Biochemical and genetic analysis of the Oka blood group antigen

The monoclonal antibody TRA-1-85 recognizes a cell surface antigen which is expressed by all human cell types tested, including red blood cells (RBCs), but not by mouse cells. All the human RBCs tested were TRA-1-85 positive except those with the rare phenotype Ok(a^–). Oka is a blood group antigen of very high frequency and only three unrelated Ok(a^–) people are known. The red cells of all three propositi were negative with the TRA-1-85 antibody. To confirm the relationship between the TRA-1-85 antibody and anti-Ok^a, the immune antibody found in the serum of Ok(a^–) individuals, Western blot analysis was used: the TRA-1-85 antibody and anti-Ok^a gave identical but complex patterns of re-activity in Western blot analysis of human cell lysates or membranes. This suggests that the anti-Oka and TRA-1-85 antibodies recognize the same cell-surface determinant and implies that Ok^a is not restricted in its expression to the surface of RBCs but is expressed on white blood cells (WBCs) of Ok(a⁺) individuals and all human cell lines tested to date. WBCs from one of the Ok(a^–) propositi were tested and found to be negative with the TRA-1-85 antibody. Finally, the species specificity of the TRA-1-85 antibody has been exploited by the use of somatic cell hybrids and DNA transfection techniques to examine the genetic control of the Ok^a antigen defined by the TRA-1-85 antibody. We report that the determinant is controlled by a single gene OK present on human chromosome 19.     

Technetium-99m-labeled antibodies

It is essential in any method for radiolabeling antibody with^99mTc that the labeling procedure is rapid and reliable, producing a highly stable^99mTc-antibody complex with minimal effect on the immunoreactivity of the antibody. In the present study, analysis of the stability and homogeneity of radiolabeled (^99mTc and¹²⁵I) antibodies (HMFG1 and PR1A3) was carried out by fast protein liquid chromatography (FPLC) using superose-6 and S-200 columns, and by polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. Superose 6 and S-200 gel filtration analysis showed the radiolabel (^99mTc or¹²⁵I) eluting with a retention time identical to that of native antibody. No peaks of relative molecular size (Mr) corresponding to possible antibody fragments were seen in either the UV or the radioactive FPLC elution profiles. PAGE analysis of^99mTc labeled antibody, however, revealed the presence of a number of radiolabeled antibody fragments (Mr<IgG) that were not detected by the same analysis of¹²⁵I labeled antibody. The stability of the radiolabeled antibodies in serum in vitro was also studied. FPLC (superose-6) analysis after 45 h incubation in normal serum in vitro revealed 3.3% (HMFG1), and 20% (PR1A3) of the^99mTc on a molecule or aggregate with a Mr greater than that of IgG. There is also the appearance of small amounts of^99mTc-labeled material with a Mr<IgG in the later fractions (2.2% for HMFG1 and 4.9% for PR1A3). Similar results were obtained using radioiodinated antibody, although the small amount of low molecular size material detected as a single peak with a longer retention time than the^99mTc equivalent corresponds to free iodide.     

A human/mouse chimeric monoclonal antibody against CA125 for radioimmunoimaging of ovarian cancer

Murine monoclonal antibody 196-14 recognizes the ovarian-cancer-associated antigen CA 125, but the epitope it recognizes is different from that of monoclonal antibody OC125. We developed a human/mouse chimeric 196-14 using the variable regions of the murine 196-14 and human heavy-chain (l) and light-chain () constant regions. Cell binding and competitive inhibition assays using chimeric 196-14 labeled with¹²⁵I,¹¹¹In or^99mTc demonstrated that the in vitro immunoreactivity of the chimeric antibody was identical to that of the parental murine monoclonal antibody. However, in mice bearing human ovarian cancer xenografts, the clearance from blood was faster and absolute levels of accumulation in the tumor were lower for the¹²⁵I-labeled or^99mTc-labeled chimeric antibody than for the murine antibody labeled with the corresponding radionuclides. The tumor-to-blood radioactivity ratio was not significantly different between the chimeric antibody and the murine antibody, regardless of the radionuclide used for labeling. Chimeric antibody 196-14 labeled with¹³¹I,¹¹¹In or^99mTc is promising for the radioimmunoimaging of ovarian cancer.     

