Pepsin digestion of a mouse monoclonal antibody of IgG1 class formed F(ab')(2) fragments in which the light chains as well as the heavy chains were truncated

In the preparation of F(ab')(2) fragments of monoclonal antibodies (mAbs) of IgG class, heavy (H) chains are truncated by pepsin and light (L) chains are remained intact. However, F(ab')(2) fragments formed by pepsin-digestion of a mouse mAb PM373, which was of the IgG1 class and raised against human prostate specific antigen (PSA), indicated that the L chains of 31 kDa were cleaved into 23-kDa fragments as well as the cleavage of H chains of 50 kDa into 28-kDa fragments. On the other hand, F(ab')(2) fragments formed by digesting the mAb by cathepsin D showed that the L chains were intact and the H chains were truncated. The immunoreactivities against PSA of the F(ab')(2) fragments containing the intact L chains and those containing the truncated L chains were almost the same as that of the parental mAb, suggesting that the truncation of the L chains does not affect the interaction of the mAb with its specific antigen.     

On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice

Methods for the production and purification of F(ab')2 fragments from BALB/c monoclonal IgG1, IgG2a, and IgG2b with pepsin and other proteases were examined. The overall susceptibility to degradation is IgG2b greater than IgG2a greater than IgG1. Stable F(ab')2 can be produced in good yield from IgG1 with pepsin at pH 3.5 to 4.0 and can be made directly by pepsin treatment of ascites fluids or cell culture supernatants containing IgG1. IgG2a is cleaved in two steps by pepsin, first to F(ab')2 and then to Fab'. With carefully chosen conditions, F(ab')2 can be obtained in acceptable yield. The primary cleavage for the IgG2a heavy chain appears to be on the COOH terminal side of the interheavy chain disulfides, and secondary cleavage is on the NH2-terminal side. For IgG2b the reverse is true, and F(ab')2 has not been obtained in useful amounts; however, the primary cleavage of IgG2b appears to be assymetric with respect to the two heavy chains, and Fab/c fragments that have one Fab plus Fc can be made. Digestion with elastase resulted in the best yield of Fab/c. This finding may provide a method for retaining cytotoxicity in monoclonal antibodies against cell surface antigens while eliminating their capacity to modulate. The cleavage patterns of the three classes of IgG are rationalized in terms of the structure of their hinge regions.     

H chain C domains influence the strength of binding of IgG for streptococcal group A carbohydrate

We have produced a panel of murine anti-streptococcal mAbs, expressing identical V domains and different H chain C domains, corresponding to the IgG3, IgG1, and IgG2b subclasses. We have used these mAb to evaluate the role of IgG subclass-specific C region determinants in modulating the interaction between antibody and the bacterial surface. We report, for the first time, that V region-identical murine IgG of different subclasses exhibit substantial differences in binding to specific Ag; IgG3 mAb binds more strongly to streptococci than the IgG1 and IgG2b mAb or IgG3-derived F(ab')2 fragments. Furthermore, the IgG3 mAB binds cooperatively to the bacteria, whereas the IgG1, IgG2b, and IgG3-derived F(ab')2 fragments do not exhibit significant cooperativity, which suggests that differences in Fc region structure can affect antibody binding to multivalent Ag by modulating the potential for cooperative binding. These results suggest a plausible mechanism by which murine IgG3 could be more effective, than other antibodies bearing identical V domains, but of different gamma-subclass, in mediating bacterial immunity.     

Single-step purification of F(ab')2 fragments of mouse monoclonal antibodies (immunoglobulins G1) by hydrophobic interaction high performance liquid chromatography using TSKgel Phenyl-5PW.

Hydrophobic interaction high performance liquid chromatography (HPLC) using TSKgel Phenyl-5PW was applicable to single-step purification of F(ab')2 fragments from pepsin digests of mouse monoclonal antibodies of IgG1 class. The digests were applied to the gel equilibrated with phosphate-buffered saline containing 1 M ammonium sulfate. F(ab')2 fragments were adsorbed onto the gel using the same buffer, and eluted by reducing the ammonium sulfate concentration to 0 M. The fraction containing F(ab')2 fragments was homogeneous (purity: higher than 98%) by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration HPLC. The recovery of the antigen binding site was 42-58%. The cycle time of the Phenyl-5PW HPLC was 45 min, and F(ab')2 of up to 2200 mg was purified in a cycle. This method could be useful especially for large scale purification of F(ab')2 fragments.     

