Membrane distribution and adsorptive endocytosis by C3b receptors on human polymorphonuclear leukocytes

C3b receptors on human polymorphonuclear leukocytes (PMN) were nonrandomly distributed in small clusters on the plasma membranes of these cells when assessed by indirect immunofluorescence at 0 degree C using monospecific rabbit Fab' or F(ab')2 anti-C3b receptor and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat IgG anti- F(ab')2. When PMN were incubated with the bivalent anti-C3b receptor at 37 rather than at 0 degree C, almost no immunofluorescence was observed, which indicates that the C3b receptor-F(ab')2 complexes had been rendered inaccessible to TRITC-IgG anti-F(ab')2. Endocytosis of the anti-C3b receptor ligand was quantitated by measuring the binding 131I-IgG anti-F(ab')2 by PMN that had previously taken up 125I-F(ab')2 anti-C3b receptor at 0 and at 37 degree C, respectively. There was a constant 2: 1 molar ratio of anti-F(ab')2 to anti-C3b receptor with PMN that had been incubated with the first antibody at 0 degree C. In contrast, when increments of F(ab')2 anti-C3b receptor were taken up by the cells at 37 degree C, there was a dose-related decline in this molar ratio to a minimum of 0.2 molecules of anti-F(ab')2 anti-F(ab')2 bound per molecule of PMN-associated anti-C3b receptor. 125I-F(ab')2 anti-C3b receptor taken up by PMN at 37 degree C was also inaccessible to release by proteolytic treatment of the cells with pronase. The rate of endocytosis of 125I-F(ab')2 anti-C3b receptor was rapid as the PMN- bound antibody fragment became inaccessible to 131I-IgG anti-F(ab')2 within 10 min during incubation of the cells at 37 degree C. In contrast to these findings, 125I-Fab' anti-C3b receptor that was taken up by PMN at 37 degree C remained accessible to both 131I-IgG anti- F(ab')2 and to proteolytic release by pronase, which suggests that monovalent interaction of ligand with C3b receptors was not sufficient for induction of endocytosis. The requirement for multivalency was also demonstrated using the C3b-OR, the normal ligand for the C3b receptor. 125I-C3b-OR was specifically bound by PMN but remained on the cell receptor. 125I-C3b-OR was specifically bound by PMN but remained on the cell surface, as determined by its accessibility to pronase, unless it was cross-linked with F(ab')2 anti-C3. Although C3b receptors on PMN do not mediate internalization of adsorptive pinocytosis of soluble ligand indicates their potential for the clearance of C3b-bearing immune complexes without recruitment of other cell surface receptors.     

Circulating immune complex-like materials which bind to heat inactivated C1q interfere with the C1q solid phase assay for immune complexes

C1q solid phase assay (C1q SP) was devised based on the fact that immune complexes (IC) and aggregated human globulin (AHG) bind to C1q. Neither IC nor AHG was found to bind to heat inactivated C1q. On the other hand, circulating immune complex (CIC)-like materials in patients' sera were able to bind to heat inactivated C1q, indicating that these CIC-like materials are not true CIC. Gel filtration analysis showed that molecular size of such CIC-like materials was almost the same as monomeric IgG, while true CIC were in heavy fractions. True CIC did not bind to heat inactivated C1q but bind only to native C1q. The CIC-like activity is not due to rheumatoid factors. About 2/3 of CIC positive sera by C1q-SP are not really CIC positive but are due to interference by the CIC-like materials.     

ELISA法检测荷有C3d的免疫复合物

将待测血清的聚乙二醇（ＰＥＧ）沉淀物加至已包被抗人ＩｇＧ的反应板微孔内，反应后再加入酶标记抗Ｃ３ｄ，建立了检测荷有Ｃ３ｄ免疫复合物的ＥＬＩＳＡ法。经对正常人、ＳＬＥ、肾病、肝病、冠心病患者检测，发现ＳＬＥ、肾病、肝病患者荷Ｃ３ｄ免疫复合物的检出率明显升高，对其意义进行了初步探讨。     

Binding of complement component C5 to model immune complexes and the use of anti-C5 antibodies for determination of C5-containing circulating immune complexes

When normal human or mouse serum is added to micro ELISA plates coated with monomeric or aggregated IgG, complement component C5 binds to IgG. C5 binding was demonstrated with a specific chicken anti-C5 antibody. Hydrazine treatment of the serum or addition of EDTA to the serum abolished the binding of C5. C5-deficient mouse serum was negative for C5 binding, whereas the same serum supplemented with human C5 restored the binding of C5. Chicken anti-C5-coated plates were used for determination of C5-containing circulating immune complexes (CIC). Increased concentrations of CIC were found in sera from patients with rheumatoid arthritis and Bell's palsy.     

