Specific Conjugation Research Articles

A streptavidin mutant containing a cysteine stretch that facilitates production of a variety of specific streptavidin conjugates.

The ability to produce specific streptavidin conjugates has been considerably enhanced by using a streptavidin mutant containing a cysteine stretch, in which sulfhydryl groups serve as unique conjugation sites. A streptavidin molecule containing five cysteine residues at its C-terminus, referred to as Stv-28, was efficiently expressed in Escherichia coli, and purified to homogeneity. Purified Stv-28 had full biotin-binding ability and formed a subunit tetramer. Reactive sulfhydryl groups of Stv-28, derived solely from the cysteine stretch, greatly facilitate the specific conjugation of partner molecules to streptavidin by simple sulfhydryl chemistry. In this manner, S-[14C]carboxymethylated streptavidin and a streptavidin-fluorescein conjugate were prepared. These conjugates contain almost twenty [14C]carboxymethyl groups and fluorescein molecules, respectively, per subunit tetramer, indicating that the sulfhydryl groups of the cysteine stretch are fully reactive. More importantly, these conjugates retain full biotin-binding ability and form subunit tetramers, suggesting that the fundamental properties of streptavidin would be unaffected by the conjugation of other partner molecules to the C-terminal cysteine stretch.

Photodynamic therapy of squamous cell carcinoma. An evaluation of a new photosensitizing agent, benzoporphyrin derivative and new photoimmunoconjugate

Photodynamic therapy for cancer depends on the relatively selective distribution of photosensitizing agents to malignant as compared with normal tissues, rendering the malignant cells more susceptible to light-mediated damage. Photodynamic therapy has been used with only moderate success to date. The purpose of this study was to compare a new photosensitizing agent, benzoporphyrin derivative (BPD), to the standard agent presently in use, photofrin II, in a hamster cheek pouch model of squamous cell carcinoma. As well we have investigated the potential of using a tumour-specific monoclonal antibody-BPD conjugate to improve the tumour localizing properties of BPD. Treatment consisted of photodynamic therapy with either photofrin II, BPD, or a tumour-specific anti-epidermal growth factor receptor-BPD conjugate. Control groups of light alone, anti-EGFr, tumour non-specific MoAb, and tumour nonspecific MoAb-BPD conjugate were included with the contralateral cheek pouch of each animal acting as a dark control. An assessment of differential delivery of BPD to tumour and to normal mucosa was undertaken using a spectrophotometric assay. Parametric statistical analysis included Student's t-tests and linear regression while non-parametric analysis was undertaken using Fisher's exact test. Animals receiving BPD alone demonstrated tumour-to-tissue levels of approximately 2:1 while animals receiving the tumour-specific anti-EGFr-BPD conjugate had significantly better tumour:tissue ratios of 26:1 ( P < 0.005). Animals treated with photofrin II had a 1 month cancer-free survival of 27% while animals treated with BPD had an improved survival of 67% ( P = 0.03) The group treated with the tumour-specific anti-EGFr-BPD conjugate at a twentieth the total dose of BPD had an 80% 1 month cancer-free survival which was not statistically different from the group treated with BPD alone. Benzoporphyrin appears to be a more effective photosensitizing agent than Photofrin II and its tumour selectivity can be improved using a tumour specific monoclonal antibody conjugate.

A quantitative ELISA for measurement of rheumatoid factor associated cross-reactive idiotypes in serum from patients with rheumatic diseases.

A sensitive ELISA for the quantitation of RF associated cross-reactive idiotypes (CRI) has been developed. CRI bearing IgM was captured by immobilized F(ab')2 fragments of monoclonal anti-CRI antibodies and revealed with isotype specific conjugates. Factors affecting sensitivity, specificity and reproducibility of the assay were assessed. The sensitivity limit was less than 10 ng/ml and the linear assay range for the CRI standard curve was from 80 to 500 ng/ml. Using this ELISA system we have quantitated serum levels of the CRI in different groups of patients with RA, early synovitis and normal individuals. Our results indicate a significant increase in the levels of the CRI in the patient groups compared to the normal subjects tested, and suggest involvement of a diverse repertoire of Ig variable region germline genes and/or somatic mutation in production of polyclonal RF in RA.

Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates.

An antigen detection system, termed immuno-polymerase chain reaction (immuno-PCR), was developed in which a specific DNA molecule is used as the marker. A streptavidin-protein A chimera that possesses tight and specific binding affinity both for biotin and immunoglobulin G was used to attach a biotinylated DNA specifically to antigen-monoclonal antibody complexes that had been immobilized on microtiter plate wells. Then, a segment of the attached DNA was amplified by PCR. Analysis of the PCR products by agarose gel electrophoresis after staining with ethidium bromide allowed as few as 580 antigen molecules (9.6 x 10(-22) moles) to be readily and reproducibly detected. Direct comparison with enzyme-linked immunosorbent assay with the use of a chimera-alkaline phosphatase conjugate demonstrates that enhancement (approximately x 10(5)) in detection sensitivity was obtained with the use of immuno-PCR. Given the enormous amplification capability and specificity of PCR, this immuno-PCR technology has a sensitivity greater than any existing antigen detection system and, in principle, could be applied to the detection of single antigen molecules.

Demonstration of a second ligand for the low affinity receptor for immunoglobulin E (CD23) using recombinant CD23 reconstituted into fluorescent liposomes.

Recombinant full-length human CD23 has been incorporated into fluorescent liposomes to demonstrate the existence of a ligand for CD23 that is different from the previously known ligand, immunoglobulin E (IgE). The novel ligand for CD23 is expressed on subsets of normal T cells and B cells as well as on some myeloma cell lines. The interaction of full-length CD23 with its ligand is specifically inhibited by anti-CD23 monoclonal antibodies and by IgE, and it is Ca2+ dependent. Moreover, tunicamycin treatment of a CD23-binding cell line, RPMI 8226, significantly reduced the binding of CD23 incorporated into fluorescent liposomes, and a sugar, fucose-1-phosphate, was found to inhibit CD23-liposome binding to RPMI 8226 cells, suggesting the contribution of sugar structures on the CD23 ligand. In addition, CD23-transfected COS cells were shown to form specific conjugates with the cell line RPMI 8226. These data demonstrate that CD23 interacts with a ligand, which is different from IgE, and that CD23 can be considered as a new surface adhesion molecule involved in cell-cell interactions.

Immunotargeting of daunomycin to localized and metastatic human colon adenocarcinoma in athymic mice.

A monoclonal antibody (designated SF25), which recognizes a protein antigen expressed on a large number of human colon carcinomas, was used for drug targeting. Daunomycin-antibody conjugates were prepared by two previously described procedures. In one, the drug was bound to the antibody through a spacer of small molecular mass (cis-aconitic acid), while in the other a dextran bridge served as the link between drug and antibody. High substitution rates of drug to antibody were obtained using the latter binding procedure. Both conjugates were tested in vitro against two human colon carcinoma cell lines, LS180 and KM-12. The efficacy of a daunomycin-dextran-SF25 antibody conjugate was tested against colon carcinoma LS180 tumors transplanted at different sites into athymic mice. The specific conjugate was significantly more inhibitory to a subcutaneous tumor growth than its components or their mixture. SF25 antibody alone showed antitumoral effects against all three forms of transplanted tumor tested, namely, local, metastatic or intrahepatic, whereas daunomycin, on its own, was effective only against the subcutaneous tumor. Binding of daunomycin to dextran partially improved its inhibitory activity against the metastatic tumor. The conjugate, daunomycin-dextran-SF25 antibody reduced the number of metastatic foci, increased the survival rate and delayed death. Yet against lymph node metastases it was not significantly better than a mixture of both constituents. However, results obtained with an intrahepatic tumor, a model that mimics the natural progression of the disease, resembled those described with the subcutaneous tumor. Daunomycin-dextran-SF25 antibody was significantly more effective than all components separately and than a mixture of drug and antibody, provided a highly drug-substituted conjugate was used.

A streptavidin-metallothionein chimera that allows specific labeling of biological materials with many different heavy metal ions.