Identification and characterization of an anti-isoaspartic acid monoclonal antibody

The deamidation and rearrangement of protein-bound asparagine residues occurs when peptides and proteins are exposed to acidic or alkaline aqueous media. Asn⁹⁹ of bovine growth hormone (bGH) is readily modified via these mechanisms. We have generated a monoclonal antibody (MAb) that interacts with a bGH fragment that contains an isoaspartyl residue. To obtain this antibody, CAF₁/J mice were immunized with [isoaspartyl⁹⁹]-bGH(96–112) conjugated to BSA. Using a competitive ELISA assay, the interaction of this MAb to [isoaspartyl⁹⁹]-bGH(96–112) has been observed to have an apparentK _m of 150 nM. The corresponding native peptide and other bGH fragments do not bind to this antibody with high affinity. For example, the binding affinities of [Asp⁹⁹]-bGH(96–112) and [Glu⁹⁹]-bGH(96–112) to this antibody are 54- and 78-fold lower than the corresponding isoaspartyl peptide. The antibody also binds to bGH that is enriched in isoaspartic acid at position 99, but not to the unmodified protein. The binding epitope of the peptide has been further characterized by comparing the binding of bGH(96–112) analogues to the MAb. Alanine substitution at residues 99, 100, 101, and 103 reduce binding affinity to the antibody by more than 10³-fold. Replacement of valine with alanine at position 102 has much less impact on antibody affinity. Further experiments suggest that the relative insensitivity to this substitution is due to the structural similarity of these sidechains. Other isoaspartic acid-containing peptides not derived from the bGH sequence do not bind to the antibody. We conclude that the epitope binding site of this MAb is highly specific for 99–103 of [isoaspartyl⁹⁹]-bGH(96–112). This strategy can be used to obtain monoclonal antibodies that selectively interact with other proteins that have been similarly modified, or that contain other chemical modifications. The potential for generating antibodies that universally recognize protein- and peptide-bound isoaspartic acid residues is discussed.     

An antibody-desferrioxamine conjugate labelled with 67Ga

The optimum conditions for producing a ⁶⁷Ga labelled antibody-desferrioxamine conjugate were determined. The mouse monoclonal antibody LICR-LON-M8 was coupled to the metal chelating compound desferrioxamine (DFO) using glutaraldehyde (GLUT). A DFO: GLUT: M8 molar ratio of 500: 150: 1 gave an immunoreactive antibody with low amounts (< 5%) of high molecular weight polymer. The labelling efficiency with ⁶⁷Ga was >90%, with a specific activity of 2–4 MBq/mg antibody. This ⁶⁷Ga labelled antibody is suitable for evaluation as a diagnostic imaging agent.     

Modulation of the activity of the transverse tubule Mg2+ATPase from frog skeletal muscle by a monoclonal antibody in vitro

We have established several hybridoma lines that produce monoclonal antibodies against transverse tubule (t-tubule) proteins from frog skeletal muscle. The specificity of these antibodies was characterized by ELISA and Western immunoblotting with purified t-tubule, sarcoplasmic reticulum and partially purified sarcolemmal membranes. One of the monoclonal antibodies (2/34.4) recognizes a band of 109 000 Da in immunoblots. When purified frog t-tubule vesicles were preincubated with this antibody we observed an increase in the rate of the Mg²⁺-ATPase enzyme (up to six fold) which was dependent on antibody concentration. Immunocytological experiments done on cryostat sections of frog muscle indicate that the antigen recognized by this antibody is localized mainly at the level of the t-tubules (I band) and to a lesser extent at the sarcolemma. These results indicate that monoclonal antibody 2/34.4 recognizes the t-tubule Mg²⁺-ATPase and modulates its activity. This antibody should be useful as a probe on studies designed to study the physiological function of the enzyme.Abbreviations t-tubules transverse-tubules - mAb monoclonal antibody - SR sarcoplasmic reticulum - SL sarcolemma     

Factors in the Fermentation-Inhibition Test for Measurement of Growth-Inhibiting Antibody to Mycoplasma pneumoniae

Factors in the fermentation-inhibition test for the measurement of growth-inhibiting antibody in serum to Mycoplasma pneumoniae were studied. The fermentation-inhibiting antibody titer, as read on the day when the color of the control cup without serum had just changed to yellow, was constant among inoculum dilutions of 10^–2 to 10^–5 (10⁶ to 10³ CFU/ml) of a M. pneumoniae broth culture. The use of 10^–3 to 10^–5 dilutions (10⁵ to 10³ CFU/ml) was adequate for inoculation, inasmuch as one day delay in reading did not result in a significant decrease in the test. Heat inactivation of the serum gave no significant effect on the titer. The test was simple and reliable. The growth-inhibiting antibody was shown to be detectable in the test, when the growth of M. pneumoniae was suppressed at least to 1/100 of the growth of the control without serum. The growth-inhibiting antibody titer rose later than the complement-fixing antibody titer in some cases after M. pneumoniae infection. It is suggested that, when an erythromycin-sensitive strain of M. pneumoniae is used, the titer transiently rises and does not show a real growth-inhibiting antibody titer in sera from patients under erythromycin administration.     