Synthesis of bifunctional antibodies for immunoassays

The synthesis of bifunctional antibodies using the principle of solid-phase synthesis is described. Two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')(2) fragments from intact immunoglobulin G (IgG) were prepared using an immobilized pepsin column. Goat, mouse, and human antibodies were digested completely within 4 h. The F(ab')(2) fragments thus produced did not contain any IgG impurities. Fab' fragments were produced by reducing the heavy interchain disulfide bonds using 2-mercaptoethylamine. Use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of bifunctional antibody preparation and the rapid optimization of reaction conditions.     

Monoclonal antibodies to Fc receptors for IgG on human mononuclear phagocytes. Antibody characterization and induction of superoxide production in a monocyte cell line

We have utilized monoclonal antibodies against the two IgG Fc receptors (p40 and p72) of U937 cells to stimulate the release of superoxide. The monoclonal antibody (mAb) specific for p40 (IV3) has been described elsewhere. A murine IgG1 mAb specific for the high affinity p72 Fc receptor (designated mAb FcR32 or simply mAb 32) bound to the same p72 precipitated by Sepharose-human IgG as shown by preclearing experiments and by identical isoelectric focussing patterns. Binding of mAb 32 to p72 was independent of the Fc region of the antibody since Fab' fragments of mAb 32 affinity adsorbed p72. The binding of both mAb 32 and human IgG1 to the intact U937 cell was not reciprocally inhibitory, indicating that mAb 32 does not interfere with the ligand binding site of p72. mAb 32 bound to human monocytes, U937, and HL60 cells, but not to granulocytes or lymphocytes. U937 cells cultured in gamma-interferon and 1,25-dihydroxycholecalciferol generated superoxide when incubated with mAb 32 or IV3 followed by cross-linking with F(ab')2 anti-murine Ig. Incubation with mAb 32 or IV3 alone or with 3 of 5 other anti-U937 mAbs cross-linked with anti-murine Ig did not result in superoxide generation. Immune complex-mediated superoxide production was inhibited 80% by IgG, but not by mAb 32 or IV3.     

Kinetics of whole serum and prepurified IgG digestion by pepsin for F(ab')2 manufacture

An alternative route for the production of polyclonal F(ab')(2) fragments that might be adopted for the facile preparation of antivenoms is assessed in this work. The method involves the digestion of whole serum by free pepsin, which results in reduction of the number of processing steps commonly in use, because it avoids the initial purification of IgG's prior to their proteolytic cleavage by the enzyme. Digestion kinetics of whole serum and caprylic acid prepurified IgG using free pepsin were monitored with SDS-PAGE followed by densitometric analysis and antigen binding activity assay of the digested samples. It was observed that with equal units of pepsin activity, caprylic acid prepurified IgG was digested more rapidly than whole serum but that the overall retention of antigen binding activity was significantly greater in the latter case. The estimated first-order digestion rate parameters were 11.8 and 4.42 microM min(-)(1) for pure IgG and whole serum, respectively. The K(m) value obtained for whole serum digestion was 33 microM and that for pure IgG digestion was 43.5 microM. Calibration with undigested whole serum and pure IgG samples of known concentrations was performed using SDS-PAGE followed by image analysis. A linear relationship was observed between the protein concentration and the respective band intensity within the range of concentrations investigated (0.63-31.2 microM IgG concentration). This technique proved to be relatively rapid, reproducible, and more precise than size-exclusion chromatography as a result of its F(ab')(2)/IgG resolving power. Staining and destaining protocols were reproduced in terms of staining and destaining times, volumes added, and compositions. Furthermore, all digestion experiments were performed in duplicate sets to monitor the extent of variation of the digestion kinetic parameters measured by this method. The results obtained from this technique confirm and quantify previous observations that pepsin digestion of whole serum is slower and easier to control than digestion of pure IgG and results in higher recovery of antigenic binding activity.     