We have designed a streptavidin-metallothionein chimeric protein in which the streptavidin moiety provides a means of binding the metallothionein moiety tightly to specific biological targets. A gene fusion of streptavidin with mouse metallothionein I was efficiently expressed in Escherichia coli, and the expressed chimeric protein was purified to homogeneity by a simple procedure. The purified chimera, consisting of four identical subunits, bound one biotin and approximately seven Cd2+ ions per subunit (19.5 kDa). This indicates that both the streptavidin and the metallothionein moieties are fully functional. The high binding affinity of the chimera both for biotin and for heavy metal ions allows the specific labeling or conjugation of any biological material containing unhindered biotin with a variety of different heavy metal ions and their isotopes, thereby opening the way for simultaneous assay systems for a large number of biological targets.

A streptavidin-protein A chimera that allows one-step production of a variety of specific antibody conjugates.

We have expressed a gene fusion of streptavidin and the two immunoglobulin G (IgG)-binding domains of protein A in Escherichia coli. The purified chimeric protein had full biotin-binding ability, and bound one IgG molecule per subunit (31.4 kD). The affinity of the chimeric protein both for biotin and IgG can be used to provide antibody molecules with additional recognition capabilities. For example, the chimera will allow any IgG molecule to be used for sensitive biotin-linked detection systems without the need for chemical modification.

Inhibition of Auxin-Induced Cell Elongation of Maize Coleoptiles by Antibodies Specific for Cell Wall Glucanases

Polyclonal antibodies were raised in rabbits in response to the administration of purified exo- and endoglucanases extracted from cell walls of maize (Zea mays L. B37 x Mo17) coleoptiles. Since the antibodies formed specific conjugates when challenged with the glucanase antigens in immunoblot assays they were employed to evaluate the participation of glucanases in tissue growth. Indole-3-acetic acid induced cell elongation of abraded coleoptile segments was inhibited when the antibodies were supplied as a short term pretreatment (25-200 microgram/milliliter of serum protein). The extent of inhibition of IAA induced cell elongation was additive when endo- and exoglucanase antibodies were applied together. The results suggest that both enzymes have a role in mediating IAA-induced cell elongation. Pretreatment with exo- and endoglucanases antibodies also inhibited IAA induced degradation of noncellulosic beta-d-glucans and the increased level of cellulosic polymers in maize coleoptiles. Antibodies also inhibited the expression of the autohydrolytic degradation of glucans in isolated cell walls. The extent of inhibition was dependent on the antibody concentration applied. The results support the contention that enzymatic processes mediated by exo- and endoglucanases are responsible for cell wall autolytic reactions and that these reactions are linked to the mechanism for expressing auxin induced cell elongation in maize coleoptiles.

Antigen targetting with monoclonal antibodies as vectors—II. Further evidence that conjugation of antigen to specific monoclonal antibodies enhances uptake by antigen presenting cells

Immunization of rats with haptenized monoclonal antibodies (mAbs) against accessory cells enhances anti-hapten antibody responses. To see whether the mAb-conjugates really targetted the antigen (hapten) to the antigen presenting cells, we have investigated the lymph node distribution of locally injected radiolabelled conjugates. Compared with control conjugates, i.e. haptenized non-binding mAbs, a much larger proportion of the specific conjugates were retained in the draining lymph nodes. Whereas control conjugates were rapidly phagocytosed and degraded by macrophages, the specific conjugates were associated with the targetted accessory cells, which were radiolabelled for extended periods. Haptenated MRC OX6 (anti-MHC class II) gave strong labelling of interdigitating cells (IDC) in the paracortex with 70% of IDC still labelled by 4 days and 15% by 16 days following injection. By Western blots intact OX6 conjugates were still detected in the draining lymph node as long as 3 days after injections, whereas control conjugates were hardly detectable even by 24 h. The findings substantiate the idea that mAbs can be exploited for vectorial transport of antigens to accessory cells.