Use of Antibody-coupled Cellulose as Immunosorbent in the Estimation of Human Immunoglobulin E (γE)

THE recent discoveries^{1, 2} of myeloma forms of IgE have made possible the development of a sensitive radio-immunosorbent method of estimating antibody IgE (reagin) in normal and pathological sera and other body fluids. The RIST (radioimmunosorbent technique³), as it has come to be known, is based on a method⁴ developed originally for the estimation of protein hormones. It offers a highly sensitive alternative to methods of assaying antibody (reaginic) IgE, being capable of detecting about 1,000 times less than the lower limit of immunoglobulin (1 µg) detectable by the single radial immunodiffusion technique⁵.     

Induction of Group-specific Interspecies Antibody in a Cat by Immunization with Disrupted Feline Leukaemia Virus

“ALL mice, cats and virtually all chickens seem to be completely refractory to developing antibody to the group-specific, gs, antigens characteristic of the RNA tumour viruses of their own species.”¹ This is explained on the basis of an immune tolerance induced in early embryonic life by the expression of these antigens before the development of immune competence. Avian group-specific (gs) antibody has been demonstrated in the sera of immunized chickens by the immunodiffusion (Ouchterlony)² and complement fixation inhibition³ tests. This report is to record the production of gs antibody in a cat which had been immunized with gs antigen from disrupted feline leukaemia virus (FeLV).     

Improved radioimmunotherapy of colorectal cancer xenografts using antibody mixtures against carcinoembryonic antigen and colon-specific antigen-p

Summary Radioimmunotherapy of GW-39 human colonic tumor xenografts grown in the hamster cheek pouch with¹³¹I-labeled NP-4 anti-(carcinoembryonic antigen) (CEA) and¹³¹I-labeled Mu-9 anti-(color-specific antigen-p) (CSAp) murine monoclonal antibodies, administered in combination, was more effective than using either antibody alone for tumor masses less than 0.5 cm³ in size. The antibody mixture had no therapeutic advantage for larger tumors. Therapeutic efficacy was determined by measuring the change in tumor size over time, quantifying the absolute number of tumors responding to radioantibody therapy, and determining the percentage growth inhibition of each treatment at various times after radioantibody administration. Several mechanisms are discussed to explain the improved tumoricidal effect of the antibody mixture noted in this model system, such as (a) the possibility that an antibody mixture could target a greater number of tumor cells, (b) the potential for antibody mixtures to provide better tumor distribution and (c) the possibility that antibodies administered in combination can increase the magnitude of tumor uptake of individual radioantibodies, thereby resulting in a greater radiation dose delivered to the tumor.     

Development of a polyclonal antibody-based AC-ELISA and its comparison with PCR for diagnosis of canine parvovirus infection

A polyclonal antibody-based antigen-capture ELISA (AC-ELISA) has been developed for detection of Canine parvovirus (CPV) antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1:1 600 and 1:400, respectively, in the check-board titration. In this study, a total of 152 samples (129 faecal samples and 23 cell culture supernatant) were tested both by AC-ELISA and by polymerase chain reaction (PCR). Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 10^2.8 TCID₅₀/mL whereas PCR sensitivity was 10^0.8 TCID₅₀/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.     

Autoradiolysis of iodinated monoclonal antibody preparations

We investigated the effects of ionizing radiation on the immunointegrity of antibody fragments (Fab) because large amounts of high specific activity ¹³¹I may damage the proteins. We found that 1000 Gy of external ¹³⁷Cs γ radiation was sufficient to destroy 80–90% of the immunointegrity of the initial preparation. This effect was also produced by internally added [¹³¹I]NaI in a quantity sufficient to provide the same radiation absorbed dose. Since radioiodinated monoclonal antibodies labeled to high specific activity are being evaluated for radioimmunotherapy, the above observation is significant since high levels of internal radiation occur with therapeutic doses of ¹³¹I-labeled antibody. Human serum albumin in low concentration (2%) added to the iodinated antibody solutions was successful in preventing loss of immunoreactivity and can be used to protect and stabilize therapeutic quantities of radiolabeled monoclonal antibody preparations.     