Pharmacokinetic studies of radiolabelled rat monoclonal antibodies recognising syngeneic sarcoma antigens

In the preceding paper it was suggested that the tumour localisation of 125I-labelled syngeneic rat monoclonal antibodies (mAbs) may be limited in immunocompetent hosts by the presence of competing endogenous serum antibodies. In syngeneic congenitally athymic (nu/nu) and cyclosporin-A-treated rats (both of which fail to mount immune responses to tumour antigens) increased uptake of mAbs in tumour tissue was obtained compared with that in immunocompetent animals. However, in the case of IgG2b and IgG1 mAbs, this appeared to be due primarily to enhanced "non-specific" localisation mediated by Fc binding, since it was abolished by the use of F(ab')2 fragments with two out of three mAbs tested. Normal tissue distribution was also influenced by host immune status: in nu/nu rats the uptake of IgG2b mAbs in the spleen was up to fivefold higher than that previously found in normal animals and the levels in liver were also increased. This effect was not seen in cyclosporin-A-treated hosts, suggesting that the reticuloendothelial system of congenitally athymic animals contains cells with enhanced IgG2b-FcR activity. This hypothesis was strengthened by the observation that splenic uptake was reduced by either the use of F(ab')2 fragments, or prior "blockade" of Fc receptors by "cold competition" with excess unlabelled IgG2b mAbs. This blockade could not be effected by mAbs of any other isotype or by IgG2b F(ab')2 fragments. The former manoeuvre resulted in higher tumour specificity ratios but usually at the expense of reduced levels of tumour associated radiolabelled mAb. The latter was found to increase "absolute" tumour localisation by up to 35%. In an attempt to characterise further and compare the Fc receptor activity of intratumour and intrasplenic host cells. The distribution of IgG2b mAbs was assayed in 3-week, 8-week and 12-week-old rats. We were able operationally to distinguish the activity of these two categories of cells, suggesting that they represent either different lineages or differentially activated subpopulations: the splenic IgG2b binding was fully expressed in weanling nu/nu rats whereas the FcR activity of cells infiltrating MC24 sarcoma was limited in 3-week-old compared with 8-12-week-old hosts. A further difference was apparent in the subclass "preference" of FcR binding: in immunodeprived rats both IgG1 and IgG2b mAbs were able to bind to tumour-infiltrating host cells, but uptake of IgG1 mAbs in the spleen was always low and not reduced further by the use of F(ab')2 fragments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of antibody Fab and F(ab')2 fragments using Gradiflow technology

The Gradiflow, a preparative electrophoresis instrument designed to separate molecules on the basis of their size and charge, was used to purify antibody Fab and F(ab')2 fragments. The method described is charge based, utilizing the difference in the pI between the antibody Fab/F(ab')2 fragments and antibody Fc fragments that occur after enzyme digestion of whole antibody molecules. This method of purification was successful across a range of monoclonal and polyclonal antibodies. In particular, F(ab')2 fragments were purified from a number of mouse monoclonal antibodies (both IgG1 and IgG2a isotypes) and Fab fragments were purified from egg yolk IgY polyclonal antibodies. This is a rapid purification method which has advantages over alternative methods that usually comprise ion exchange and gel filtration chromatography. This method may be applicable to most antibody digest preparations.     

Immunoglobulin G3 from polyclonal human immunodeficiency virus (HIV) immune globulin is more potent than other subclasses in neutralizing HIV type 1.

Passive antibody prophylaxis against human immunodeficiency virus type 1 (HIV-1) has been accomplished in primates, suggesting that this strategy may prove useful in humans. While antibody specificity is crucial for neutralization, other antibody characteristics, such as subclass, have not been explored. Our objective was to compare the efficiencies of immunoglobulin G (IgG) subclasses from polyclonal human HIV immune globulin (HIVIG) in the neutralization of HIV-1 strains differing in coreceptor tropism. IgG1, IgG2, and IgG3 were enriched from HIVIG by using protein A-Sepharose. All three subclasses bound major HIV-1 proteins, as shown by Western blot assay and enzyme-linked immunosorbent assay. In HIV-1 fusion assays using X4, R5, or X4R5 envelope-expressing effector cells, IgG3 more efficiently blocked fusion. In neutralization assays with cell-free viruses using X4 (LAI, IIIB), R5 (BaL), and X4R5 (DH123), a similar hierarchy of neutralization was found: IgG3 > IgG1 > IgG2. IgG3 has a longer, more flexible hinge region than the other subclasses. To test whether this is important, IgG1 and IgG3 were digested with pepsin to generate F(ab')(2) fragments or with papain to generate Fab fragments. IgG3 F(ab')(2) fragments were still more efficient in neutralization than F(ab')(2) of IgG1. However, Fab fragments of IgG3 and IgG1 demonstrated equivalent neutralization capacities and the IgG3 advantage was lost. These results suggest that the IgG3 hinge region confers enhanced HIV-neutralizing ability. Enrichment and stabilization of IgG3 may therefore lead to improved HIVIG preparations. The results of this study have implications for the improvement of passive immunization with polyclonal or monoclonal antibodies and suggest that HIV-1 vaccines which induce high-titer IgG3 responses could be advantageous.     