Use of polymyxin-coated polyester cloth in the enzyme immunoassay of Salmonella lipopolysaccharide antigens

Polyester cloth coated with polymyxin B was used to capture Salmonella typhimurium lipopolysaccharide antigens which were then quantitatively or qualitatively assayed using a specific antibody-peroxidase conjugate. This simple, rapid method can be used to assay a large number of samples by employing a large sheet of the polymyxin-coated cloth onto which multiple samples can be blotted. The method is reproducible and economical, since polymyxin B is relatively inexpensive, stable and available in pure form.

An ELISA employing a Haemophilus influenzae type b oligosaccharide-human serum albumin conjugate correlates with the radioantigen binding assay

An ELISA measuring total Ig antibodies to the capsular polysaccharide of Haemophilus influenzae type b (HbPs) in human sera using an antigen composed of Haemophilus b oligosaccharides conjugated to human serum albumin (HbO-HA) was shown to have an excellent correlation to the radioantigen binding assay (RABA). When 214 sera with different anti-HbPs levels were assayed for total Ig by HbO-HA ELISA and by RABA the correlation coefficient was 0.917 and the paired t test p values was 0.575. Use of competitive ELISA employing soluble HbPs, HbO-HA and human albumin as competitors, showed that the HbO-HA ELISA was specific for antibodies to HbPs. The HbO-HA ELISA yielded reproducible results both within and between laboratories. The HbO-HA ELISA can also be used to determine the isotype of anti-HbPs antibodies by using isotype specific enzyme conjugates. The sum of the IgG, IgA and IgM HbO-HA ELISA results had excellent correlation to the RABA results (correlation coefficient = 0.976). Thus, the HbO-HA ELISA can be substituted for the classical RABA and also be utilized for quantitating the isotype of the anti-HbPs antibodies.

Use of conjugates made between a cytolytic T cell clone and target cells to study the redistribution of membrane molecules in cell contact areas

In many models of cell-cell adhesion, it was reported that some cell membrane molecules might be redistributed into contact areas. However, this phenomenon was not subjected to precise quantification. In the present work, fluorescence microscopy, immunolabelling and digital image processing were combined to analyse quantitatively the spatial organization of specific or nonspecific conjugates made with a cytolytic T (CTL) lymphocyte clone (BM3.3) and target cells (EL4 or RDM4). Binding was achieved under calcium-free conditions to study the earliest steps of cell interaction, preceding CTL activation. Fluorescent antibodies were used to label class I histocompatibility molecules on both killer and target cells, and T cell receptor, CD3, CD8 and LFA-1 (CD18/CD11a) on the killer cells. Membrane bilayers were stained with a fluorescent phospholipid, glycoconjugates were labelled with periodic oxidation and Lucifer Yellow uptake, and polymerized actin was revealed with a fluorescent phallacidin derivative. Also, the fine geometry of killer-target interaction area was studied with electron microscopy and computer-assisted contour analysis. It is concluded that: (1) qualitative examination of fluorescence photomicrographs cannot permit accurate comparison between different fluorescence densities. (2) The cell-cell contact area was about fourfold higher in specific conjugates than in non-specific ones. (3) The surface density of adhesion molecules exhibited similar increases (between 30 and 80%) in the contact areas of both specific and nonspecific conjugates. (4) However, the amount of redistributed surface molecules was higher when cell-cell interaction was enhanced either by specific immunological recognition (in specific conjugates) or periodate oxidation. (5) Since redistribution did not require extracellular calcium and it was detected on nonspecific conjugates, this did not require full lymphocyte activation. Spatial reorganization of cell surface molecules may thus be a general consequence of adhesion, cell surface mobility and intermolecular forces.

