scholarly journals Standardization of Licorice and TCM Formulations Using Eastern Blot Fingerprinting Analysis

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Yukihiro Shoyama
Keyword(s):  
Cross Reactivity ◽  
Glycyrrhetic Acid ◽  
Spleen Cells ◽  
Pvdf Membrane ◽  
Polyvinylidene Difluoride ◽  
Sugar Moiety ◽  
Eastern Blotting ◽  
Licorice Extract ◽  
Specific Reactivity ◽  
Protein Enzyme

To prepare the antiglycyrrhizin (GC) monoclonal antibody (MAb), GC was treated with NaIO4resulting in aldehyde which can be combined with carrier protein. An antigen conjugate was performed by a matrix-assisted laser desorption/ionization TOF mass spectrometry to determine the hapten numbers in the conjugate. Anti-GC MAb was prepared from a hybridoma which was fixed from the spleen cells producing anti-GC MAb and the myeloma cells after immunization. The TCM and licorice extract were developed by TLC and blotted to a polyvinylidene difluoride (PVDF) membrane. The membrane was treated by NaIO4and protein, enzyme labeled secondary MAb, and finally substrate was added. Clear spot appeared on PVDF membrane identifying GC against a background containing large amount of impurities. In eastern blotting, the GC molecule was divided into two functions. The aglycone part is recognized as an epitope and the sugar moiety can be combined to membrane. The specific reactivity of sugar moiety in the GC molecule against anti-GC MAb might be modified by the NaIO4treatment on the membrane because glycyrrhetic acid 3-O-glucuronide can be stained although the cross-reactivity is only 4.3%. Eastern blotting for GC can not only apply for the standardization of licorice and TCM, but also it can open for the other bioactive products.

Immunoaffinity chromatography of bovine FSH using monoclonal antibodies

1987 ◽  
Vol 115 (2) ◽  
pp. 283-288 ◽  
Author(s):  
K. F. Miller ◽  
R. A. Goldsby ◽  
D. J. Bolt
Keyword(s):  
Monoclonal Antibodies ◽  
Pituitary Hormones ◽  
Immunoaffinity Chromatography ◽  
Cross Reactivity ◽  
Scatchard Analysis ◽  
Spleen Cells ◽  
Chromatographic Techniques ◽  
One Step ◽  
Kappa Light Chains ◽  
Pituitary Homogenate

ABSTRACT Bovine FSH (bFSH) was used to immunize BALB/c mice. Spleen cells were fused to the SP 2/0 cell line to produce hybridomas that secreted monoclonal antibodies to bFSH. One of these antibodies (USDA-bFSH-MC28) was extensively characterized and found to be a gamma 1 with kappa light chains, having extremely low cross-reactivity with other bovine pituitary hormones and with ovine and porcine FSH. The dissociation constant as measured by Scatchard analysis was 4·3 nmol/l, and proved to be in a very useful range for affinity chromatography. In an essentially one-step immunoaffinity chromatography procedure, bFSH was easily isolated in a single chromatographic step from crude anterior pituitary homogenate with better yield and with the same purity as classical chromatographic techniques. J. Endocr. (1987) 115, 283–288

scholarly journals ALLOGENEIC THYMUS GRAFTS AND THE RESTORATION OF IMMUNE FUNCTION IN IRRADIATED THYMECTOMIZED MICE

1970 ◽  
Vol 131 (2) ◽  
pp. 275-286 ◽  
Author(s):  
Alan C. Aisenberg
Keyword(s):  
Cross Reactivity ◽  
Peripheral Lymphocyte ◽  
Third Party ◽  
Spleen Cells ◽  
Immune Restoration ◽  
Immunological Parameters ◽  
Cba Mice ◽  
Skin Homograft ◽  
Skin Donors ◽  
Thymectomized Mice

Irradiated and thymectomized CBA mice are markedly depressed in several immunological parameters (skin homograft rejection, graft-vs.-host activity and hemolytic plaque-forming cells of the spleen, hemolysin and hemagglutinin formation, and peripheral lymphocyte counts). In the present experiments the ability of homografts of neonatal thymus placed beneath the kidney capsule to restore immunological capacity of such animals was studied. Thymus homografts which share the same H-2 locus with the CBA mouse were permanently tolerated and immunological restoration was complete. Skin from the thymus donor was specifically retained, but third party skin with even minor (non-H-2) incompatibility was normally rejected and hemolytic plaque-forming cells of the spleen were restored. Thymus homografts which differ at the H-2 locus were promptly rejected and led to accelerated rejection of skin subsequently grafted from the thymus donor. With such H-2 incompatible thymus grafts, third party skin with minor histo-incompatibility was retained while there was slight to moderate restoration of rejection of skin with major (H-2) incompatibility. Graft-vs.-host activity was restored, but there was no return of plaque-forming spleen cells, hemolysins, hemagglutinins, or peripheral lymphocyte counts. In view of the cross-reactivity at the H-2 locus in CBA mice between thymus and third party skin donors, it was felt that restoration of skin rejection and graft-vs.-host activity could be adequately explained on the basis of immunization by the thymus graft and did not require the postulation of true immune restoration or a thymus hormone.

