anti-R Antibodies Research Articles

Electrochemical Biosensor for the Determination of Specific Antibodies against SARS-CoV-2 Spike Protein

In this article, we report the development of an electrochemical biosensor for the determination of the SARS-CoV-2 spike protein (rS). A gold disc electrode was electrochemically modified to form the nanocrystalline gold structure on the surface. Then, it was further altered by a self-assembling monolayer based on a mixture of two alkane thiols: 11-mercaptoundecanoic acid (11-MUA) and 6-mercapto-1-hexanol (6-MCOH) (SAMmix). After activating carboxyl groups using a N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloride and N-hydroxysuccinimide mixture, the rS protein was covalently immobilized on the top of the SAMmix. This electrode was used to design an electrochemical sensor suitable for determining antibodies against the SARS-CoV-2 rS protein (anti-rS). We assessed the association between the immobilized rS protein and the anti-rS antibody present in the blood serum of a SARS-CoV-2 infected person using three electrochemical methods: cyclic voltammetry, differential pulse voltammetry, and potential pulsed amperometry. The results demonstrated that differential pulse voltammetry and potential pulsed amperometry measurements displayed similar sensitivity. In contrast, the measurements performed by cyclic voltammetry suggest that this method is the most sensitive out of the three methods applied in this research.

The biology of multiple myeloma

Epstein-Barr virus immediate early genes BZLF1 and BRLF1 encode the Z and R proteins respectively. Elevation of the anti-Z immunoglobulin (Ig) antibody titer is a common feature of nasopharyngeal carcinoma (NPC) in areas where the disease endemic, whereas R levels have not been examined. This study was performed to analyze antibody titers against Z and R in Japan. Sera from 14 patients with newly diagnosed NPC, 7 with NPC in complete remission, 15 with head and neck squamous cell carcinoma (HNSCC), and 31 controls were evaluated by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Both anti-Z and anti-R Ig were significantly increased in sera from patients with NPC compared with those in remission, those with HNSCC, and controls (Z, P = 0.0016, = 0.0344, = 0.0002; R, P = 0.0015, = 0.0004, <0.0001 respectively). By immunoblot analysis, anti-Z and anti-R Ig were detected in 9 of 9 cases of NPC, 1 of 3 cases NPC in remission, and 2 of 13 cases of HNSCC. Anti-Z and anti-R antibody titers were nine times higher in NPC than in other groups. None of 15 control cases showed positive reactivity against either Z or R. Coupled evaluation of anti-Z and anti-R Ig by ELISA might be a useful system for screening NPC.

Cystic Fibrosis Gene and Protein Expression during Fetal Lung Development

The cystic fibrosis transmembrane conductance regulator (CFTR) transports Cl- in the apical membrane of secretory epithelial cells. Normal lung development is critically dependent on active Cl- secretion by developing airway epithelia. In this study, the polymerase chain reaction (PCR) was used to detect CFTR mRNA expression in rabbit lung at different stages of development. Using nested amplification in exons 7 and 11, a 494-bp fragment was isolated from cDNA prepared from lungs obtained at gestational days 22 (pseudoglandular) and 29 (terminal sac). Nucleotide sequencing verified the identification of rabbit CFTR sequences. At day 22 of gestation, CFTR was detected in fetal liver, kidney, heart, intestine, and brain. CFTR could not be amplified from adult rabbit brain. CFTR protein was detected using a polyclonal antibody raised in chickens against a synthetic peptide from the R domain conjugated to thyroglobulin. Rabbit CFTR migrated as a 165-kD protein on 5% sodium dodecyl sulfate polyacrylamide gels when detected either by chicken anti-R IgG or a previously characterized rabbit anti-R antibody. In frozen sections of fetal rabbit lung at day 22 of gestation, CFTR was localized to the membrane regions of primitive tubular epithelial cells and absent from surrounding connective tissue. At day 29, CFTR was concentrated in the apical region of bronchiolar epithelial cells and absent from alveoli and vessels. These results suggest that CFTR gene expression begins very early in lung development and, with differentiation of the airways, becomes confined to differentiated bronchiolar epithelium, the presumed site for cAMP-mediated Cl- secretion.

