广州管圆线虫单抗结合抗原定位分布及在免疫诊断上的应用

目的 研制广州管圆线虫单克隆抗体诊断循环抗原提高诊断的特异性.方法 将广州管圆线虫分泌性抗原免疫小鼠,免疫鼠脾细胞与骨髓瘤细胞融合为杂交瘤细胞,用广州管圆线虫阳性患者血清筛选阳性杂交瘤细胞,培养阳性的杂交瘤细胞分离制备单克隆抗体命名为12D5和21B7,用免疫组织化学的方法分析12D5和21B7单抗结合抗原在广州管圆线虫体内的分布,并用筛选的双12D5和21B7单抗进行抗体夹心ELISA检测实验感染广州管圆线虫的大鼠、广州管圆线虫感染病人血清循环抗原(CAg),用其他寄生虫抗原鉴别单抗的特异性,并与抗体检测比较其敏感性和特异性.结果 经鉴定单抗12D5为IgG1,21 B7为IgM,两株单抗同时识别广州管圆线虫成虫相对分子质量为55 × 103的蛋白,两个单抗针对的抗原分布在虫体肠表面,12D5和21 B7双抗体夹心ELISA法对实验感染的广州管圆线虫的大鼠血清中CAg检出率为100%(48/48),广州管圆线虫感染病人血清CAg检出率为100%(32/32),与日本血吸虫、肝吸虫、肺吸虫、旋毛虫、蛔虫、包虫病人血清无交叉反应,与健康人血清无反应;而用抗原检测32个广州管圆线虫感染病人的抗体检出率为75%(24/32),同时抗体检测与其他寄生虫出现一定的交叉反应.结论 12D5和21 B7单抗结合的抗原为肠相关抗原,双抗体夹心ELISA法对感染广州管圆线虫人和动物血清中CAg检测的特异性强,敏感性高,优于抗体检测试剂,并能够确定现症感染.

Abstract：

Objective To detect infection of Angiostrongylus cantonensis and examine effection of treatment to prepare monoclonal antibodies(McAbs). Methods Six-week-old BALB/c mice were imrnunized by the intraperitoneal injection of e/s antigens of Angiostrongylus cantonensis. Fusion of splecn cells from immunized mice with prepared SP2/0-Ag14 myeloma cells was performed in RPMI 1640. Fused cells were suspended in RPMI 1640 containing 1% HAT and 20% fetal calf serum and dispensed into 96-well cell culture plates. The supernatants of clones were screened by ELISA with sera of patients of angiostrongyliasis.Distribution of cohere antigen of 12D5 and 21B7 monoclonal antibodies was analyzed with immunohistochemistry. Two McAbs ( 12D5 and 21B7) were applied to detect the circulating antigen (CAg) in the sera of rats infected with A. cantonensis and angiostrongyliasis patients respectively by double antibody sandwich ELISA.Results 12D5 McAb was identified as IgG1 and 21 B7 McAb was IgM. Western blot result showed two McAbs could used to identified 55 × 103 protein of adult worms of A. cantonensis. Cohere antigen of 12D5 and 21B7 monoclonal antibodies were distributed on intestine surface of A. cantonensis. The detection rates of CAg in the sera of infected rats 100% (48/48), the detection rates of CAg in the sera of angiostrongyliasis patients was 100% (32/32). No cross-reaction to sera of patients with other infection of parasites, such as clonochiasis, fasiolopsiasis, ancylostomiasis, trichinosis, anisakiasis as well as schsitosomiasis, and health srea did not reacted with 12D5 and 21B7 McAbs,and detaction rate of antibody of angiostrongyliasis patients only reached 75% (24/32) with antigen of A. cantonensis. Conclusion Cohere antigen of 12D5 and 21B7monoclonal antibodies were antigens of enteric epithelium. Sandwich ELISA with 12D5 and 21B7 McAbs showed high specificity act as detecting CAg of A. cantonensis in sera of infection animal and patients. It is apparent that Sandwich ELISA with 12D5 and 21 B7 is not only rapid and simple without requirement of special instrument, but also rather sensitive and specific for the detection of current infection with A. cantonensis.     