Determination of complement activating circulating immune complexes and an example for antigen specific immune complex assays

For the screening of complement activating circulating immune complexes a new fluorescence linked immunosorbent assay was developed. Anti-human C3 or anti-human C4 F(ab')2 fragments were coupled to a nitrocellulose matrix. Nitrocellulose pieces of definite size covered with anti-C3 and anti-C4 were incubated with serum samples or standards for 15 min followed by a reaction with a fluorescence labelled anti-human IgG or anti-human-IgM. The nitrocellulose disks were then washed and the remaining fluorescence was read by a solid-phase fluorometer. The method was standardized by a WHO Tetanus toxoid anti-Tetanus immune complex reference substandard. (This standard and further controls were used to prove the reproducibility of the system.) Patients suffering from chronic inflammatory diseases were compared to healthy controls. The best discrimination between patients and controls was demonstrated by the determination of C3-IgG aggregates, followed by C3-IgM, C4-IgG, and C4-IgM aggregates. The easy performance, the stability of necessary chemical substances, the reliable standardization by a reference standard, and the clear-cut differences between patients and healthy control groups proved this test to be suitable for routine purposes. Furthermore, it could be demonstrated by means of an artificial model immune complex that this system can be expanded--by slight modifications--to antigen-specific CIC-assays. A CEA-anti-CEA CIC test is described as an example of an antigen specific CIC test not limited to complement activating CIC, and the data of 127 patients with colorectal carcinoma are given.     

Quantification of circulating immune complexes by chicken anti-C4 micro ELISA

A quantitative assay for C4-containing immune complexes (IC) by a solid phase anti-C4 micro ELISA is described. It is based upon the use of an affinity purified chicken anti-human C4 antibody to capture the immune complex, and protein A-alkaline phosphatase for detection. The chicken antibody was chosen as capture antibody because it does not react with rheumatoid factor, does not activate the human complement system and is not detected by anti-mammalian IgG antibodies or protein A. Increased levels of C4 containing circulating immune complexes (CIC) were detected in sera from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and lung cancer, when compared with normal sera. Normal levels of C4 containing immune complexes were found in sera from patients with Bell's palsy.     

Antigen/antibody content of circulating immune complexes in HIV-infected patients

Dual infection with HIV and hepatitis B virus (HBV) is not an uncommon feature. Immunity impairment due to HIV infection can be the cause of a higher rate of HBV replication with less intensive liver damage and less effective immune response to HBV. Many HIV-infected patients have an elevated level of circulating immune complexes (CIC) in serum, throughout all stages of illness evolution. The aim of our study was to estimate p24 and HBsAg content of CIC in dually infected patients, and the prevalence of major classes of complexed antibodies (IgM and IgG). We examined 146 samples of sera from 105 HIV positive patients of the Institute for Infectious and Tropical Diseases during 1992 and 1993. On those sera we performed p24Ag and HbsAg detection, with and without prior dissociation of CIC, we determined serum level of CIC and immunoglobulin classes IgM and IgG level in sera and in polyethilenglycol (PEG) precipitates of sera. Acid dissociation of immune complexes revealed a high proportion of HIV antigen positive sera in all stages of HIV disease progression. HbsAg in serum of HIV positive patients was also found coupled in immune complexes much more frequently than in the HIV negative control group. In many instances both antigens were simultaneously found coupled in CIC. Immune complexes detected have been shown to contain both IgM and IgG immunoglobulins, while IgM antibodies were associated to immune complexes in higher proportion than IgG, compared to total serum immunoglobulins.     

Anti-F(ab'')2 antibodies in thrombocytopenic patients at risk for acquired immunodeficiency syndrome.