CAMP-dependent protein kinase isozymes in porcine follicles and corpora lutea

Our purpose was to identify regulatory (R) subunits and their associations with catalytic (C) subunits to form cAMP-dependent protein kinase (A-kinase) holoenzymes in select porcine ovarian tissues during follicular differentiation. Soluble extracts of small and preovulatory follicles and corpora lutea (CL) were separated on DEAE-cellulose chromatography. R subunits were labeled with 8-N 3[ 32P]cAMP or with [α- 32P]ATP under RII autophosphorylation conditions and were identified by molecular weight ( M r) determination on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as well as cross-reactivity of unlabeled subunits with anti-R antibodies. A-kinase holoenzymes and C subunit-free R (free R) subunits were distinguished on the basis of DEAE elution position and sedimentation position on sucrose density gradient centrifugation of phosphotransferase and [ 3H]cAMP binding activities. In small and preovulatory follicles and CL we identified a minor peak of type I A-kinase containing RIα ( M r = 47,000) and a major peak of type II A-kinase holoenzyme containing two RII isoforms ( M r = 52,000 and 56,000). Notable amounts of free RIα eluted between the type I and II holoenzymes in all three tissues. Neither of the holoenzymes nor free RIα was regulated as a function of follicular differentiation or CL formation. In contrast, free RII subunits were moderately increased in preovulatory follicles relative to levels in small follicles and CL. We conclude that only the RII subunits are hormonally regulated in developing follicles, and in tissues which express both RI and RII subunits, the RII subunits preferentially associate with C subunits to form the dominant holoenzyme despite the presence of significant amounts of RI.

Cloning of the crystalline cell wall protein gene of Bacillus licheniformis NM 105.

A protein with a tetragonal pattern, defined as RS protein, was found on the wall surface of an alkaline phosphatase secretion-deficient mutant (NM 105) of Bacillus licheniformis 749/C. The protein was present on the wall surface of the exponential-growth-phase cells, but at the stationary growth phase it was overproduced and hypersecreted. This protein was precipitated to homogeneity from the culture fluid by 80% ammonium sulfate saturation and chilled acetone. The molecular mass of the protein was 98 kilodaltons, and it had a single subunit in a sodium dodecyl sulfate gel. Specific anti-RS antibody was generated in rabbits and used to immunolabel the RS protein on the cells at different growth phases. In early-exponential-growth-phase cells, the outside surface of the wall, the cytoplasm, and the inside surface of the cytoplasmic membrane were labeled. In stationary-growth-phase cells, the cytoplasm was poorly labeled, but the labeling on the outside surface of the wall was high. AB. licheniformis NM 105 gene library was made by using the lambda phage EMBL3. The RS protein expression from this gene library was detected by a modified autoradiographic procedure. One of the amplified RS protein-positive plaques (4213-1) containing recombinant DNA was chosen, and the restriction map of this DNA was prepared. The RS protein expressed in Escherichia coli NM 539 infected with 4213-1 recombinant phage had a lower molecular mass than the purified authentic RS protein. The 4.5-kilobase-pair (kbp) SalI-EcoRI fragment of the recombinant DNA was cloned in the shuttle plasmid pMK4 to construct pMK462, which was expressed in B. subtilis MI112 and produced the RS protein identical in molecular mass to the purified authentic RS protein. The RS protein expression was also demonstrated in cryosections of transformed E. coli and B. subtilis cells by immunoelectron microscopy. The 1.2-kbp SalI-HindIII and 1.8-kbp HindIII-HindIII recombinant DNA restriction enzyme fragments, respectively, from the right of the restriction map produced anti-RS antibody cross-reacting proteins. The expression of the 1.2-kbp SalI-HindIII DNA fragment cloned in pUC8 could be induced with isopropyl-beta-D-thiogalactopyranoside. The 1.8-kbp DNA restriction fragment hybridized with both the chromosomal DNA of strain NM 105 and the recombinant phage 4213-1 DNA. The RS gene expression was finally demonstrated in transformed E. coli 539 cells by in situ hybridization of frozen thin sections with the 1.8-kbp HindIII biotin-dATP probe and immunolabeling these with anti-biotin immunoglobulin G and protein A-gold.