Production and partial characterization of monoclonal antibodies against 3,3',5-triiodo-L-thyronine

Spleen cells of Biozzi HL mouse (selection V) immunized with bovine albumin-triiodothyronine conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Thirteen monoclonal antibodies, selected by an enzyme-linked immunosorbent assay and a radioimmunoassay, were produced in mouse ascites fluid, purified and analyzed. All of them were IgG1 (kappa). The cross-reactivity of all these monoclonal antibodies with thyroxine (T4) was less than 0.2%. The association constant determined by Scatchard analysis ranged from 1.5 x 10(9) M-1 to 2.7 x 10(10) M-1.     

The use of monoclonal antibodies to define levels of cystatin C in normal human serum

We established four hybridoma cell lines which secreted monoclonal antibodies against human cystatin C. These monoclonal antibodies were of IgA(kappa), IgG2a(lambda), IgG1(kappa), and IgG1(lambda) isotypes, respectively. The association constants (Ka) of the three IgG monoclonal antibodies ranged from 3.6 x 10(9) M-1 to 7.3 x 10(10) M-1. An ELISA technique for the measurement of cystatin C was established by using one of these monoclonal antibodies. The assay procedure was highly specific, sensitive, and reproducible. The lower detectable limit of cystatin C by this procedure was 1.9 ng/ml. The level of cystatin C in normal human serum was 1.16 +/- 0.91 micrograms/ml (mean +/- S.D., n = 274).     

The production of high affinity monoclonal antibodies to human chorionic gonadotropin and their application to immunoradiometric assay

Production of monoclonal antibodies against hCG has been studied using hCG as the antigen. This study reports the successful isolation of hybrid clones secreting monoclonal antibodies specific for hCG with an affinity constant higher than 10(10) M-1. Of 23 fusions, only 17 fusions have produced positive clones which secrete antibodies giving high levels of binding with 125I-labelled hCG in the supernatant. Finally, 6 different monoclonal antibodies have been isolated; 4 of them, specific for the beta-subunit, with a Ka approximately 1.1-4.0 X 10(11) M-1 and 2 others, specific for the alpha-subunit, presenting an affinity of 2.5 X 10(10) M-1. When the antibodies specific for the beta-subunit are used, specific and highly sensitive radioimmunoassays are obtained after only 3 hrs of incubation. Using iodinated monoclonal antibodies specific for the alpha-subunit and tubes coated with antibodies against the beta-subunit, we have developed sensitive immunoradiometric assays.     

Monoclonal antibodies specific for rat IgG1, IgG2a, and IgG2b subclasses, and kappa chain monotypic and allotypic determinants: reagents for use with rat monoclonal antibodies

Mouse monoclonal antibodies to rat IgG were obtained by fusion of immune SJL mouse spleen cells to NSI myeloma cells. Seven monoclonal antibodies have been labeled with 125I and studied as to specificity and avidity by using a panel of rat monoclonal antibodies both as inhibitors and target antigens in soft well plate and indirect cell binding assays. All MAb were selected for high avidity of 4 X 10(7) to greater than or equal to 2 X 10(9) M-1. Four MAb were subclass-specific. RG11/39, RG7/1, and RG7/11 were absolutely specific for the Fc' region of IgG1, IgG2a, and IgG2b, respectively. RG9/6 showed specificity for the Fab' region of IgG2a but crossreacted with lower avidity with IgG2c. Three MAb reacted with rat kappa chains. RG7/9 defined a monotypic (common) kappa chain determinant. RG11/15 and RG7/7 were specific for allelic kappa 1a and kappa 1b determinants, respectively. The monotypic and kappa 1a allotypic determinants are topographically separated. The antibodies can be used as screening reagents in indirect cell binding assays. They have sensitivity similar to affinity-purified rabbit anti-rat IgG and more defined specificity. They do not crossreact with mouse or human IgG, making them particularly suitable companion reagents for rat anti-mouse or anti-human MAb. One Mab, RG7/7, strongly crossreacts with Syrian hamster IgG.     

Monoclonal antibodies to 52-kilodalton protein of Salmonella typhi.

Ten monoclonal antibodies (6 immunoglobulin G1 kappa [IgG1 kappa] and 4 IgG2b kappa) from six hybrid clones specific for Salmonella typhi antigen were produced by immunizing BALB/cJ mice with affinity-purified S. typhi proteins (Bp). The latter were prepared by passing crude S. typhi Bp through an affinity column made from Sepharose conjugated to IgG antibodies against partially purified S. typhi Bp. The eluent was subsequently used as the immunogen for the production of monoclonal antibody. All 10 monoclonal antibodies reacted specifically with a 52-kilodalton (kDa) protein of S. typhi and were species specific. The presence of IgM antibody to the 52-kDa antigen in the sera of a majority of patients with acute typhoid fever suggested that this 52-kDa protein is also a good immunogen for humans. The potential usefulness of this antigen in the early diagnosis of typhoid fever is discussed.     