Immunochemical Features of Complementarity Determining Region (CDR) Peptide in Anti Hemin Monoclonal Antibody

Messenger RNA purified from the anti hemin monoclonal antibody (1D3) secreting hybridoma was amplified by RT‐PCR and the nueleotide and amino acid sequences of the antibody were determined. The role of complementarity determining regions (CDRs) in porphyrin recognition and its immunochemical feature of the antibody were investigated by using ELISA, fluorescence measurement and computational calculation of the conformation. All CDR peptides of the heavy chain of the antibody were synthesized and their affinity constants to porphyrins were determined. The value of CDR2 of heavy chain (CDRH2) of 1D3 was 1.5×10⁵/M for protoporphyrin and 7×10⁷/M for TCPP, respectively, while that of the whole antibody showed to be 1.2×10⁷/M for TCPP. Though CDRH2 is a 17 meric peptide, it showed higher affinity than the whole antibody (1D3). Porphyrins can be considered to firmly bind with CDRH2, while CDRH3 is not involved in the antigen binding. CDR‐1 may participate in the recognition with a small contribution. By the computational analysis of steric conformation, it was suggested that CDRH1 and CDRH2 co‐operatively function in the recognition of porphyrin. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Mammary tumor immunoscintigraphy in rats: the use of 131I-anti-estriol 3-sulfate antibody

The scintigraphic imaging of mammary tumors with anti-estriol 3-sulfate (E₃ 3-S) antibody was studied in rats. A chemical carcinogen, 7,12-dimethylbenz(a)anthracine (DMBA), induced mammary tumors in Sprague-Dawley female rats. Highly specific anti-E₃ 3-S antibody was prepared and radioiodinated by [¹³¹I]NaI using the chloramine-T method. At 24 h after administration of ¹³¹ I-anti-E₃ 3-S antibody, goat anti-guinea pig immunogloblin G (IgG) was injected as the second antibody (SA) and nuclear scintigraphy was performed. Mammary tumors were clearly visualized following SA injection.     

Identification of Le antigens in meconium of a phenotypically a Le(ab) non-secretor individual

Blood group active fucolipids of human meconium have been shown to correlate to the ABH and Lewis blood groups and to the secretor status of the corresponding children. Using a monoclonal anti-Le^b antibody and an antibody to chromatogram binding assayy the presence of Le^b antigens in meconium of a phenotypically A Le(a⁺b⁻) non-secretor indivual is here demonstrated. Phenotype was determined on cord blood and saliva obtained 2 years after birth.     

Kinase suppressor of Ras transphosphorylates c-Raf-1

Whether kinase suppressor of Ras1 (KSR1) is an active kinase that phosphorylates c-Raf-1 or a scaffold that coordinates signaling along the Ras/ERK1 signaling module is actively debated. In this study, we generated a monoclonal antibody against a c-Raf-1 peptide containing phosphorylated Thr²⁶⁹, the putative target for KSR1 kinase activity. We show that this antibody detects Thr²⁶⁹-phosphorylated c-Raf-1 in A431 cells upon epidermal growth factor (EGF) stimulation, preceding MEK1 activation. Furthermore, this antibody detects in vitro phosphorylation of FLAG-c-Raf-1 and kinase-dead FLAG-c-Raf-1(K375M) by immunopurified KSR1, but fails to detect phosphorylation of FLAG-c-Raf-1(K375M/T269V), engineered with a Thr²⁶⁹ to valine substitution. To provide unequivocal evidence that KSR1 is a legitimate kinase, we purified KSR1 to homogeneity, confirmed by mass spectrometry, renatured it in-gel, and demonstrated that it phosphorylates BSA-conjugated c-Raf-1 peptide at Thr²⁶⁹. These studies add to emerging data validating KSR1 as a kinase that phosphorylates c-Raf-1.     