22 homosexual or narcotic addict patients at risk for acquired immunodeficiency syndrome (AIDS) or with AIDS, were studied for the presence of antiimmunoglobulin antibodies and circulating immune complexes (20 were thrombocytopenic, 6 had AIDS). Circulating immune complex levels were 10-fold higher than levels in normal subjects. IgG anti-F(ab')2 antibodies were noted in homosexual as well as narcotic addict patients. Of 16 homosexual patients, 7 had IgG anti-F(ab')2 antibody of moderate to marked titer with broad reactivity against autologous, homologous, and control F(ab')2 fragments. Three others demonstrated limited reactivity against one or two F(ab')2 fragments. The remaining six patients were negative. Six of six narcotic addict patients had IgG anti-F(ab')2 antibody, five with limited reactivity, one with broad reactivity. In contrast, neither elevated circulating immune complexes nor anti-F(ab')2 antibodies were detectable in six autoimmune thrombocytopenic patients. Anti-F(ab')2 antibody could be affinity purified from serum or circulating immune complexes. Anti-F(ab')2 reactivity correlated with circulating immune complex levels, r = 0.83, P less than 0.01.     

Circulating immune complex levels measured by new ELISA kits utilizing monoclonal anti-C1q and anti-C3d antibodies correlate with clinical activities of SLE but not with those of RA

The authors studied sera from both patients with SLE and from those with RA to evaluate clinical usefulness and significance of circulating immune complexes (CIC) detected with new ELISA kits utilizing monoclonal anti-C1q and anti-C3d antibodies. CIC values of patients with SLE significantly correlated to severity of disease activities evaluated by clinical symptoms and laboratory tests, especially for serum complement levels. Since substances detected with the ELISA kits were closely related to serum complement components, it was determined that a direct relationship exists between clinical activities and CIC values appearing in SLE patients with hypocomplementemia. In RA patients, CIC values did not correlate to clinical activities evaluated by Lansbury's index, anatomical bone damage with X-ray or functional assessment of activities of daily living, but did significantly correlate to levels of IgM-RF, serum IgG concentrations and some markers of systemic inflammation. Detection of IC after fractionation of RA sera revealed a broad range of molecular sizes detectable with the ELISA kits, which indicated that CIC in vivo were heterogenic and complicated in formation, degradation and interaction with serum complements.     

Generation of a monoclonal antibody to mouse C5 application in an ELISA assay for detection of anti-C5 antibodies

We have generated a monoclonal antibody with specificity for the fifth component of mouse complement (C5). This antibody precipitates the two chains of C5 from normal mouse serum and inhibits C5-dependent hemolysis in a functional complement test. In this study we describe its application in an enzyme-linked immunoadsorbent assay (ELISA assay) for the detection of anti-C5 antibodies in serum. Monoclonal anti-C5 coupled to wells of an ELISA plate specifically binds C5 from unfractionated normal mouse serum. This subsequently serves as antigen to bind anti-C5 serum antibodies. By this approach we have circumvented the need for extensive purification of C5 from serum which would be required if C5 was directly coupled to ELISA plates as antigen. Serum antibodies from C5-immunized mice bound with high avidity to wells containing normal serum as antigen source in amounts representing 1 microgram to 250 ng C5. There was no antibody binding to wells containing C5-deficient serum as antigen source. The immune reaction was detected by development with enzyme-coupled goat-anti mouse Ig antibodies specific for various mouse Ig subclasses. This method allows the qualitative characterization of immune responses to mouse C5 which is an ideal model for a natural self antigen in studies of immunological tolerance.     

Studies on the mechanism of solubilization of immune precipitates by serum

Antigen (Ag)-antibody (Ab) aggregates prepared with several different antigens are solubilized by fresh serum at 37 degrees C (complex-release activity of serum or CRA). The rate of solubilization varies in different systems and is strongly influenced by the affinity of Ab for the Ag in the immune precipitate. With a given Ag-Ab precipitate, the maximum amount of complex that can be solubilized by individual sera is independent of the initial concentration of complexes and cannot be increased by prolonged incubation. CRA occurs in the absence of C2 and C4, but not in the absence of C3 and factor B of the properdin pathway. Addition of C2 to C2-deficient serum or C4 to C4-deficient serum enhances CRA. Solubilization does not involve extensive degradation of the complexed antibody, as might be detected by acrylamide gel electrophoresis of released antibody after reduction and alkylation to separate H and L chains. Immune precipitates can also be solubilized by incubation with monovalent fragments (Fab or Fab') of antibodies against determinants of the Ab molecules in the immune precipitate. In contrast, F(ab')2 fragments decrease the solubility of the immune precipitates. In view of these findings, we propose that CRA is mediated by the binding of functionally monovalent C fragments (C3 and C4) onto Ab molecules in the precipitates.     