Pan natural killer cell monoclonal antibodies and their relationship to the NK1.1 antigen

The study of natural killer (NK) has been difficult because they account for a small percentage of peripheral blood and splenic lymphocytes and the paucity of NK specific antigens that have been identified. We have isolated pure populations of C57BL/6 (H-2b) NK cells using the IgG2b monoclonal antibody PK136 (anti-NK1.1). These NK1.1+ cells were used to immunize 129/J (H-2b) mice, and in this report, we describe three new NK specific monoclonal antibodies (SW3A4(IgM), SW4B12(IgG1), and SW2B4(IgG2b] and their relationship to the known murine NK antigen NK1.1. We have further characterized the NK1.1 antigen as a 39 kd molecule which is coded for by a gene which appears to map to chromosome 6.     

Epitopes on sialoglycoprotein alpha: evidence for heterogeneity in the molecule.

The binding of 15 125I-labelled mouse monoclonal antibodies to cell-surface sialoglycoprotein alpha (SGP alpha: synonym Glycophorin A) was studied using intact IgG and Fab fragments. It was estimated that the number of sialoglycoprotein alpha (SGP alpha) molecules per red cell is of the order of 1 x 10(6) and the number of sialoglycoprotein delta (SGP delta; synonym Glycophorin B) molecules per red cell is of the order of 1.7-2.5 x 10(5). Competitive binding assays showed that antibodies of the same blood group specificity (four anti-Ns reacting with SGP alpha and SGP delta (BRIC 33, BRIC 115, BRIC 120, BRIC 123) and four anti-Wrbs reacting with SGP alpha (R7, BRIC 14, BRIC 89, BRIC 93) inhibited binding of each other to red cells. Two antibodies (R1.3 reacting with SGP alpha and SGP delta and R18 reacting with SGP alpha) recognized distinct epitopes, and the remaining five antibodies (BRIC 116, BRIC 117, BRIC 119, BRIC 127, R10 reacting with SGP alpha) partially inhibited binding of each other to red cells. This latter observation and the finding that four of these antibodies (BRIC 116, BRIC 117, BRIC 119, BRIC 127) bind to a considerably smaller number of antigen sites (1.69-2.71 x 10(5) for intact IgG) than the maximum value obtained, suggests heterogeneity of glycosylation within SGP alpha molecules. The functional affinities of the IgG antibodies ranged from 1 x 10(5) to 4 x 10(7)M-1.     

Characterization of human monoclonal antibodies directed against the pp65-kD matrix antigen of human cytomegalovirus.

Human monoclonal antibodies specific for human cytomegalovirus (CMV) antigens have been established using peripheral blood lymphocytes from a seropositive donor. Immortalization of antigen-specific B cells was achieved by Epstein-Barr virus transformation followed by somatic cell fusion of antigen-specific lymphoblastoid cells. Four clones producing high-affinity antibodies (0.2-7 x 10(9) M-1) specific for the viral matrix protein pp65 have been further characterized with respect to epitope specificity of secreted antibodies. The studied antigen represents a major protein produced by in vitro-cultivated virus, and is important in the serodiagnosis of CMV infection. The human monoclonal antibodies recognized different epitopes, some of which proved to be overlapping. The fine specificity of these antibodies was evaluated using synthetic peptides covering the sequence of pp65. The antibody MO58 recognized a linear epitope (residues 283-288) whereas antibody MO53 recognized a discontinuous epitope involving residues 208-216 and 280-285. Despite the close proximity of these epitopes, the antibodies did not compete with each other for the same binding site on intact antigen.     

Establishment of hybrid cell lines producing monoclonal antibodies to a synthetic peptide from the E1 region of the hepatitis C virus

We aimed at establishing hybridoma cells secreting monoclonal antibodies (mAbs) against E1 synthetic peptide of HCV. BALB/c mice were immunized with HCV E1-synthetic peptide (GHRMAWDMM) and its spleenocytes were fused with the P3NS1 myeloma cell line. Two highly reactive and specific mAbs (10C7 IgG2b mAb, and 10B2 IgG1 mAb) were generated. The target HCV E1 antigen was identified at approximately 38 kDa in serum of infected individuals. A newly developed ELISA detected the target antigen in 90% of sera from HCV RNA infected individuals with a specificity of 84%. So, the generated mAbs may provide promising probes for serodiagnosis of HCV infection.     

