Founder's Award, Society for Biomaterials. Sixth World Biomaterials Congress 2000, Kamuela, HI,May 15-20, 2000. Really smart bioconjugates of smart polymers and receptor proteins.

Over the past 18 years we have been deeply involved with the synthesis and applications of stimuli-responsive polymer systems, especially polymer-biomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymer-protein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article.

Application of a thermally-reversible polymer-antibody conjugate in a novel membrane-based immunoassay.

We have developed a novel method to immobilize antibodies onto a cellulose acetate membrane using a conjugate of an N-isopropylacrylamide polymer covalently bound to the antibody. When compared with the unconjugated antibody, over 30-fold increase in retention of the antibody on the membrane was observed when it was conjugated to poly (N-isopropylacrylamide). Studies of the polymer-membrane interaction suggest a combination of hydrophobic and ionic forces, especially the former, is responsible for the high retention. We applied this novel immobilization technology in the development of a membrane-based immunoassay.

Phase-separation immunoassays.

Solid-phase-based immunoassays have traditionally been plagued by nonspecific binding to the solid phase and by slow reaction kinetics relative to reactants that are free to diffuse in solution. We have developed two novel immunoassays in which the solid phase is generated in situ after the specific binding reaction has occurred, thereby enhancing reaction kinetics and minimizing the opportunities for non-specific binding. In the first system, the capture antibody is conjugated to an organic monomer, polymerization of which to form insoluble polymer particles is initiated by a reaction involving free radicals. The amount of signal-labeled antibody incorporated into the resulting particles is directly proportional to the concentration of antigen. The principle is illustrated for the simultaneous assay of IgG and IgM in a single sample. In the second system, capture antibody is conjugated to a polymer, the solubility of which is a function of temperature. Specific binding is conducted below the critical solution temperature of the polymer, which is then separated from solution by increasing the temperature above the critical temperature. The incorporation of signal-labeled antibody into the precipitated polymer is directly proportional to the concentration of antigen. This principle is illustrated for the assay of hepatitis B surface antigen and Chlamydia trachomatis.

Characterization of monoclonal antibodies to Treponema pallidum.

Thirteen hybrid cell lines which produce mouse monoclonal antibodies to Treponema pallidum, the causative agent of syphilis, have been established. All of the monoclonal antibodies react with T. pallidum, Nichols strain, in ELISA and in immunofluorescence assays, but do not react with normal rabbit testicular tissue in the ELISA. Two of these antibodies were demonstrated to react with the nonpathogenic treponemes T. phagedenis, biotype Reiter, T. refringens (Noguchi strain), T. vincentii, and T. denticola (strains 11 and W), as well as with Borrelia recurrentis, Leptospira interrogans, serogroup Canicola, and the swine pathogen T. hyodysenteriae. The remaining 11 antibodies react with four recently isolated strains of T. pallidum, but with none of the related nonpathogens nor with Borrelia or Leptospira. Thus, our results to date indicate that these monoclonal antibodies may identify antigenic determinants that are specific either for T. pallidum alone or for those treponemes which are pathogenic for humans. The molecular specificities of six of the 13 antibodies were determined by Western blotting. We anticipate potential usefulness of these antibodies in the investigation of the antigenic structure of T. pallidum, the taxonomic study of the pathogenic and nonpathogenic treponemes, and in the diagnosis of syphilis.

Serological classification of Neisseria gonorrhoeae with use of monoclonal antibodies to gonococcal outer membrane protein I.

Identification of strain-specific markers on Neisseria gonorrhoeae that are capable of differentiating gonococci into a large number of distinct classes could facilitate analysis of patterns of gonorrhea transmission and application of gonorrhea control measures. A panel of 12 monoclonal antibodies to gonococcal outer membrane protein IA (PrIA) and IB (PrIB) was used to classify 1,433 strains serologically in a worldwide survey. Eighteen PrIA and 28 PrIB serovars were identified, and a nomenclature is proposed. Gonococcal strains were classified further by auxotyping. Auxotyping and serotyping served to classify the 1,433 isolates into 107 unique auxotype/serovar classes. Dual classification by auxotype and serovar can be used to identify epidemiologically related gonococcal infections in order to test the effectiveness of innovative, focused measures to control gonorrhea.

Culture-independent diagnosis of Chlamydia trachomatis using monoclonal antibodies.

To simplify the diagnosis of chlamydial genital infection, we used a fluorescein-conjugated monoclonal antibody in immunofluorescence tests on smears prepared from urethral or cervical secretions obtained directly from patients. This direct test, requiring less than 30 minutes to perform, was based on the detection of extracellular chlamydial elementary bodies. A comparison of the direct test with cultures stained with iodine on specimens from 926 patients demonstrated a sensitivity of 93 per cent and a specificity of 96 per cent. The direct test provides a rapid, simple, and sensitive method for the diagnosis of chlamydial infection, which can be performed in laboratories that do not have tissue-culture capability.