Studies of possible antiidiotypic control mechanisms in Graves' disease

Serum from 55 patients with active Graves' disease and 55 patients who had received successful treatment (in whom the disease was inactive) were examined for the presence of possible antiidiotypic antibodies with an enzyme-linked immunosorbent assay (ELISA) for anti-F(ab')2. Murine IgG monoclonal antibodies (Mabs) against human thyroid-stimulating hormone (TSH) and human TSH receptors were also used as antigens in parallel ELISA assays. Patients with active and patients with inactive Graves' disease showed elevations of IgG anti-F(ab')2 antibodies when compared with normal controls. Similarly, both active and inactive Graves' disease sera showed higher levels of IgG anti-LE4, a mouse Mab to human TSH, than was seen with normal controls. However, F(ab')2 isolated from sera reacting with LE4 in the ELISA did not inhibit binding of the LE4 Mab with labeled TSH in a fluid phase competition assay. Patients with inactive Graves' disease showed higher ELISA reactivity with two different murine anti-TSH receptor Mabs than was recorded with either active Graves' or normal controls. A rough inverse correlation was noted between strongly positive ELISA reactions against these two Mabs with anti-TSH receptor specificity and the ability of immunoglobulins from inactive Graves' sera to stimulate increases in cyclic adenosine monophosphate (cAMP) in the normal rat thyroid cell line assay. Untreated Graves' sera showing high cAMP release only rarely showed elevated ELISA reactivity against Mabs with anti-TSH receptor activity.     

Analysis of sera from ovarian cancer patients for immune complexes

In contrast to other malignancies, circulating immune complexes (CICS) are usually not detected by conventional assays in the sera of ovarian cancer patients. However, a polyethylene glycol (PEG) precipitation assay has been reported to detect putative CICS in ovarian cancer. To determine if CICS were indeed present, we analyzed sera from 12 women with ovarian cancer. All were negative for CICS by the Raji cell assay; 5 (42%) were positive by the PEG assay. However, the PEG precipitate did not possess characteristics of immune complexes. IgG in sera or in the precipitate sedimented in sucrose gradients solely at the same rate as 7S monomeric IgG. In addition, the precipitates were not able to activate the complement system and the four IgG subclasses were present in the same relative concentration as that found in normal serum. The results suggest that it is probably a misnomer to label the material detected in ovarian cancer sera by the PEG precipitation assay as CICS. Instead a non-immune interaction of IgG with other components, possibly membrane fragments, is probably being measured.     

Autoimmune anti-HIV-1gp120 antibody with antiidiotype-like activity in sera and immune complexes of HIV-1-related immunologic thrombocytopenia.

Autoimmune antiidiotype-like antibody (Ab2) directed against anti-HIV-1gp120 (Ab1) was found in high titer in the sera of 10 consecutive homosexual and 11 narcotic addict HIV-1-related immunologic thrombocytopenia (HIV-1-ITP) patients, was barely detectable in 10 nonthrombocytopenic HIV-1 sero-positive individuals, and was not detectable in 5 normal subjects by use of a solid-phase RIA. Reactivity of autologous Ab2 for Ab1 was 4-120-fold greater than Ab2 for homologous Ab1. Affinity-purified Ab2 did not block the binding of affinity-purified Ab1 to its HIV-1gp120 epitopes on immunoblot, indicating the absence of "internal image" antiidiotype. Both Ab1 and Ab2 are precipitable from sera with polyethylene glycol (PEG) and present in a macromolecular complex that is excluded by gel filtration on G200 and contains IgG, IgM, C3, and the anti-F(ab')2 antiidiotype-like complex. PEG-precipitable complexes bind to platelets in a saturation-dependent manner. Neither affinity-purified Ab1 nor Ab2 binds to platelets. However, the combination of Ab1 and Ab2 (preincubated for 2 h at 22 degrees C) binds to platelets in a saturation-dependent manner at an optimum ratio range of 10-20:1. Ab2 reactivity correlates with serum PEG-precipitable immune complex level (r = 0.91; P less than 0.001) and with thrombocytopenia (r = 0.89; P less than 0.001). We suggest that the anti-HIV-1gp120 antiidiotype-like complex contributes to the markedly elevated platelet Ig and C3 level of HIV-1-ITP patients and propose that this may contribute to their thrombocytopenia.     