Mouse monoclonal antibodies at the red cell surface--II. Effect of hapten density on complement fixation and activation

Complement (C)-dependent hemolytic dose-response curves of anti-TNP IgG2b and IgM monoclonal antibodies as a function of TNP density were analyzed: sheep red cells coupled with TNP served as targets. Under conditions when equal numbers of either IgG2b or IgM anti-TNP antibodies were taken up by cells with various TNP densities, both antibodies showed optimal activity at a hapten density of approximately 10(6) TNP/E with regard to C-mediated lysis. These results were similar to those obtained with polyclonal antibodies. The effectiveness of monoclonal antibodies in utilizing guinea pig or mouse C was also investigated. IgM anti-TNP monoclonal antibodies lysed E-TNP in the presence of guinea pig C, but failed to produce lysis in the presence of mouse C. Two monoclonal IgG1 (anti-TNP and anti-SRBC) and an IgG2a anti-TNP antibody failed to produce hemolysis in the presence of guinea pig and mouse C. IgG2b monoclonal antibodies, whether directed against TNP or Forssman antigen, activated guinea pig and, to a lesser extent, mouse C. Finally, monoclonal anti-TNP IgG3 antibodies exhibited a low but measurable hemolytic activity with guinea pig C (10-20 times below that of IgG2b).     

Specific IgG antibody subclasses to Angiostrongylus cantonensis in patients with angiostrongyliasis

Total IgG, IgG1, IgG2, IgG3, IgG4, IgA and IgM specific antibodies against Angiostrongylus cantonensis somatic antigen were determined by enzyme-linked immunosorbent assay (ELISA) in sera from proven human angiostrongyliasis (PA) cases, clinically suspected angiostrongyliasis cases with eosinophilic meningitis (EM) and healthy control (HC). The specific IgA antibody in each of the patient groups was significantly higher than those of the HC group (p < 0.05). The mean ELISA value of the specific IgM in the PA group was not significantly different from that of the HC group (p > 0.05). However, the mean specific IgM ELISA value in the EM group was significantly higher than that of the HC group (p < 0.05). The levels of the specific IgG and IgG subclasses in both patient groups were significantly higher than in the healthy control (HC) group (p < 0.001). Major differences were evident in the distribution of the IgG subclass antibodies between the patient groups. The IgG1 antibody demonstrated the highest sensitivity and specificity while the IgM and IgA responses were generally poor in both patient groups. The levels of the specific IgG antibody subclasses possibly explain immune responses to the parasite.     

Production and characterization of monoclonal antibodies to anti-human chorionic somatomammotropin by immunization with two free synthetic peptides

The peptides corresponding to the fragments 135-140 and 166-174 of human chorionic somatomammotropin (hCS) were synthesized, and used to raise monoclonal antibodies to the native hCS molecule. The synthetic peptides were injected into BALB/c mice in the free form, i.e. not conjugated to a carrier, and the spleens were fused with Sp2/01Ag8 myeloma line to produce monoclonal antibodies. The antibodies produced belonged to the IgM and IgG classes and, once purified by affinity chromatography on hCS-Sepharose, they were covalently coupled to macroporous polystyrene beads and characterized by competitive radioimmunoassay. Their affinity constants were determined by elaborating the radioimmunoassay data by nonlinear regression analysis and they were found to range from 10(5) to 10(6) M-1. The evaluation of the affinity constant of the antibodies produced is always important as a measure of the immunogenicity of an antigen, particularly when synthetic peptides are used as immunogens.     

Human monoclonal antibodies specific to hepatitis B virus generated in a human/mouse radiation chimera: the Trimera system.

An approach to develop fully human monoclonal antibodies in a human/mouse radiation chimera, the Trimera system, is described. In this system, functional human lymphocytes are engrafted in normal strains of mice which are rendered immuno-incompetent by lethal total body irradiation followed by radioprotection with severe combined immunodeficient (SCID) mouse bone marrow. Following transplantation, human lymphocytes colonize murine lymphatic organs and secrete human immunoglobulins. We have established this system as a tool to develop fully human monoclonal antibodies, and applied it for the generation of monoclonal antibodies specific for hepatitis B virus surface antigen. A strong memory response to hepatitis B surface antigen was elicited in Trimera engrafted with lymphocytes from human donors positive for antibodies to hepatitis B surface antigen. The human specific antibody fraction in the Trimera was 10(2)-10(3)-fold higher as compared with that found in the donors. Spleens were harvested from Trimera mice showing high specific-antibody titres and cells were fused to a human-mouse heteromyeloma fusion partner. Several stable hybridoma clones were isolated and characterized. These hybridomas produce high-affinity, IgG, anti-hepatitis B surface antigen antibodies demonstrating the potential of the Trimera system for generating fully human monoclonal antibodies. The biological function and the neutralizing activity of these antibodies are currently being tested.     