Targeting weak antigens to CD64 elicits potent humoral responses in human CD64 transgenic mice

Previous studies have documented that targeting foreign Ags to IgG FcgammaR leads to enhanced Ag-specific responses in vitro and in vivo. However, the ability to overcome immunologic nonresponsiveness by targeting poorly immunogenic Ags to FcgammaR has not been investigated. To address this question in a simple model, we immunized transgenic mice expressing human CD64 (FcgammaRI) and their nontransgenic littermates with Fab' derived from the murine anti-human CD64 mAb m22. The m22 Fab' served as both the targeting molecule and the Ag. We found that only CD64-expressing mice developed anti-Id titers to m22. Furthermore, chemically linked multimers of m22 Fab', which mediated efficient internalization of the human CD64, were significantly more potent than monomeric m22 F(ab')(2) at inducing anti-Id responses. In all cases, the humoral responses were specific for m22 Id and did not react with other murine IgG1 Fab' fragments. Chemical addition of a second murine Fab' (520C9 anti-human HER2/neu) to m22 Fab' multimers demonstrated that IgG1 and IgG2a anti-Id titers could be generated to 520C9 only in the CD64-expressing mice. These results show that targeting to CD64 can overcome immunological nonresponsiveness to a weak immunogen. Therefore, targeting to CD64 may be an effective method to enhance the activity of nonimmunogenic tumor vaccines.     

Purification of antibody and antibody-fragment from E. coli homogenate using 6,9-diamino-2-ethoxyacridine lactate as precipitation agent

To obtain a more efficient purification process for antibody fragments from an Escherichia coli homogenate, the precipitant, Ethodin (6,9-diamino-2-ethoxyacridine lactate) was introduced to the homogenate. By adding the precipitant a drastic reduction of host cell protein was obtained. The majority of the proteins were recovered in a precipitate with the cell debris, while the antibody or antibody-fragment was recovered in the clarified supernatant. In addition, DNA was also efficiently precipitated when using Ethodin as a precipitation agent. The improved purity of the clarified extract obtained by using the precipitant allows for the use of smaller chromatography columns and may reduce the number of chromatographic steps required in the recovery process. The effect of Ethodin concentration, pH, temperature, and conductivity were investigated. The investigation was performed on two different antibody-fragments, e.g., F(ab')(2) molecules and a full-length antibody produced in E. coli. The two F(ab')(2) proteins were F(ab')(2)A and F(ab')(2)B, which have a similar molecular mass (100 kDa) but different isoelectric points (pIs), i.e., 8.9 and 7.5, respectively. The full-length antibody, Ab (the full IgG form of F(ab')(2)B) has a pI of 7.8 and molecular mass of 150 kDa. The investigation showed that the highest purification factors were obtained at neutral pH, low conductivity, and Ethodin concentrations of 0.6%.     

Rapid determination of bovine lactoferrin in dairy products by an automated quantitative agglutination assay based on latex beads coated with F(ab′)2 fragments

This study evaluated an automated immunoassay for bovine lactoferrin (LF) in dairy products based on latex beads coated with F(ab')2 fragments. Methods: F(ab')2 fragments were obtained by pepsin digestion of rabbit anti-bovine LF (IgG fraction) and polystyrene latex beads were coated with the F(ab')2 fragments. We used the beads to develop a rapid and homogeneous light scatter immunoassay employing an autoanalyzer (the Automated Latex assay). The Automated Latex assay was easy to perform and could rapidly determine bovine lactoferrin in lactoferrin-supplemented products. It was sensitive enough for testing products and showed good precision.     

Down-regulation of cell surface CD4 molecule expression induced by anti-CD4 antibodies in human T lymphocytes.

