Peter J. Lachmann

Bispecific antibody: a tool for diagnosis and treatment of disease

Antibodies with two distinet binding specificities have great potential for a wide range of clinical applications as targeting agents for in vitro and in vivo immunodiagnosis and therapy, and for improving immunoassays. They have shown great promise for targeting eytotoxic effector cells, delivering radinuclides, toxins or cytotoxic drugs to specific targets, particularly tumour cells. We discuss potential applications of bispecitic antibodies, the theoretical basis and problems associated with their production and purification. Cell fusion and chemical conjugation techniques, and propose a new manufacturing strategy by genetic engineering. This approach has enormous potential applications for producing tailor‐made bispecific antibodies, and will enable widespread clinical uses of these antibodies both for diagnostic purposes and therapy.

A comparative study of IgG subclass antibodies in patients allergic to wasp or bee venom

IgG1 and IgG4 antivenom antibody responses were compared in groups of patients who had experienced systemic reactions to wasp (Vespula spp.) or bee stings. Pretreatment serum IgG4 antibody levels were low in both groups, but IgG4 antibodies were significantly raised in bee‐allergic patients (P<0.002), probably reflecting their greater exposure to stings than wasp‐reactive patients. No direct or indirect relationships were found, in untreated bee or wasp patients, between IgG1, IgG4, or IgE antibody levels and the severity of a patients last systemic reaction to a sting. After a 12‐week course of venom immunotherapy (VIT), IgG1 antibodies increased significantly only in wasp‐sensitive patients (P<0.001), although both groups responded with marked increases in venom‐specific IgG4 (P<0.01). Wasp‐allergic subjects who responded to VIT with high production of specific IgG4 showed the greatest increases (pre‐ to post‐VIT) in IgE antibodies (P<0.05). This group also demonstrated a direct correlation (P<0.05) between post‐VIT levels of IgE and IgG1 antibodies, a finding contrary to an IgE‐immunoregulatory role for IgG 1. High levels of venom‐specific IgG1 alone, or in combination with IgG4, were not protective in three patients who suffered repeated adverse reactions to bee VIT, showing that absolute levels of IgG subclass antivaenom antibodies are not reliably indicative of clinical responsiveness in individual patients.

A competitive inhibition ELISA for the quantification of human interferon-γ

A competitive enzyme immunoassay has been developed for the measurement of human interferon-gamma (IFN-gamma) in cell culture supernatants. The assay is based on the dose-dependent inhibitory effect of liquid phase IFN-gamma on the binding of a specific monoclonal antibody to recombinant IFN-gamma (rIFN-gamma) immobilized on microtitre plate wells. The extent of monoclonal anti-IFN-gamma inhibition was determined by the uptake of alkaline phosphatase-conjugated goat anti-mouse IgG and the subsequent development of enzyme substrate colour. Absorbance readings were taken and results for test samples were extrapolated from standard rIFN-gamma inhibition curves constructed as logit-log plots. Assay performance was assessed using three different monoclonal antibodies (clones 20G7, H-22 and GZ-4). Optimum sensitivity was achieved with the antibodies of higher affinity, 20G7 and H-22, which gave reliable quantification of IFN-gamma over a wide range of concentrations from 0.4 ng/ml (3.4 IU/ml), or less, to 250 ng/ml approximately 2000 IU/ml). The inhibition assay incorporates the advantages of specificity, reproducibility and convenience of performance which are the hallmarks of monoclonal antibody-based ELISAs. However, compared to the sandwich ELISAs previously described for human IFN-gamma, it is considerably more economical in its use of monoclonal anti-IFN-gamma, requiring < 50 ng of a single antibody per 96 well plate. It also uses relatively small volumes of test samples (50 microliters/well) which is particularly advantageous where limited amounts of cell culture supernatant are available for cytokine assays.

TREATMENT OF SYSTEMIC CAPILLARY LEAK SYNDROME

access by conventional techniques. All patients had poor vasculature, multiple failed access procedures, failed chronic ambulatory peritoneal dialysis because of peritonitis, and recurrent sepsis from temporary subclavian vein cannulation. Two patients have had CVC as their sole method of access, without problem for 7 and 14 months, respectively. The third patient had her silastic catheter removed after 5 months after successful maturation of a reconstructed upper limb arteriovenous fistula.

The profiles of interleukin (IL)‐2, IL‐6, and interferon‐gamma production by peripheral blood mononuclear cells from house‐dust‐mite‐allergic patients: a role for IL‐6 in allergic disease

We have developed a model to measure cytokine production by peripheral blood mononuclear cells (PBMC) in vitro. In this report, we examine the production of interleukin‐2 (IL‐2), IL‐6, and interferon‐gamma (IFN‐γ) by PBMC of house‐dust‐mite (Dermatophagoides pteronyssinus)‐allergic subjects. When stimulated with specific allergen (D. pteronyssinus), PBMC of patients produced significant levels of IL‐2 and high levels of IL‐6, but little or no IFN‐γ. Nonatopic control PBMC also produced IL‐6, although at lower levels, but no IL‐2 or IFN‐γ. A ubiquitous antigen, streptokinase/streptodornase (SKSD), induced high levels of IL‐2 in patients, but only low levels of IFN‐γ and IL‐6. Nonatopic controls produced similar levels of IL‐2 and IL‐6, but high levels of IFN‐γ to SKSD. IL‐2 and IFN‐γ levels induced by the T‐cell mitogen phytohaemagglutinin (PHA) were similar in patient and control groups, but IL‐6 levels were significantly lower in the patients. IgE synthesis in vitro was shown only in atopic PBMC cultures stimulated with specific allergen. The major points can be summarized as 1) IL‐2 production by atopic patients in response to allergen; 2) IL‐6 production to allergen by both atopic and nonatopic patients, but significantly increased in atopic patients; and 3) defective IFN‐γ production by atopic patients to both allergen and antigen. These findings suggest that IL‐6 may be important in the immune response to inhalent allergens such as D. pteronyssinus, possibly by creating a cytokine environment favourable to a TH2 response, and that atopic patients exhibit a generalized defect of IFN‐γ production, not related to the response to allergen.