抗AIV(H9)独特型单克隆抗体的制备及其免疫原性的研究

用H9N2亚型AIV作为抗原免疫接种SPF鸡制备高免血清,用饱和硫酸铵法从该血清中提取免疫球蛋白G(IgG)。以该IgG制备弗氏佐剂疫苗免疫SPFBALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞NS-1在聚乙二醇(PEG)作用下融合,采用间接ELISA检测法,筛选获得3株(2H1D1、2H1G4、3H2D7)分泌特异性单克隆抗体的杂交瘤细胞。3株融合细胞培养上清液中的单克隆抗体效价均为2-4,小鼠腹水中的单抗效价是细胞培养上清液中的300～500倍。经检测表明,3株细胞系产生的单克隆抗体仅与相应的鸡抗H9N2亚型AIV血清发生特异性反应。用2H1D1分泌的抗独特型单克隆抗体制备疫苗与抗AIV灭活疫苗同时免疫SPF鸡收获的高免血清与AIV在MDCK细胞上进行中和实验,中和效价分别为10-1.8/10μl、10-1.6/10μl。由此表明,该抗独特型抗体疫苗具有良好的免疫原性。     

IgG subclasses in human chronic schistosomiasis: over-production of schistosome-specific and non-specific IgG4

IgG subclasses were determined quantitatively in sera from 63 Egyptian men who were infected with Schistosoma mansoni. Total and antigen-specific IgG was measured pre- and post-treatment. Total IgG subclass antibodies were determined by immunoradiometric assay (IRMA) using monoclonal antibodies (MoAbs). The anti-worm and anti-egg specific S. mansoni IgG subclass antibodies were quantitatively measured by ELISA using specific MoAbs and standards obtained by affinity chromatography. Our data show that total IgG of the patients was elevated in the range of two to three times above normal. The magnitude of increase differed markedly among the four subclasses of IgG. The IgG1, IgG2 and IgG3 concentrations were approximately two to four times higher than normal, whereas the IgG4 concentrations was 20 times normal (9000 mg/l). IgG1 and IgG4 tended to dominate the IgG subclass distribution of anti-soluble worm antigen preparation (SWAP) antibodies followed by IgG2 and IgG3. On the other hand, IgG1 and IgG2 dominated the IgG subclass distribution of anti-soluble egg antigen (SEA) antibodies. As with IgG1, IgG2 and IgG3, most IgG4 was non-specific. The role of IgG subclasses in the pathogenesis of schistosomiasis is not clear. However, the high concentration of IgG4 might act as IgE blocking antibody, possibly as anti-idiotypes that may play a role in down-regulation of the immune system when it is challenged with an excess of antigen.     

The quantification of erythrocyte antigen sites with monoclonal antibodies.

The application of monoclonal antibodies to the quantification of blood group antigen sites on erythrocytes was examined. A second antibody technique using labelled anti-mouse IgG could not be used as it was not possible to predict the binding ratio between this and the monoclonal antibody. A series of monoclonal antibodies (R10, R18, BRIC 13, BRIC 14) to the erythrocyte sialoglycoprotein alpha (syn: glycophorin A) showed the number of antigen sites to be from 0.3 X 10(6) to 1.2 X 10(6) per erythrocyte and supported the conclusion that the Wrb antigen is located on this protein. An antibody with a specificity related to the Rh blood group system (R6A) showed 4.6 - 10.4 X 10(4) binding sites to be present on cells of phenotype cCDEe. On cells of phenotype -D- 1.24 X 10(4) binding sites were present but protease treatment increased the number of available sites to 1.3 X 10(5). An antibody to a Kell-related antigen (BRIC 18) recognized 2.5 - 5.9 X 10(3) sites per erythrocyte on cells of phenotype Kk. However, a similar number also appeared to be present on cells of the McLeod and Ko phenotypes although the affinity for the antigen on these cells was very much reduced. The potential of using monoclonal antibodies for this purpose and the value of this in the study of blood group systems has been demonstrated.     