Antigenic modulation was defined as the down-regulation of a cell surface antigen expression induced by exposure to specific antibody. We investigated the modulation of CD4 surface expression in human peripheral blood lymphocytes incubated in vitro with anti-CD4 monoclonal antibodies (mAbs). Modulation of surface CD4 was achieved at 37 degrees C, but not at 4 degrees C, with five different murine anti-CD4 mAbs of IgG1 and IgG2a subclasses, with different epitope specificities. Modulation was dose dependent with a maximum at nonsaturating mAb concentration. It was reversible upon culture in mAb-free medium. It was accelerated and amplified in the presence of monocytes or after cross-linking of anti-CD4 mAbs. It could be induced with solid phase anti-CD4 mAbs, but not with soluble F(ab')2 fragments. Its magnitude was identical on all CD4+ lymphocytes. It was associated with a moderate down-regulation of CD2 and CD3 but not of CD8 and HLA class I surface expression. Modulation was slightly augmented by addition of inhibitors of the endosome/lysosome pathway but not by protein synthesis inhibitors. The anti-CD4 mAb initially bound to cell surface was no longer detectable after 24 hr of culture. Most of surface CD4 proteins complexed with antibody were rapidly internalized and transiently replaced by CD4 from an intracytoplasmic pool and then no longer were expressed. CD4 mRNA was moderately decreased in cells incubated with anti-CD4 mAb while beta-actin and beta 2-microglobulin mRNAs remained at stable levels. It was concluded that down-regulation of CD4 surface expression induced by anti-CD4 mAb concerned only a part of CD4 molecules and was associated with a decreased synthesis. The delay required to achieve maximal modulation is likely to reflect exhaustion of the intracytoplasmic recycling pool of CD4 molecules.     

Monoclonal Antibodies to Human Myelin Basic Protein

SJL/J and (SJL X PL) F1 hybrid mice were immunized with intact human myelin basic protein (MBP) or the three major peptic fragments of MBP, residues 1-38, 39-89, and 90-170. Immune spleen cells were fused with mouse myeloma P3 X 63Ag8 (NS1) cells in the presence of polyethylene glycol. Hybridoma supernatant culture fluids were screened for antibody to MBP by a solid-phase radioimmunoassay (RIA). The specificity of the monoclonal antibody (mAb) was characterized by RIA using the three major MBP peptic fragments and subfragments as well as MBP and MBP fragments of different species with known amino acid sequence differences. Six MBP mAbs were generated, one of them IgM isotype and the remainder IgG isotypes. One mAb each reacted against regions of residues 22-38, 39-69, 70-89, 90-116, and two reacted against residues 118-157. Immunoblots also showed that the five IgG mAbs were reactive against MBP and the peptic fragment of MBP containing the epitope. Immunohistochemical studies showed the IgG mAbs specifically stained myelinated fiber tracts in human brain tissue.     

天然产物莪术醇单克隆抗体的制备

为制备小分子化合物莪术醇的单克隆抗体,先将莪术醇(curcumol)与载体蛋白牛血清蛋白(BSA)偶联形成完全抗原,用基质辅助激光解吸飞行时间质谱法(MALDI-TOF-MS)鉴定莪术醇人工抗原的偶联率,然后采用杂交瘤技术获得杂交瘤株,并对其进行小鼠腹水的制备与纯化.结果表明:莪术醇半抗原与载体的偶联比为19.6,单克...     

Involvement of Fc gamma RI (CD64) in the mechanism of HIV-1 inhibition by polyclonal IgG purified from infected patients in cultured monocyte-derived macrophages

The aim of this study was to investigate the mechanism of HIV-1 neutralization using monocyte-derived macrophages (MDM) in comparison to PBMC as target cells. For this purpose, we analyzed neutralizing activities of different human polyclonal IgG samples purified from sera of HIV-1-infected individuals using a single cycle infection assay. We found an increase of the neutralizing titer when macrophages vs PBMC were used as target cells. Moreover, polyclonal IgG from HIV-1-infected patients that are not able to neutralize virus when PBMC are used as target cells strongly inhibit MDM infection. Similar results were obtained with neutralizing mAbs. To explore the participation of FcgammaRs in HIV-1 inhibition, F(ab')(2) and Fab of these Igs were produced. Results indicated that both F(ab')(2) and Fab are less effective to inhibit virus replication in MDM. Moreover, competition experiments with Fc fragments of IgG from healthy donors or with purified monoclonal anti-human FcgammaRs Ab strengthen the participation of the FcgammaRs, and in particular of FcgammaRI (CD64) in HIV-1 inhibition on MDM. Mechanisms by which HIV-specific IgG inhibit virus replication in cultured macrophages are proposed and the benefit of inducing such Abs by vaccination is discussed.     