Maleimidobenzoyl-G-actin: structural properties and interaction with skeletal myosin subfragment-1

We have investigated various structural and interaction properties of maleimidobenzoyl-G-actin (MBS-actin), a new, internally cross-linked G-actin derivative that does not exhibit, at moderate protein concentration, the salt--and myosin subfragment 1 (S-1)-induced polymerizations of G-actin and reacts reversibly and covalently in solution with S-1 at or near the F-actin binding region of the heavy chain (Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032). The far-ultraviolet CD spectrum and alpha-helix content of the MBS-actin were identical with those displayed by native G-actin. 45Ca2+ measurements showed the same content of tightly bound Ca2+ in MBS-actin as in G-actin and the EDTA treatment of the modified protein promoted the same red shift of the intrinsic fluorescence spectrum as observed with native G-actin. Incubation of concentrated MBS-actin solutions with 100 mM KCl + 5 mM MgCl2 led to the polymerization of the actin derivative when the critical monomer concentration reached 1.6 mg/mL, at 25 degrees C, pH 8.0. The MBS-F-actin formed activated the Mg2(+)-ATPase of S-1 to the same extent as native F-actin. The MBS-G-actin exhibited a DNase I inhibitor activity very close to that found with native G-actin and was not to be at all affected by its specific covalent conjugation to S-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of methotrexate-carcinoembryonic-antigen-antibody immunoconjugates on GW-39 human tumors in nude mice.

Methotrexate (MTX) was conjugated to an anti-carcinoembryonic antigen monoclonal antibody (NP2) by using amino-dextran as an intermediate carrier. The drug was chemically linked to amino-dextran (average Mr = 40,000), and the resulting MTX-dextran was then site-specifically attached to the carbohydrate moiety of the antibody. Athymic nude mice that carried human colonic GW-39 tumors (s.c.) were treated with the immunoconjugate. In this study, the specific conjugate caused a greater inhibition of the tumor growth than either free MTX or its conjugate with dextran and an irrelevant antibody. The intermediate MTX-dextran and the unlinked mixture of MTX-dextran with NP2 were both relatively ineffective in inhibiting tumor growth. The greatly reduced host toxicity permitted the use of the MTX-dextran-NP2 in a high-dose therapy of this tumor system.

Influence of Campylobacter jejuni Cecal Colonization on Immunoglobulin Response in Chickens

The immunoglobulin response of chickens to colonization by Campylobacter jejuni isolates B-540 and Clin-1 was monitored. Chicken humoral IgG and biliary secretory IgA (sIgA) responses were assessed by enzyme-linked immunosorbent assay (ELISA). Samples were taken from 128 C. jejuni-colonized chickens and 104 uncolonized chickens housed in a controlled environment. An indirect ELISA was performed using the homologous isolate of C. jejuni as the capture antigen and was developed with the specific goat anti-chicken IgG or IgA alkaline phosphatase conjugates. The ELISA absorbance values of the test samples at 405 nm (serum diluted 1:32 and bile diluted 1:10) were normalized in direct proportion to standard sera and bile sample values. In the colonized chickens, humoral IgG activities were highest at hatch, dropped to their lowest level after 2 weeks, and increased by 8 weeks to levels similar to those detected at hatch. The sIgA activity was lowest at hatch and increased by 4 weeks in colonized chickens while remaining lower in the control chickens. Chickens colonized with isolate B-540 showed a primary sIgA response during the first 4 weeks and reached a plateau over the final 4 weeks. In spite of these limited humoral and secretory immunoglobulin responses, once the chicken ceca was colonized by C. jejuni, the organism persisted throughout the 8-week experiment.

Practical detection of wasabi strain of tobacco mosaic virus by using "cocktail" monoclonal antibodies.

Nine monoclonal antibodies (MABs) were examined for their reactivities to wasabi strain of tobacco mosaic virus (TMV-W) in two indirect ELISA and competitive ELISA. Based on the results, seven epitopes were discriminated on the surface of a particle of TMV-W and one cryptotope was found inside of the virus particle. For practical detection of TMV-W, mixture of different types of MABs, i.e., cocktail MABs, were examined in double sandwich antibody form ELISA (DAS-ELISA). Five MABs with high titers, which recognized different epitopes were chosen to compound the cocktail MABs. Among the combinations of five MABs, that with the highest sensitivity was selected for coating γ-globulin. To obtain the sensitive and specific conjugates, four MABs were used for compounding cocktail conjugate. When the MABs which did not compete in the reaction with antigens were compounded, the highest sensitivity was obtained. When the cocktail γ-globulin and cocktail conjugates were used for detection of TMV-W from infected plants in the DAS-ELISA, the sensitivity was almost the same as that used polyclonal antibodies. Furthermore, the cocktail MABs distinguished TMV-W from crucifer strain of TMV, the differentiation of which was difficult by using polyclonal antibodies.