scholarly journals Use of a synthetic homologue of human fibrinopeptide A for production of a monoclonal antibody specific for the free peptide

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 1036-1044 ◽  
Author(s):  
B Kudryk ◽  
M Gidlund ◽  
A Rohoza ◽  
M Ahadi ◽  
D Coiffe ◽  
...  
Keyword(s):  
Cross Reactivity ◽  
Fibrinopeptide A ◽  
Spleen Cells ◽  
Model Studies ◽  
Free Peptide ◽  
Thrombosis Model ◽  
Patients At Risk ◽  
Alpha 7 ◽  
Clinical Tools ◽  
Equilibrium Dissociation

Abstract It has been shown that epitopes reactive with one group of rabbit antibodies to human fibrinopeptide A (hFPA, A alpha 1–16) are included in its COOH-terminal region (A alpha 7–16). It was further established that Asp-7, Phe-8, and Arg-16 contribute to immunoreactivity and that intact fibrinogen and hFPA-containing fragments react poorly with such antibodies. The purpose of this investigation was to prepare a synthetic peptide corresponding to A alpha 7–16 and use it for generation of FPA-specific monoclonal antibodies (MoAbs). Such probes would allow for development of assays that could measure hFPA directly in plasma. In our approach, an ovalbumin-conjugate of the hFPA homologue served as immunogen. Mouse spleen cells were fused with the immunoglobulin nonsecretor myeloma (P3X63Ag8.653). A hybridoma (8C2–5) has been isolated that secretes an antibody (MoAb/8C2–5) with the following characteristics: (a) IgG1, kappa isotype; (b) equilibrium dissociation constant of 1.5 +/- 0.2 x 10(7) L/mol with the [125I]- labeled N-tyrosyl derivative of hFPA [( 125I] Tyr-hFPA) as ligand; (c) reacts with hFPA and dog FPA (dFPA) but not with the des Arg (A alpha 1– 15) or shorter peptides; (d) does not react with intact fibrinogen or A alpha-chain of human or dog origin; (e) does not react with the elastase-generated hFPA-containing peptide A alpha 1–21. Enzyme-based immunoassays (EIAs) have been developed for measuring plasma hFPA levels in the range 3 x 10(-8) to 5 x 10(-7) mol/L. Since it has already been shown by a number of investigators that hFPA levels in patients with overt defibrination fall into this range, we propose that the MoAb/8C2–5-based assays may serve as useful clinical tools in screening patients at risk of thrombosis. The 8C2–5 antibody may also be helpful in studies dealing with congenital dysfibrinogenemias, particularly in identifying heterozygous propositi with amino acid substitutions at any position within the A alpha 7–16 region. Finally, due to its cross-reactivity with dFPA, assays using this antibody should also be valuable in the canine experimental thrombosis model studies.

scholarly journals Anti-Carbamylated Fibrinogen Antibodies Might Be Associated With a Specific Rheumatoid Phenotype and Include a Subset Recognizing In Vivo Epitopes of Its γ Chain One of Which Is Not Cross Reactive With Anti-Citrullinated Protein Antibodies

2021 ◽  
Vol 12 ◽  
Author(s):  
Pauline Brevet ◽  
Claire Lattard ◽  
Clément Guillou ◽  
Pascal Rottenberg ◽  
Patrice Fardellone ◽  
...  
Keyword(s):  
Prognostic Value ◽  
Cross Reactivity ◽  
Mass Spectrometry Analysis ◽  
Radiological Progression ◽  
Erosive Disease ◽  
Spectrometry Analysis ◽  
Specific Reactivity ◽  
Very Early Ra ◽  
Citrullinated Protein