Complement Deficiency and the Pathogenesis of Autoimmune Immune Complex Disease

In the case of the autoimmune immune complex disease it is, however, apparently the effector side that is at fault and this does suggest that it may be advisable to treat asymptomatic patients with deficiencies of the early components of the complement pathway before they develop disease and perhaps to try to avoid prolonged complement depression occurring in disease although it must be confessed that safe and effective methods of so doing are not yet available

Close linkage between mouse genes determining the two forms of complement component C6 and component C7, and cis action of a C6 Regulatory Gene

Two forms of mouse complement component C6, with molecular weights (Mrs) of 90 and 100 kilodaltons (kd), are present in the sera from certain inbred strains such as the CBA strain; other strains, such as the BALB/c and DBA/2 strains, have only the 90 kd C6A form. The present work was undertaken to determine whether the two Mr forms were the products of genes coding at separate loci. We screened sera from mice from a number of inbred strains by isoelectric focusing and found one strain, AKR, exhibiting allotypic structural variations of C6 forms. To distinguish the various types, we designated the 90 kd types from CBA and AKR mice C6A1 and C6A2, respectively, and the corresponding 100 kd types C6B 1 and C6B2, respectively. Mice possessing only one Mr form were all typed as C6A1. Results of breeding experiments strongly suggested that the two Mr forms of C6 are coded for at two closely linked loci. Sera from a number of inbred strains were also screened for a complement C7 polymorphism by means of isoelectric focusing and functional overlay. C7 from all strains, excepting the AKR strain, produced identical C7 band patterns. AKR C7 produced a unique band pattern, and results of breeding experiments with AKR and BALB/c mice showed the C6 and C7 loci to be closely linked. In addition, we identified a regulatory gene for C6 production. The gene apparently requires androgen to facilitate C6 production in the majority of strains. In these strains C6 activity is virtually absent from female sera. However, we observed moderate levels of C6 activity in sera from IS/Cam females, indicating that, in this strain, male physiological androgen levels are not necessary for C6 production. IS/Cam possess one form of circulating C6 which appears identical with BALB/c C6A1, and therefore IS/Cam mice differ from AKR mice at both the C6 structural and regulatory loci. These two strains were thus suitable for use in breeding experiments to determine the manner of action of the regulatory gene. Results showed that it acted in a cis manner.

C7 M/N protein polymorphism typing applied to inherited deficiencies of human complement proteins C6 and C7

C7 M/N typing, the determination of the complement component C7 M/N phenotypes, was successfully used in family studies to trace haplotypes bearing C7 deficiency genes. Furthermore, it was shown to be preferable to CT allotyping based on isoelectric focusing (IEF) since it distinguishes two common alleles (C7*M and C7*N), whereas one common C7IEF allele (C7*I) predominates in most populations. It is also the more sensitive method, as it enabled detection of very low amounts of abnormal C7 molecules in the third generation of a combined subtotal C6/C7‐deficient subject and thus confirmed that this partial deficiency gene is not silent in heterozygotes. In this respect C7 M/N typing is even more informative than DNA restriction fragment length polymorphism typing which will assess the presence but not necessarily the functional status of a gene. C6 and C7 genes are tightly linked and therefore C7 M/N typing was also applied to tracing C6 deficiency genes in families.C6/C7 haplotype analysis of South African C6‐deficient tC6Q0) subjects revealed a strong allelic association of C6*Q0 and C7*M.

Complement components on human lymphocytes

Abstract Lymphocytes isolated from normal human EDTA-blood were tested for the presence of cell membrane-bound complement components by reaction with specific antibodies coupled to trypsin-treated ox erythrocytes. The highest proportions of positively reacting cells were found to have C4 and β1H-globulin, which were present on 11 and 9% (mean values) of lymphocytes, respectively. Other complement components, C1q, C3, and C6, were detected on approximately 3% lymphocytes. C4 was also demonstrated on tonsil and lymph node lymphocytes. All of the cells carrying complement were C3-receptor-bearing, Ig-positive, B lymphocytes, a proportion of which also had receptors for IgG-Fc.

Enzyme immunoassay detection of circulating immune complexes by monoclonal antibodies to C3 neoepitopes with special reference to IgG concentration and to interfering anti-immunoglobulin antibodies

A sensitive solid-phase anti-C3 enzyme immunoassay for detection of circulating immune complexes (CIC) is described. A mixture of the monoclonal antibodies (MoAbs) bH6 and Clone 9 specific for neoepitopes on C3 activation products was used as capture reagent. MoAb bH6 recognized C3b, iC3b and C3c, and Clone 9 recognized iC3b and C3dg. Detection antibody was a polyclonal peroxidase-conjugated rabbit anti-human Ig antiserum. A quantitative assay was constructed using serum incubated with heat aggregated IgG (HAG) as standard. The lower detection limit was 5 micrograms/ml of HAG. Interassay and intra-assay coefficient of variation was 15% and 5%, respectively. Anti-animal immunoglobulin antibodies were detected both in normal and pathological sera. This activity was efficiently absorbed by nonimmune immunoglobulins added to the samples. The present assay was compared with a polyethylene glycol precipitation assay for CIC determination. The latter assay was strongly influenced by the IgG concentration (rs = 0.78; P = 0.006), whereas no such correlation was seen for the anti-C3 immune complex assay (rs = -0.30; P = 0.20).