Absence of auto-antiidiotypic activity between the IgM and IgG fractions of human mixed cryoglobulins

Experimental animal models and observations in humans suggest that levels of Id and auto-anti-Id fluctuate reciprocally after Ag stimulation. In human monoclonal B cell disorders, however, the co-existence of paraprotein Id and its auto-anti-Id has been described in essential mixed cryoglobulinemia and in association with acquired C1 inhibitor deficiency. Because the majority of cryoglobulin IgM possess rheumatoid factor activity and thus bind the Fc region of IgG, we examined potential idiotypic interactions between cryoglobulin IgM and F(ab')2 fragments of autologous cryoglobulin IgG fractions. A rabbit antibody to the pepsin agglutinator site of human F(ab')2 was used as detection reagent. By recognizing epitopes exposed on F(ab')2 after the removal of Fc determinants by pepsin digestion, this reagent eliminates the detection of contaminating intact IgG. In a sensitive assay, we were unable to detect idiotypic interactions between the separated IgM and pepsin-digested IgG fractions of 10 mixed cryoglobulins. On the basis of these results, we suggest that in mixed cryoglobulinemia, the coexistence of paraprotein Id and its auto-anti-Id is unlikely.     

Mechanism of human monocyte activation via the 40-kDa Fc receptor for IgG

It is shown that a mAb specific for the human 40-kDa FcR (FcRII) leads to activation of human monocytic cells but that extensive cross-linking of the receptor is required. Calcium mobilization can be induced in immature monocytic cells (undifferentiated U937 cells) and peripheral blood monocytes with an intact IgG1 anti-FcRII antibody (CIKM5) but not by F(ab')2 fragments of this antibody. The intact antibody can bind in a tripartite manner by its two F(ab') sites and its Fc-binding site whereas the F(ab')2 fragments of this antibody can only bind in a divalent fashion. A rise in intracellular free calcium ion concentration occurs when F(ab')2 fragments are cross-linked with F(ab')2 anti-mouse Ig indicating that more extensive cross-linking of FcRII is required rather than an obligatory requirement for an Fc-FcRII interaction. Calcium mobilization in response to intact or cross-linked F(ab')2 fragments of CIKM5 is associated with superoxide production only in IFN-gamma-primed peripheral blood monocytes and IFN-gamma differentiated U937 cells indicating that the activation signal produced via FcRII is inadequate to fully stimulate non-"primed" cells. A second mAb reactive with FcRII (2E1) does not cause calcium mobilization in monocytes or U937 cells, and partially blocks the effects of CIKM5. 2E1 also blocks CIKM5 superoxide production in IFN-gamma-primed monocytes and differentiated U937 cells. This may be explained in part by the fact that 2E1 is an IgG2a antibody and can only participate in bipartite binding with FcRII. When 2E1 is cross-linked with F(ab')2 anti-mouse Ig there is a small calcium response. This does not cause superoxide generation in IFN-primed monocytes but does do so in IFN-gamma differentiated U937 cells. FcRII is also expressed on granulocytes and some B cells but the effects of cross-linking the receptor on these cells differ from those seen in monocytes.     

Localization of biologic functions of toxic shock syndrome toxin-1 by use of monoclonal antibodies and cyanogen bromide-generated toxin fragments

Monoclonal antibodies (mAb) against toxic shock syndrome toxin-1 (TSST-1) were generated that block two of the most important biologic activities of TSST-1, nonspecific T lymphocyte mitogenicity and the suppression of immunoglobulin synthesis. Fourteen hybridomas producing antibody against TSST-1 were isolated independently. The culture supernatant and ascitic fluids from each were analyzed to determine the mAb isotypes. Seven of the mAb were IgG1, and the remaining seven were IgM; all the mAb had kappa light chains. Immunoglobulin was partially purified from hybridoma-generated ascitic fluid by ammonium sulfate precipitation and tested for the ability to block TSST-1-induced mitogenicity and immunosuppression. Three mAb (all IgG1) were shown to block both the toxin-induced mitogenicity and the suppression. None of the mAb tested inhibited just one of the two toxin activities. The neutralizing mAb were then used in Western analysis with previously mapped cyanogen bromide (CNBr)-generated toxin fragments to localize the aforementioned biologic functions. The Western blot analysis showed that the mitogenic and the suppressive functions of TSST-1 were located on a 14,000 dalton internal CNBr fragment.