified components, and the cryoprecipitate. The relative affinity of the radiolabeled [125I]IgM kappa for IgG was 3.6 x 10(3) liters/mol at 37 degrees C as assessed by sucrose density gradient ultracentrifugation, and increased to 1.7 x 10(4) liters/mol at 4 degrees C. The addition of Ca produced no change in the affinity at 37 degrees C and 4 degrees C. The absence of a direct effect of Ca on the Ag/antibody reaction was confirmed in experiments using polyethylene glycol as precipitating agent. In conclusion, two independent steps were responsible for the precipitation of this cryoglobulin. The first step was an efficient formation of soluble immune complexes as produced by a drop in temperature. The second step was caused by a change in the physicochemical conditions--the presence of Ca--which induced polymerization of the IgM kappa/IgG complexes.