Inhibition of allergic reactions with monoclonal antibody to the high affinity IgE receptor.

A mAb that reacts with the high affinity IgE-R on the rat basophilic leukemia cells (RBL-2H3) was used to inhibit allergic reactions. In vitro, the intact mAb BA3 and its Fab fragment inhibited radiolabeled IgE binding to the RBL-2H3 cells. The mAb binds to the IgE-R with a higher affinity than does IgE. Whereas the intact mAb released histamine from the RBL-2H3 cells, the Fab was inactive. The addition of the Fab fragments to RBL-2H3 inhibited the IgE-mediated histamine release reaction. The Fab fragments also inhibited in vivo passive cutaneous reactions in rats when injected intradermally either before or after IgE. The injection of the mAb Fab i.v. before the injection of the IgE into the skin sites also inhibited reactions, although it was less effective. The results demonstrate that anti-R antibodies can be used as a model for inhibiting immediate hypersensitivity reactions.

The biologic activity of anti-T cell receptor V region monoclonal antibodies is determined by the epitope recognized.

We have used 10 independently isolated mAb reactive with the Ag R on a cloned Th cell line to map three distinct epitopes and three subepitopes on the R. One of these epitopes is clearly on the V beta 8 region, as it is defined by the antibodies KJ-16 and F23.1, known to react with the V beta 8 family of variable regions, and a functional rearranged V beta 8 gene has been cloned from this cell line. Antibodies directed at a second epitope, believed to be on V alpha because it is unaffected by anti-V beta antibodies, are completely inhibited from binding by monoclonal anti-CD3 epsilon-chain antibodies. Because the cloned Th cell line used, D10.G4.1, responds to soluble monoclonal anti-TCR antibodies, it has been possible to compare the binding of anti-R antibodies with their ability to activate this cloned T cell line. We find that for antibodies all specific for the same or a closely related epitope, activation is proportional to binding, by using antibodies that differ by greater than 100-fold in avidity for the R. By contrast, antibodies directed at different epitopes on the R differ markedly in their ability to activate the D10.G4.1 cell line. We have tested whether these differences reflect differences in the orientation of cross-linking the TCR or possible conformational changes induced in the R by the antibodies, and our data support the latter hypothesis as an explanation for the differences in activation potency between antibodies.

Activation of cyclic AMP-dependent protein kinase isoenzymes: Studies using specific antisera

A novel method for rapidly determining the amount and degree of association-dissociation of the Type I and Type II cAMP-dependent protein kinases has been developed and validated. Antibodies directed against the regulatory subunits of Type I and Type II cAMP-dependent protein kinases were used. The antibodies formed complexes with holoenzymes and regulatory subunits which were precipitated by goat anti-rabbit IgG (immunoglobulin G). These complexes bound [ 3H]cAMP with an apparent K b of 20 n m for protein kinase I and 80 n m for protein kinase II. Immunoprecipitated protein kinases I and II were catalytically active when incubated with cAMP, [γ- 32P]ATP, and histone H2B. When mixtures of the two kinase isoenzymes or cytosol were incubated with various amounts of [ 3H]cAMP and the isoenzymes were separated by precipitation with antisera specific for each isoenzyme, the amount of [ 3H]cAMP associated with immunoprecipitates was proportional to the concentration of [ 3H]cAMP. In contrast, the catalytic activity that was immunoprecipitated varied inversely with the concentration of [ 3H]cAMP, showing that the activation of protein kinase could be assessed by the disappearance of catalytic activity from the immunoprecipitates. In the absence of MgATP protein kinase I was activated by a 10-fold lower concentration of cAMP than protein kinase II. However, when MgATP was added to the incubation, there was no significant difference in the binding of [ 3H]cAMP or dissociation of catalytic subunits of the two isoenzymes. The anti-R antibodies were also used to rapidly quantitate the concentration of regulatory subunits and the relative ratio of protein kinases I and II in tissue cytosols.