Monoclonal antibodies to herpes simplex viruses: use in antigenic typing and rapid diagnosis.

Four monoclonal antibodies that react specifically with cells infected with herpes simplex viruses (HSV) are described. One of these antibodies (3-G11) reacts with a glyco-protein C complex (molecular weight, 80,000 and 120,000) that is specific for HSV type 1, while the other three antibodies (6-A6, 6-E12, and 6-H11) react with proteins that are specific for HSV type 2 and that have molecular weights of 140,000, 55,000, and 38,000, respectively. The monoclonal antibodies possessed sufficient specificity to allow rapid serotyping of HSV obtained either from infected cells in culture or directly from patients. In immunofluorescence tests the antibodies were used to serotype 263 culture isolates of HSV, while in preliminary tests performed with specimens obtained directly from patients, the antibodies demonstrated 88% of the sensitivity of tissue culture isolation.

Typing of clinical herpes simplex virus isolates with mouse monoclonal antibodies to herpes simplex virus types 1 and 2: comparison with type-specific rabbit antisera and restriction endonuclease analysis of viral DNA.

A total of 122 clinical isolates of herpes simplex virus (HSV) from 107 patients were typed by using an indirect immunoperoxidase technique with commercially available type-specific rabbit antisera, recently developed mouse monoclonal antibodies to HSV types 1 and 2, and restriction endonuclease analysis of viral DNA. With the commercially available type-specific rabbit antisera, 34% of clinical HSV isolates were of indeterminate type; 63% of them were typed as HSV type 1 and 37% as HSV type 2 by using monoclonal antibody and restriction enzyme typing systems. Typing by immunofluorescence assay with the monoclonal antibodies gave identical results to those obtained by restriction enzyme analysis. Simultaneous infection with both HSV types was demonstrated by monoclonal antibody typing in five isolates from three patients. These findings were subsequently confirmed by plaque purification and restriction endonuclease analysis of viral DNA. Monoclonal antibodies were as sensitive as restriction enzyme analysis for the typing of clinical HSV isolates. Because of their simplicity, they are more amenable to use in clinical laboratories than is restriction endonuclease analysis.

Monoclonal antibodies to cytomegalovirus: rapid identification of clinical isolates and preliminary use in diagnosis of cytomegalovirus pneumonia.

Two monoclonal antibodies which react specifically with cells infected by cytomegalovirus (CMV) are described. One antibody, 6-E3, reacts with a 72,000-dalton protein that appears early in infection and remains localized in the cell nucleus. The other antibody, 6-C5, reacts with an 80,000-dalton protein that appears late in infection and remains localized in cytoplasmic inclusion bodies. Both monoclonal antibodies react with conventional laboratory strains of CMV and can be used in immunofluorescence assays to identify clinical isolates of CMV in culture. Preliminary tests on lung tissues from patients with CMV pneumonia show that only antibody 6-C5 detects CMV infection in primary clinical specimens. A comparison of culture, histological, and immunological methods demonstrates that the monoclonal antibodies possess sufficient specificity and sensitivity to warrant their continued development as immunodiagnostic tools for the detection of CMV infection in both tissue culture and tissues obtained directly from patients.

Serological classification of Neisseria gonorrhoeae with monoclonal antibodies.

Hybrid cells producing monoclonal antibodies against antigens of Neisseria gonorrhoeae were obtained by the polyethylene glycol-mediated fusion of mouse myeloma cells and lymphocytes from mice immunized with gonococcal protein I or outer membrane proteins. From four fusions, 16 phenotypically stable, independently cloned hybrid cell lines were selected for continued study. Each of the cell lines produced a characteristically different monoclonal antibody which reacted in immunoprecipitation assays with a unique antigenic determinant on protein I of the outer membrane complex of the bacteria. In antibody binding, immunofluorescence, and coagglutination assays these antibodies each reacted with a restricted group of N. gonorrhoeae strains. None of the monoclonal antibodies reacted with 17 other different species of Neisseria or with Branhamella catarrhalis. When tested on 34 N. gonorrhoeae reference serotyping strains, the monoclonal antibodies demonstrated serological relationships between the strains which paralleled those observed with conventional polyvalent antisera. These antibodies now provide standardized reagents for the rapid and precise serological characterization of many strains of N. gonorrhoeae.

Monoclonal antibodies to Chlamydia trachomatis: antibody specificities and antigen characterization.