Immunoglobulins from the sera of immunologically activated persons with pairs of electrophoretic restricted bands show a greater tendency to aggregate than normal

From in vitro data, it has been speculated that pairs of endogenous restricted bands migrating in close proximity in the gamma region upon high resolution serum electrophoresis (HRE) represent circulating immune complexes (CIC). Using a polyethylene glycol (PEG) method to separate CIC, we found a very high correlation between the presence of such band pairs and elevated levels of CIC (CHI2 = 25.7, p less than 0.001) in 51 sera. HRE appears to be a good screening technique to identify, with a high degree of certainty, samples with elevated levels of CIC for delineation by more specific methods. Yet, examination by gel-filtration chromatography and precipitation with PEG indicated that the molecules comprising the band patterns were not CIC, but polyclonal 7S IgG. These bands are usually found in patients with chronic activation of the immune system. Fractionation of the sera from 5 such patients with various cuts of PEG indicated that the average IgG concentration in the 2.5-5%, and 5-7.5% cuts from patients was 3.99 g/l and 2.2 g/l, while from healthy subjects the concentrations were 0.68 g/l and 2.88 g/l. This reversed precipitation pattern was seen both for absolute levels of IgG and for percent of total IgG. On the average the amount of precipitation of IgG in the 2.5-5% fraction of patients was about 5-fold above that seen in the healthy subjects. The endogenous bands were not associated with any specific cut of PEG, but appeared to be proportionally distributed in accord with the levels of IgG. The data is consistent with the idea that immunologically activated patients exhibit a greater tendency for immunoglobulins to associate than normal. This propensity to aggregate may cause CIC to form in situ in local compartments even though CIC do not appear to be present upon analysis by biochemical techniques.     

Anti-C3d Antiglobulin Reagents. I. Characteristics of the Anti-C3c and Anti-C3d Responses During Hyperimmunization in Rabbits

Antiglobulin sera designed to detect erythrocyte coating by the third component of complement (C3) should contain primarily antibody to the C3d (α2D) antigenic grouping on the C3 molecule. During immunization, animals respond independently to the C3c (βlA) and C3d antigens. The present study reports quantitative serial precipitin and antiglobulin measurements of anti-C3c and anti-C3d responses in 12 rabbits during a four and one-half month hyperimmunization program and a subsequent six month follow-up period. Anti-C3c precipitin responses occurred earlier and were sustained longer than were anti-C3d precipitin responses. There was a general correlation of the strength of anti-C3d precipitin and antiglobulin responses among serum samples from a single rabbit, but not among sera from different rabbits. While most rabbits developed strong anti-C3c and anti-C3d precipitin and antiglobulin reactivities at the peak of their immune response, five of five commercially manufactured goat anti-C3 sera exhibited strong anti-C3c reactivity with little or no anti-C3d reactivity. The implications of these observations for production of anti-C3 antiglobulin reagents are discussed.     

Circulating Immune Complexes in the Serum in Systemic Lupus Erythematosus and in Carriers of Hepatitis B Antigen QUANTITATION BY BINDING TO RADIOLABELED Clq

A sensitive and reproducible procedure for the detection of soluble immune complexes in sera from patients with various immunopathological disorders is reported. Radiolabeled C1q is reacted with sera containing immune complexes. Separation of free from complex bound [(125)I]C1q is achieved by selective precipitation with polyethylene glycol (PEG). The method is based on both the large molecular size and the C1q-binding property characterizing immune complexes. The minimal amount of aggregated immunoglobulins thus detected is about 10 mug and that of soluble human IgG-anti-IgG complexes is about 3 mug of complexed antibody. Some immune complexes formed in large antigen excess (Ag(2)Ab) can still be detected by this radiolabeled C1q binding assay. The specificity of the radiolabeled C1q binding test was documented by the inability of antigen-F(ab')(2) antibody complexes to lead to a precipitation of [(125)I]C1q in PEG.In a second step, this radiolabeled C1q binding assay was applied to an experimental model of immune complex disease and was shown to be efficient for the detection of in vivo formed immune complexes.Finally, the technique could be applied to the study of sera from patients with systemic lupus erythematosus (SLE) or to carriers of the hepatitis B antigen (HB-Ag). Significantly increased [(125)I]-C1q binding values were observed in 52 sera from SLE patients when compared to values obtained with healthy blood donors (P<0.001). Particularly high values were seen in active disease, a finding which was confirmed by follow-up studies performed with four SLE patients.No increased [(125)I]C1q binding was seen in 18 healthy carriers of the HB-Ag; whereas, sera from carriers with hepatitis appear to precipitate increased [(125)I]C1q percentages: 7/24 cases with acute transient and 4/7 cases with chronic persistent hepatitis were found to increasingly bind [(125)I]C1q. The results were also used for a correlative study of [(125)I]C1q binding to IgG levels in the sera but increased [(125)I]C1q binding could not be attributed to high serum IgG levels which are likely to account for gammaglobulin aggregates.These examples suggest the utility of the radiolabeled C1q binding assay for the evaluation of immune complex diseases in human pathology.     