Isolation of a low molecular weight constituent from Kentucky blue grass pollen possessing carrier activity of high molecular weight constituent(s).

An immunologically active fraction, designated as C-I-3 was isolated from the dialysate of the aqueous extract of defatted Kentucky Blue Grass pollen by differential dialysis and gel filtration. This fraction was shown to consist of a glycoprotein with a molecular size corresponding to 4,800 daltons and with a pI value of 3.6, and to contain all amino acids except cysteine. The intraperitoneal administration of C-I-3 in saline into mice or rats prior to their immunization with the nondialyzable constituents of the aqueous extract of Kentucky Blue Grass (i.e., the retentate, R) in presence of Al(OH)3 resulted in the enhanced production of anti-R IgE antibodies. The enhancement of the anti-R murine IgE response was shown in adoptive cell transfer experiments to be due to the generation of R-specific T helper cells. Hence, it is suggested that the structural elucidation of C-I-3 may prove useful in establishing the nature of carrier determinant(s) of the major allergenic components of Kentucky Blue Grass pollen.

Human secretory component—I. Evidence for a new antigenic specificity

In the present work, we have studied the antigenically reactive regions of S.C. with immune sera of well-defined specificity. Immunochemical analyses conducted with an I.S. containing only precipitating antibodies against a hidden S.C. group of determinants, unravels the existence of a new antigenic specificity that we chose to call provisionally “R” (from mercapto-resistant). R was shown to be completely independent from Brandtzaeg's I and A 1 by its resistance to strong reduction and to be distinct from A 2 by the fact that anti-R antibodies do not precipitate sIgA or tryptic S.C. The experiments described show that R occurs unaltered on spontaneously degraded S.C. as well as on the 35.000 daltons fragment from peptic digestion. By crossed passive hemagglutination inhibition two distinct parts of the R specificity were found to occur on sIgA and tryptic S.C. respectively. The R specificity could be located on the S.C. near the site(s) where this molecule is fixed on IgA.

Identification of anti-A + M and anti-R antibodies in cattle vaccinated with rough strain Brucella abortus

Identification of anti-A + M and anti-R antibodies in cattle vaccinated with rough strain Brucella abortus

Transplantation Tolerance at the T-Lymphocyte Receptor Level II.

CBA T lymphocytes deprived temporarily of receptors for alloantigens A[RS(A)] cultivated &lt;i&gt;in vitro &lt;/i&gt;for 30 h with anti-receptor antibody-forming (AxCBA)F&lt;sub&gt;1&lt;/sub&gt; spleen cells were capable of resynthesizing RS(A) if primed F&lt;sub&gt;1&lt;/sub&gt; cells exceeded parental T cells by a factor of 25 or less, but not if the excess was 50-fold or more. This indicated that resynthesis of CBA T-cell RS(A) was successful if primed F&lt;sub&gt;1&lt;/sub&gt; cells formed insufficient amounts of anti-CBA T-cell RS(A) antibody. Abortive or successful receptor resynthesis was measured by two parameters, (a) reappearing RS(A) formed PAR together with A alloantigens of (A&amp;#215;CBA)F&lt;sub&gt;1&lt;/sub&gt; spleen cells and (b) budding receptors bound anti-receptor antibody. CBA B lymphocytes did not interfere with these reactions. A search for putative T suppressor cells in the F&lt;sub&gt;1&lt;/sub&gt;cell population was unsuccessful. PAR formation and anti-RS antibody consumption by reappearing receptors differed temporally: receptors forming PAR were present after a delay lasting 8 h; receptor structures fixed anti-RS antibody as early as 5 h after being cultivated. With due caution, these results might reflect processes operating in maintenance of transplantation tolerance, suggesting that this condition is a serum-mediated suppression of long duration. The suppression would encompass continued neutralisation of receptors for the alloantigen to be tolerated by anti-T-cell receptor antibody formed by the F&lt;sub&gt;1&lt;/sub&gt; chimeric cells within an animal with acquired transplantation tolerance.