Affinity of anti-peptide antibodies measured by resonance energy transfer

Because of the increasing use of monoclonal anti-peptide antibodies we have undertaken to formulate a general method for the measurement of intrinsic association constants characterizing complex formation between peptide and antibody. The method is based on the phenomenon of resonance energy transfer between tryptophan-excited antibody and an appropriate fluorophor conjugated to the amino terminus of the peptide. The fluorophor we have employed is 8-(2-N-succinylaminoethylamino)-1-naphthalene-sulfonic acid with an absorption maximum at 344 nm and an emission maximum at 500 nm. The model peptide used was the sequence corresponding to residues 48-60 of the regulatory subunit of aspartyltranscarbamoylase. Three IgG and two IgM affinity-purified monoclonal anti-peptide antibodies were used in the fluorescence titration experiments. A maximum value of 2.1 X 10(6) M-1 was found for the IgG antibodies and a maximum of 2.7 X 10(4) M-1 for the IgM antibodies. These limited results suggest similar behavior for the anti-peptide B cell response with respect to affinity maturation as observed for other specificities. In particular it is likely that the IgM affinities are restricted to the potential available in the germline repertoire of variable region genes and, therefore, express only germline affinities.     

Differences between the activities of human monoclonal IgG1 and IgG3 anti-D antibodies of the Rh blood group system in their abilities to mediate effector functions of monocytes.

Seven IgG1 and seven IgG3 human monoclonal antibodies derived from heterohybridoma or Epstein-Barr virus-transformed lymphocytes and specific for the D antigen of the human Rh blood group system were tested for their ability to bring about red cell attachment to and phagocytosis by monocytes. The antibodies produced by the heterohybridomas were also investigated for their potency to mediate antibody-dependent cellular cytotoxicity (ADCC) by monocytes. When red cells were sensitized with any of the IgG1 anti-D antibodies, most of them were ingested by the phagocytes. By contrast, many of the red cells coated with any of the IgG3 antibodies remained attached to the monocyte surface while only few underwent phagocytosis. Some of the attached red cells remained on the phagocyte exterior for a considerable length of time. The ADCC activities of the IgG3 anti-D antibodies was greater than that of the IgG1 anti-D antibodies. The results mean that in vitro IgG1 anti-D mediates red cell destruction mainly by phagocytosis, while IgG3 anti-D causes their destruction predominantly by prolonged cytolysis. These differences between the effector functions of human monoclonal IgG1 and IgG3 anti-D antibodies might have important implications for their use in the prophylaxis of haemolytic disease of the new-born.     

Characterization of a new monoclonal anti-Fc gamma RII antibody, AT10, and its incorporation into a bispecific F(ab')2 derivative for recruitment of cytotoxic effectors.

Fc gamma RII (CDw32) on monocytes is capable of triggering both phagocytosis and lysis of chick red blood cells (CRBC) coated with antibody of the appropriate isotype. In this report we describe the production and characterization of a mouse monoclonal IgG1 antibody specific for Fc gamma RII and compare its activity in binding studies, tissue distribution and redirected cellular cytotoxicity (RCC), with the previously identified anti-Fc gamma RII antibodies KB61 and IV.3. Immunohistochemical and flow cytometry analyses demonstrated that AT10 binds very strongly to Fc gamma RII on normal monocytes, but only weakly to that expressed on lymphocytes. This pattern does not correspond to the staining seen with either KB61 or IV.3, and appears to give an intermediate profile. The binding constant (Ka) for the Fab' fragment of AT10 was calculated at 5.3 x 10(8) M-1, four times higher than that for KB61 (1.4 x 10(8) M-1). Bispecific F(ab')2 antibodies were constructed from Fab' fragments of AT10 or KB61 thioether-linked to Fab' from an anti-CRBC monoclonal antibody. These bispecific derivatives directed monocyte cytotoxicity against CRBC as efficiently as either a monoclonal or polyclonal anti-chick erythrocyte antibody. The bispecific F(ab')2 antibodies had a distinct advantage over the conventional reagents, in that they were not blocked in the presence of human Fc gamma at 3.5 mg/ml (a concentration comparable with that provided by IgG in serum). Therefore, bispecific derivatives constructed with the high affinity anti-Fc gamma RII antibody, AT10, may be used as therapeutic reagents for targeting tumour cell lysis in vivo.