Nineteen independent hybrid cell lines that produce monoclonal antibodies to Chlamydia trachomatis surface antigens were prepared by the fusion of mouse myeloma cells with lymphocytes of mice that were immunized with C. trachomatis immunotypes B, C, and L2. Seven serologically distinct reaction patterns were detected by microimmunofluorescence (micro-IF) of elementary body (EB) preparations when culture fluids were tested against a panel of 18 chlamydial serotyping reference strains. These reaction patterns demonstrated genus-, species-, subspecies-, and type-specific distributions. Additionally, these antibodies were tested in parallel against reticulate body (RB) preparations of several chlamydial strains. Monoclonal antibodies that reacted with genus-specific antigens reacted preferentially with RB, whereas antibodies that reacted to species-, subspecies-, or type-specific antigens reacted equivalently to both RB and EB. Physiochemical characterization of antigens recognized by the different monoclonal antibodies was assessed by heat treatment, pronase digestion, periodate oxidation, and immuno-blot techniques. The genus-specific antigen was a heat-stable, pronase-resistant, and relatively periodate-sensitive component of less than 10,000 m.w. The species-, subspecies-, and type-specific antigens were heat stable, pronase sensitive, and periodate resistant. The antibodies that detected species- and subspecies-specific antigens predominantly reacted in immuno-blots with the 40,000 m.w. major outer membrane protein. These monoclonal antibodies now provide a new approach for the precise serologic classification and detection of different C. trachomatis strains.

Alteration of lymphoid cells in AKR mice by treatment with monoclonal antibody against Thy-1 antigen.

AKR/J mice treated with high levels of monoclonal anti-Thy-1.1 antibody and C' remained healthy without apparent side effects. Examination of the lymphatic organs of these mice demonstrated a selective depletion of T cells in lymph nodes and spleen. Following cessation of treatment of the levels of anti-Thy-1.1 antibody in the circulation fell and the peripheral lymphatic organs gradually became repopulated with T cells. Depletion occurred in lymph nodes whether or not C' was infused along with the antibody. Although the thymocytes of these mice were coated with anti-Thy-1.1 antibody they were not eliminated by the treatment. The elimination of peripheral but not thymic T cells suggests either a thymic barrier to the penetration of cofactors (possibly antibody-dependent effector cells) or that antibody acts by interfering with the normal traffic of peripheral T cells which normally "home" to the lymph nodes and spleen.

Serological and biochemical analysis of the PlA1 alloantigen of human platelets.

A solid phase platelet antibody assay has been developed which rapidly and sensitively detects PlA1 antibodies. The three-step assay is performed by: (1) adhering platelets to the wheels of a microtitre plate, (2) incubating the platelets with test serum, and (3) adding radiolabelled Staphylococcal protein A which binds to the Fc domain of IgG antibodies. Immune reactions are detected by overnight autoradiography. Characterization of the PlA1 antigen was performed by using PlA1 antisera in immune precipitation assays. A 90 000 dalton molecular weight species was precipitated from PlA1 positive human and dog platelets.

Structure of retroviral RNAs produced by cell lines derived from spontaneous lymphomas of AKR mice.

The retrovirus expression of eight independent lymphoid cell lines derived from spontaneous thymomas of AKR mice was investigated. The RNase T1 fingerprints of viral 70S RNA produced by these cell lines were compared with genome structures of the non-leukemogenic Akv virus and with two types of cloned leukemogenic viruses derived from one of the thymoma cell lines. Viral RNAs from three cell lines, SL3, 4, and 7, were indistinguishable from one another. The fingerprint patterns indicated that these cell lines produce equal amounts of two prototype, leukomogenic SL viruses that were previously isolated from the SL3 cell line. Viral RNA produced by the SL1 and SL2 cell lines contained similar components, but at a different ratio. Two other cell lines (SL5 and SL11) produced viral RNAs that resemble those of AKR mink cell focus-forming viruses. One additional line, SL9, produced viral RNA of a novel structure. The complex pattern of viral RNA expression observed for these lymphoid cell lines can be interpreted in terms of recombination among three types of endogenous viral sequences: the Akv virus, a xenotropic virus, and an SL (for spontaneous leukemia) virus.

Monoclonal antibodies recognizing normal human T lymphocytes and malignant human B lymphocytes: a comparative study.

We have undertaken a direct comparison of 8 different murine monoclonal antibodies that recognize antigens shared by human T cells and certain malignant B cells. Immune precipitation, lysostripping, and competitive binding experiments indicate that the antigenic determinants detected by antibodies Leu 1, T101, 17F12, SC1, A50, OKT1, and 10.2 are closely associated on the same molecular species and may in fact be identical. The antigenic determinant recognized by antibody 12.1, which has properties similar to the 1 detected by other antibodies, is present on a distinct cell-surface molecule. Our results emphasize the importance of a quantitative direct comparison of different monoclonal antibodies in distinguishing newly reported antibodies from those already described. Such a comparison can resolve apparent discrepancies that arise either from methodologic variations or from differences in antibody avidity.

Cell-specific cytotoxicity expressed by a conjugate of ricin and murine monoclonal antibody directed against thy 1.1 antigen.

The isolation and characterization are described of a conjugate between ricin, and a thy 1.1-specific monoclonal IgG2a murine antibody synthesized using N-succinimidyl-3-(2-pyridyldithio)propionate. The conjugate selectively inhibited protein synthesis in Thy 1.1-positive (AKR SL3) mouse leukemia cells compared to Thy 1.2-positive (AKR/Cu SL1) cells.