两种血清循环免疫复合物测定方法的比较

目的　对测定血清循环免疫复合物 ( circulating immune complexes,CIC)的两种方法 ( PEG沉淀法及 CIC-C1 q ELISA法 )进行评价 ,进一步探讨 CIC测定的临床意义。方法　采用 PEG沉淀法和 CIC-C1 q ELISA法检测肾脏损害、系统性红斑狼疮 ( SLE)、类风湿性关节炎 ( RA)等 86例患者的血清 CIC。结果　 86例患者中 PEG沉淀法检测阳性 1 7例 ( 1 9.77% ) ,CIC-C1 q ELISA法检测阳性 3 8例 ( 4 4.1 9% ) ,差异有显著性 ( P<0 .0 5 )。CIC-C1 q ELISA法检测 SLE、RA和肾脏损害患者的 CIC阳性率分别为 1 1 /1 5、5 /8和 2 0 /61 ( 3 2 .79% )。结论　与 PEG沉淀法相比 ,CIC-C1 q ELISA法较敏感 ,适宜于临床推广应用。CIC的阳性与 SLE、RA和肾脏损害有关。     

The humoral response to human factor VIII in hemophilia A mice

BACKGROUND: Inhibitory antibodies (Abs) to factor VIII (FVIII inhibitors) constitute the most significant complication in the management of hemophilia A. The analysis of FVIII inhibitors is confounded by polyclonality and the size of FVIII. OBJECTIVES: The goal of this study was to dissect the polyclonal response to human FVIII in hemophilia A mice undergoing a dosage schedule that mimics human use. METHODS: Splenic B-cell hybridomas were obtained following serial i.v. injections of submicrogram doses of FVIII. Results of a novel, anti-FVIII domain-specific enzyme-linked immunosorbent assay were compared to Ab isotype and anti-FVIII inhibitory activity. RESULTS: The robust immune response resulted in the production of approximately 300 hybridomas per spleen. We characterized Abs from 506 hybridomas, representing the most comprehensive analysis of a protein antigen to date. Similar to the human response to FVIII, anti-A2 and anti-C2 Abs constituted the majority of inhibitors. A novel epitope was identified in the A2 domain by competition ELISA. Anti-A2 and anti-C2 Abs were significantly associated with IgG(1) and IgG(2a) isotypes, respectively. Because the IgG(2a) isotype is associated with enhanced Fc receptor-mediated effector mechanisms, this result suggests that anti-C2 Abs and inflammation may be linked. Additionally, we identified a novel class of Abs with dual specificity for the A1 and A3 domains. Forty per cent of the Abs had no detectable inhibitory activity, indicating that they are prominent and potentially pathologically significant. CONCLUSION: The expanded delineation of the humoral response to FVIII may lead to improved management of hemophilia A through mutagenesis of FVIII B-cell epitopes.     

QUANTITATIVE EXPERIMENTS WITH ANTIBODIES TO A SPECIFIC PRECIPITATE. I

1. Rabbits were injected with the washed specific precipitate from Type II antipneumococcus horse serum. Antibody in the resulting antiserum was determined by the quantitative agglutinin method using various specific precipitates as antigens. 2. Suspensions of Types I and II antipneumococcus horse specific precipitates, as well as the specific precipitates derived from Type VIII Pn (anti-C portion), and H. influenzae horse antisera were found to remove the same amount of antibody from the immune rabbit serum. 3. Purified antibody solutions prepared by dissociation methods from Types I and II antipneumococcus horse sera were found to remove the same quantity of antibody as did the homologous specific precipitates. 4. Specific precipitates from anti-crystalline egg albumin and anti-diphtheria horse sera were found to remove only a fraction of the antibody. The reasons for this are discussed. 5. A specific precipitate prepared from pepsin-digested Type I anti-pneumococcus horse serum removed all of the antibody to the homologous antigen from the rabbit anti-precipitate serum, but followed a different quantitative course. 6. From the quantitative course of these reactions and from experiments with specific precipitates from anti-Pn rabbit and pig sera it is concluded that the only antigenic specificity demonstrable for the antibodies investigated was that due to their common origin, and that the groupings responsible for their antibody function constitute either a small part of the total protein molecule or else are non-antigenic.