Spontaneous release of T cell receptors for alloantigens. II. Induction of antibodies to T cell receptors.

Spleen cells from normal mice, when cultivated in vitro, released receptors or recognition structures (RS) for alloantigens into the surrounding medium. The spontaneous shedding of receptors was revealed by their ability to induce the formation of anti-recognition structure(anti-RS) antibodies upon injection into appropriate F1 hybrid recipients. Cell suspensions containing T and B lymphocytes and those containing T lymphocytes were capable of inducing anti-RS antibody formation, whereas suspensions devoid of T cells were incapable of doing so. Receptors shed from such cell suspensions during a 24-hour cultivation period gave exactly the same results. Cell-free culture supernatants, however, incited higher anti-RS antibody titers, presumably because of an accumulation of RS. The capacity of released T cell receptors to recognize alloantigens as determined in the PAR assay and their ability to induce anti-RS antibodies went roughly parallel.

Activation of T and B thymus cells to recognize histocompatibility antigens

Lethally irradiated (A × CBA) F 1 or (A × C57BL/6) F 1 mice were injected with 10 7 A strain thymus cells in attempts to activate donor cells to recognize CBA or C57BL/6 histocompatibility antigens, respectively. Activation could be revealed by injecting activated thymus cells (day 5 irradiated F 1 hybrid spleen cells) into corresponding unirradiated F 1 hybrid hosts. The alloantibody titers formed by these cells and the antirecognition structure (anti-RS) antibody titers induced by them were similar to those observed after injection of normal parental strain spleen cells, indicating that thymus cells had become endowed with recognition structures (RS). Alloantibodies, but no anti-RS antibodies, were present in the serum of F 1 mice given activated thymus cells treated with anti-θ and complement. It, therefore, appeared that activated thymus cells contained sufficient B cells differentiated into antibody-forming cells to give a measurable alloantibody response. On the other hand, receptors responsible for anti-RS antibody induction presumably were located on T cells. Specificity and restriction of antigenic recognition were revealed by negative results obtained when activated thymus cells were injected into F 1 hosts not containing the antigens against which activation had been directed.

Factors affecting the chain length of group A streptococci. II. Quantative M-anti-M relationships in the long chain test.

Minute amounts of M protein were detected in culture supernates of virulent Group A streptococci by type-specific inhibition of the long chain and the bactericidal tests for anti-M antibody. The amount of M protein that was detected by the inhibition of these biological systems was less than could be demonstrated by precipitation tests. All strains of streptococci rich in M protein which were studied formed long chains when grown in sufficient concentrations of anti-M antibody. Very low concentrations of anti-M antibody escaped detection by the long chain test when strains of excessive M protein content were employed. Under such conditions the bactericidal test detected anti-M antibody more sensitively than the long chain test owing to the smaller inoculum employed in the former method. The scission of streptococcal chains may be inhibited by union of antibodies with surface antigens other than M protein. Long chains were formed when M-negative, R-positive strains were grown in sera containing anti-R antibody.

IN VITRO TRANSFORMATION OF PNEUMOCOCCAL TYPES

1. Type-specific S pneumococci may be transformed from one specific S type into other specific S types entirely by in vitro methods. 2. R forms of pneumococci, derived from S forms of one specific type, may be transformed into S forms of other specific types by the following in vitro procedure:-the growth of small inocula of R forms in media containing vaccines prepared from heterologous S cultures. 3. Transformation of type may be effected in this procedure by the use of small quantities of S vaccine,-quantities representing the bacteria from as little as 0.1 cc. of the original culture. 4. Transformation of type, as induced by this procedure, is most readily effected by employing anti-R serum in the culture medium. Transformation of type may be effected, however, in media which do not contain anti-R antibodies. 5. Previous findings on the thermal characteristics of the property of S vaccines responsible for transformation of type have been confirmed and extended.