Development and Implementation of a Single-Chain Fv Antibody for Specific Detection of Bacillus anthracis Spores

A single-chain Fv (scFv) antibody was developed and applied for efficient and specific detection of Bacillus anthracis spores. The antibody was isolated from a phage display library prepared from spleens of mice immunized with a water-soluble extract of the outer membrane of the B. anthracis spore (exosporium). The library (7 × 10⁶ PFU) was biopanned against live, native B. anthracis ATCC Δ14185 spores suspended in solution, resulting in the isolation of a unique soluble scFv antibody. The antibody was affinity purified and its affinity constant (3 × 10⁸ ± 1 × 10⁸ M⁻¹) determined via flow cytometry (FCM). Preliminary characterization of scFv specificity indicated that the scFv antibody does not cross-react with representatives of some phylogenetically related Bacillus spores. The potential use of scFv antibodies in detection platforms was demonstrated by the successful application of the soluble purified scFv antibody in enzyme-linked immunosorbent assays, immunofluorescence assays, and FCM.     

An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody

Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody–hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen‐binding fragment (Fab) derived from the TN1 antibody (TN1‐Fab). To clarify the mechanism by which hTPO is recognized by TN1‐Fab the conformation of free TN1‐Fab was determined to a resolution of 2.0 Å using X‐ray crystallography and compared with the hTPO‐bound form of TN1‐Fab determined by a previous study. This structural comparison revealed that the conformation of TN1‐Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen‐binding site (paratope) of TN1‐Fab upon hTPO complexation. Interestingly, the heat capacity change (ΔCp) measured by ITC (?1.52 ± 0.05 kJ mol^?1 K^?1) differed significantly from calculations based upon the X‐ray structure data of the hTPO‐bound and unbound forms of TN1‐Fab (?1.02 ～ 0.25 kJ mol^?1 K^?1) suggesting that hTPO undergoes an induced‐fit conformational change combined with significant desolvation upon TN1‐Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition.     

Functional significance of the complement receptors on B lymphocytes

The functional role of complement receptor (CR⁺) lymphocytes in antibody responses was investigated. Initially it was found that in the spleens of 6–8-week-old CBA/H mice only approximately 40% of the B cells were CR⁺. The CR⁺ and CR^? splenocytes were then separated by a recently described fractionation procedure (Parish, C. R., et al., Eur. J. Immunol.4, 808, 1974) and assayed alone or in combination for their ability to transfer a range of antibody responses to irradiated recipients. All of the antigens studied, irrespective of their structure or T-cell dependence, were capable of activating CR⁺ B cells to synthesize antibody. However, only repeating determinant antigens, such as horse red blood cells (HRBC) and dinitrophenyl-polymerized flagellin (DNP-POL), were capable of activating CR^? B cells, the soluble antigen DNP-flagellin (DNP-MON) being unable to trigger these cells. Repeating determinant nature rather than T-cell dependence appeared to be the factor that determined whether an antigen could provoke the CR^? B cells to synthesize antibody, as HRBC and DNP-POL differ widely in their T-cell dependence. The same phenomenon was observed with direct and indirect PFC responses and also with primary and secondary antibody responses. In addition, there was no evidence for collaboration between CR⁺ and CR^? B cells in the induction of antibody responses to the T-dependent antigens, HRBC and DNP-MON. Furthermore, no CR⁺ helper T cells were detected.It is postulated that complement receptors facilitate T-B interaction by stabilizing the union of soluble antigens with antigen-specific receptors on CR⁺ B cells. In contrast, repeating determinant antigens can avidly bind to antigen-specific receptors on B lymphocytes without involvement of the complement receptors.     

Cytolysis of human erythrocytes by a covalent antibody-selenium immunoconjugate

A selenium (Se)-containing immunoconjugate of a human anti-erythrocyte membrane antibody (Ab-Se) has been synthesized via oxidation of the carbohydrate moieties of the antibody and covalent coupling with selenocystamine. The isolated Ab-Se immunoconjugate is shown to be more hemolytic than is selenocystamine when expressed on equivalent selenium basis. Native antibody preincubated with the human erythrocytes prevented hemolysis induced by the Ab-Se immunoconjugate. As observed microscopically, the Ab-Se immunoconjugate caused extensive damage to the erythrocyte membrane and lysis. The cytotoxicity of Se toward the human erythrocytes is believed to be caused initially by the localized generation of superoxide (O₂^·-) within the cell membrane. This is the first demonstration of site-directed immunoselectivity of Se cytotoxicity and demonstrates the potential for a free radical pharmacology based on localized Se-generated O₂^·-.