Comparison of T cell sites recognized by thymic and blood lymphocytes in Myasthenia Gravis patients

We investigated potential therapeutic effects of a conjugate of BAFF receptor specific-monoclonal antibody and short interference RNA in a mouse model of myasthenia gravis (EAMG). Whereas high-dose siRNA conjugate resulted in significant accumulation of Fas expressing CD19+/B220+ cells and concurrent expression of type 1 interferon in lymph nodes, low-dose conjugate did not induce FAS expression but caused marked BAFF receptor deficiency in lymph nodes that was further associated with improved MG symptoms. Unexpectedly, despite inhibiting BAFF receptor significantly in PBMCs and secondary lymphoid organs, conjugate treatment did not reduce the levels of autoantibody. Rather, at high dose, it caused robust increase in high affinity anti-AChR antibody and increased levels of serum IL10 and IL-4 cytokines. Our findings reveal a previously undocumented, dose dependent, immunomodulatory distant effect resulting from BAFF receptor specific mAb-siRNA conjugate treatment in an in vivo model of autoimmune disease.

Quantitation of conjugate formation between human polymorphonuclear leukocytes and antibody-coated target cells by flow cytometry: the role of Fc receptor and LFA-1 antigen.

The specific binding of human polymorphonuclear leukocytes (PMN) to antibody-coated target cells was characterized by flow cytometry. PMN were labeled with phycoerythrin-E (PE) via a granulocyte-specific monoclonal antibody (leu-M1) and mixed with fluorescein isothiocyanate-labeled K562 tumor cells sensitized with rabbit antiserum. Specific conjugates were formed as analyzed by two-color fluorescence in a flow cytometer. The formation of stable conjugates was dependent on initiation of contact, temperature, time, and antiserum concentration. Studies with inhibitors implicate that microfilaments, but not microtubules, Ca2+, Mg2+, or energy-dependent processes were a prerequisite for binding of PMN to the antibody-coated target cells. No conjugates were formed when uncoated target cells were used or when the experiment was performed in the presence of protein A, indicating that binding was specifically mediated through Fc receptors (FcR). Monoclonal antibodies against the FcRII and FcRIII were used to address the role of these receptors in conjugation. One of the two anti-FcRIII antibodies and an anti-FcRII antibody effectively prevented conjugation. A monoclonal antibody directed against the common beta-chain of the adhesion molecule family and a combination of antibodies against the alpha-chain of LFA-1 and Mo-1 also blocked conjugation when target cells were sensitized under suboptimal conditions. The antibody against the beta-chain also diminished killing of antibody-coated K562, as measured by chromium release when included in the cytotoxicity assay. These results indicate that flow cytometry permits accurate quantitation and characterization of the binding between PMN and antibody-coated target cells, which in principle, can be prevented by monoclonal antibodies against surface receptors. Binding is primarily established by both the FcRII and FcRIII. Adhesion-associated molecules on the PMN surface contribute to optimal binding.

Coupling of nonpolymerizable monomeric actin to the F-actin binding region of the myosin head.

Polymerizations of skeletal G-actin induced by salt and myosin subfragment 1 (S-1) were suppressed by reaction of G-actin with m-maleimidobenzoyl-N-hydroxysuccinimide ester. The G-actin derivative, containing few intramolecular crosslinks and a free maleimide group, was covalently coupled in solution to the S-1 heavy chain. The resulting complex could no longer bind to F-actin. The SH-1 and SH-2 thiols of S-1 were not involved in the complexation and the covalent link was shown to be exclusively on the 50-kDa segment of the S-1 heavy chain. The specific conjugation of the two proteins followed formation of a reversibly associated pyrophosphate-sensitive binary complex which was characterized by different approaches. Potentially, these complexes may be useful in developing the crystallography of actin-bound S-1.