To identify the targets recognized by anti-carbamylated protein antibodies (anti-CarP) in patients with early Rheumatoid Arthritis (RA), to study the cross-reactivity between anti-CarP and anti-citrullinated protein antibodies (ACPA) and to evaluate their prognostic value. 331 patients (184 RA and 147 other rheumatisms) from the Very Early Arthritis (VErA) French cohort were analyzed. We performed mass spectrometry analysis of RA sera displaying anti-CarP activity and epitope mapping of the carbamylated fibrinogen γ chain to identify immunodominant peptides. The specificity of these targets was studied using competition assays with the major antigens recognized by ACPA. The prognostic value of anti-carbamylated fibrinogen IgG antibodies (ACa-Fib IgG) was compared to that of anti-cyclic citrullinated peptide antibodies (anti-CCP) and anti-CarP using an in-house ELISA. Besides the α chain, the γ chain of fibrinogen, particularly one immunodominant epitope that has a specific reactivity, was identified as a circulating carbamylated target in sera. The prevalence of ACa-Fib was 37% at baseline and 10.9% for anti-CCP-negative RA. In anti-CCP-negative patients, ACa-Fib positivity was associated with a more inflammatory and erosive disease at baseline but not with rapid radiological progression, which remains strongly related to anti-CCP antibodies. Fibrinogen seems to be one of the antigens recognized in vivo by the anti-CarP response, particularly 2 epitopes of the γ chain, one of which is not cross reactive with ACPA. This specificity might be associated with a distinct clinical phenotype since ACa-Fib IgG were shown to be linked to systemic inflammation in very early RA but not to rapid radiological progression.

scholarly journals Use of a synthetic homologue of human fibrinopeptide A for production of a monoclonal antibody specific for the free peptide

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 1036-1044
Author(s):  
B Kudryk ◽  
M Gidlund ◽  
A Rohoza ◽  
M Ahadi ◽  
D Coiffe ◽  
...  
Keyword(s):  
Cross Reactivity ◽  
Fibrinopeptide A ◽  
Spleen Cells ◽  
Model Studies ◽  
Free Peptide ◽  
Thrombosis Model ◽  
Patients At Risk ◽  
Alpha 7 ◽  
Clinical Tools ◽  
Equilibrium Dissociation

It has been shown that epitopes reactive with one group of rabbit antibodies to human fibrinopeptide A (hFPA, A alpha 1–16) are included in its COOH-terminal region (A alpha 7–16). It was further established that Asp-7, Phe-8, and Arg-16 contribute to immunoreactivity and that intact fibrinogen and hFPA-containing fragments react poorly with such antibodies. The purpose of this investigation was to prepare a synthetic peptide corresponding to A alpha 7–16 and use it for generation of FPA-specific monoclonal antibodies (MoAbs). Such probes would allow for development of assays that could measure hFPA directly in plasma. In our approach, an ovalbumin-conjugate of the hFPA homologue served as immunogen. Mouse spleen cells were fused with the immunoglobulin nonsecretor myeloma (P3X63Ag8.653). A hybridoma (8C2–5) has been isolated that secretes an antibody (MoAb/8C2–5) with the following characteristics: (a) IgG1, kappa isotype; (b) equilibrium dissociation constant of 1.5 +/- 0.2 x 10(7) L/mol with the [125I]- labeled N-tyrosyl derivative of hFPA [( 125I] Tyr-hFPA) as ligand; (c) reacts with hFPA and dog FPA (dFPA) but not with the des Arg (A alpha 1– 15) or shorter peptides; (d) does not react with intact fibrinogen or A alpha-chain of human or dog origin; (e) does not react with the elastase-generated hFPA-containing peptide A alpha 1–21. Enzyme-based immunoassays (EIAs) have been developed for measuring plasma hFPA levels in the range 3 x 10(-8) to 5 x 10(-7) mol/L. Since it has already been shown by a number of investigators that hFPA levels in patients with overt defibrination fall into this range, we propose that the MoAb/8C2–5-based assays may serve as useful clinical tools in screening patients at risk of thrombosis. The 8C2–5 antibody may also be helpful in studies dealing with congenital dysfibrinogenemias, particularly in identifying heterozygous propositi with amino acid substitutions at any position within the A alpha 7–16 region. Finally, due to its cross-reactivity with dFPA, assays using this antibody should also be valuable in the canine experimental thrombosis model studies.

scholarly journals A single full-length VAR2CSA ectodomain variant purifies broadly neutralizing antibodies against placental malaria isolates

2021 ◽  
Author(s):  
Justin Y.A. Doritchamou ◽  
Jonathan P. Renn ◽  
Bethany Jenkins ◽  
Michal Fried ◽  
Patrick E. Duffy
Keyword(s):  
Surface Antigen ◽  
Neutralizing Antibodies ◽  
Placental Malaria ◽  
Neutralizing Antibody ◽  
Cross Reactivity ◽  
Full Length ◽  
Broadly Neutralizing Antibodies ◽  
Protective Antibodies ◽  
Broadly Neutralizing Antibody ◽  
Specific Reactivity

Placental malaria (PM) is a deadly syndrome most frequent and severe in first pregnancies. PM results from accumulation of Plasmodium falciparum (Pf)-infected erythrocytes (IE) that express the surface antigen VAR2CSA and bind to chondroitin sulfate A (CSA) in the placenta. Women become PM-resistant over successive pregnancies as they develop anti adhesion and anti-VAR2CSA antibodies, supporting VAR2CSA as the leading PM-vaccine candidate. However, the first VAR2CSA subunit vaccines failed to induce broadly neutralizing antibody and it is still unclear whether naturally acquired protective antibodies target variant or conserved epitopes. This is crucial to determine whether effective vaccines will require incorporation of many or only a single VAR2CSA allele. Here, IgG from multigravidae was sequentially purified on five full-length VAR2CSA ectodomain variants, thereby depleting IgG reactivity to each. The five VAR2CSA variants purified ~0.7% of total IgG and yielded both strain-transcending and strain-specific reactivity to VAR2CSA and IE-surface antigen. IgG purified on the first VAR2CSA antigen displayed broad cross-reactivity to both recombinant and native VAR2CSA variants, and inhibited binding of all isolates to CSA. IgG remaining after depletion on all variants showed significantly reduced binding-inhibition activity compared to initial total IgG. These findings demonstrate that a single VAR2CSA ectodomain variant displays neutralizing epitopes shared by multiple parasites, including maternal isolates, and suggest that a broadly effective PM-vaccine can be achieved with a limited number of VAR2CSA variants.

scholarly journals Application of Monoclonal Antibodies against Bioactive Natural Products: Eastern Blotting and Preparation of Knockout Extract

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Hiroyuki Tanaka ◽  
Osamu Morinaga ◽  
Takuhiro Uto ◽  
Shunsuke Fuji ◽  
Frederick Asare Aboagye ◽  
...  
Keyword(s):  
No Production ◽  
Laser Desorption Ionization ◽  
Double Staining ◽  
Immunoaffinity Column ◽  
New Techniques ◽  
Pvdf Membrane ◽  
Bioactive Natural Products ◽  
Eastern Blotting ◽  
Complete Identification ◽  
Blotting Technique

Matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry was used for the confirmation of hapten number in synthesized antigen. As application of MAb, the MAbs against ginsenosides and glycyrrhizin have been prepared resulting in the development of two new techniques that we named the eastern blotting method and the knockout extract preparation. In eastern blotting technique, glycosides like ginsenosides and glycyrrhizin separated by silica gel TLC were blotted to PVDF membrane that was treated with a NaIO4solution followed by BSA resulted in glycoside-BSA conjugate on a PVDF membrane. The blotted spots were stained by MAb. Double staining of eastern blotting for ginsenosides using antiginsenoside Rb1and Rg1MAbs promoted complete identification of ginsenosides inPanaxspecies. The immunoaffinity concentration of glycyrrhizin was determined by immunoaffinity column conjugated with antiglycyrrhizin MAb resulting in the glycyrrhizin-knockout extract, which was determined by the synergic effect with glycyrrhizin on NO production using the cell line.

scholarly journals Migratory behavior of lymphocytes with specific reactivity to alloantigens. II. Selective recruitment to lymphoid cell allografts and their draining lymph nodes.

1978 ◽  
Vol 147 (1) ◽  
pp. 13-24 ◽  
Author(s):  
E E Emeson
Keyword(s):  
Lymph Nodes ◽  
Tumor Immunology ◽  
Migratory Behavior ◽  
Spleen Cells ◽  
Dual Isotope ◽  
The Mean ◽  
Specific Reactivity ◽  
Multiple Groups ◽  
The Right ◽  
Draining Lymph Nodes

A dual-antigen, dual-isotope assay has been used to monitor the migratory behavior of selectively labeled antiallogeneic lymphocytes in mice challenged subcutaneously in all four foot pads with semiallogeneic spleen cells. 3H-labeled anti-C3H and 14C-labeled anti-C57BL lymphocytes of DBA/2J origin were pooled and adoptively transferred to multiple groups of previously challenged DBA/2J recipients. In some of the studies, separate groups of recipients were challenged with either CDF or BDF spleen cells in all four paws, whereas in others CDF spleen cells were used to challenge the right paws of each mouse in the group and BDF spleen cells to challenge the left paws of each mouse in the group. At intervals varying from 24 to 96 h after challenge, a subgroup of four mice from each appropriate group was sacrificed and the relative numbers of anti-C3H and anti-C57BL lymphocytes present in the challenged paws, draining lymph nodes, and other tissues of each mouse were inferred from the mean 3H/14C ratios of the respective tissues of that subgroup. The results of these studies firmly establish that specific antiallogeneic lymphocytes are selectively recruited to the paws and draining lymph nodes of mice challenged subcutaneously in the foot pads with semiallogeneic spleen cells and are deleted from their circulating blood and nondraining lymph nodes. A mechanism for antigen-induced selective recruitment and its possible functional significance in tumor immunology are discussed.

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