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Enhancement of anti-phospholipid antibody activity by Tween 20

Ming CH, Fan YS

J Immunol Methods, 1988 May 09;109(2):253-5.
PMID: 3361136

Addition of the non-ionic detergent Tween 20 to the serum diluent enhances anti-cardiolipin binding reactivity in an ELISA system. Maximal enhancement was obtained using a concentration of 0.05% Tween 20 in the diluent. Non-specific interactions were also considerably reduced.

Matched MeSH terms: Antibodies/immunology*
HLA-DRB genes and antiribosomal P antibodies in systemic lupus erythematosus

Teh LS, Doherty DG, Williams BD

Br J Rheumatol, 1994 Dec;33(12):1125-6.
PMID: 8000739

Antibodies to the ribosomal P protein are specific for SLE but their prevalence varies in different ethnic groups. In a group of Chinese SLE patients from Malaysia who have a high prevalence of this antibody, we have found an increased frequency of an uncharacterized HLA-DRB gene allele, DR16X, in patients who are positive for anti-P antibodies compared to antibody negative patients (31.3% vs 3.2%, P < 0.01, Pcorr not significant, relative risk = 13.6). DR16X has only been found in south east Asian populations and may be a genetic factor which influences the high prevalence of anti-P antibodies in Chinese.

Matched MeSH terms: Antibodies/immunology
Fulltext Acquired haemophilia--a therapeutic challenge

Teh A, Leong KW, Bosco JJ, Koong PL, Jayaranee S

Med J Malaysia, 1995 Jun;50(2):166-70.
PMID: 7565188

Acquired haemophilia is a rare clinical condition arising from the spontaneous development of inhibitors to factor VIII. We describe two cases encountered in the University Hospital over the past five years. We also review the literature and discuss the therapeutic difficulties faced in dealing with patients with high levels of inhibitors. In one of these patients we also describe, for the first time in this region, a novel method in managing the acute bleeding episode in acquired haemophilia using recombinant factor VIIa.

Matched MeSH terms: Antibodies/immunology
L-arginine-dependent nitric oxide production of a murine macrophage-like RAW 264.7 cell line stimulated with Porphyromonas gingivalis lipopolysaccharide

Sosroseno W, Herminajeng E, Bird PS, Seymour GJ

Oral Microbiol. Immunol., 2004 Apr;19(2):65-70.
PMID: 14871343

The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg-LPS). RAW 264.7 cells were incubated with i) various concentrations of Pg-LPS or Salmonella typhosa LPS (St-LPS), ii) Pg-LPS with or without L-arginine and/or NG-monomethyl-L-arginine (NMMA), an arginine analog or iii) Pg-LPS and interferon-gamma (IFN-gamma) with or without anti-IFN-gamma antibodies or interleukin-10 (IL-10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg-LPS, but was observed after stimulation with St-LPS. Exogenous L-arginine restored the ability of Pg-LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg-LPS with exogenous L-arginine was abolished by NMMA. IFN-gamma induced independent NO production by Pg-LPS-stimulated macrophages and this stimulatory effect of IFN-gamma could be completely suppressed by anti-IFN-gamma antibodies and IL-10. These results suggest that Pg-LPS is able to stimulate NO production in the RAW 264.7 macrophage cell model in an L-arginine-dependent mechanism which is itself independent of the action of IFN-gamma.

Matched MeSH terms: Antibodies/immunology
Effect of colchicine on the murine immune response induced by Aggregatibacter actinomycetemcomitans

Sosroseno W

Biomed Pharmacother, 2009 Mar;63(3):221-7.
PMID: 18534811 DOI: 10.1016/j.biopha.2008.04.004

The aim of the present study was to test the hypothesis that colchicine may alter Aggregatibacter actinomycetemcomitans-induced immune response and abscess formation in mice. BALB/c mice were either sham-immunized or immunized with heat-killed A. actinomycetemcomitans. Spleen cells were stimulated with heat-killed A. actinomycetemcomitans in the presence or absence of colchicine. Specific IgG subclass antibodies, interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and cell proliferation were determined. The animals were sham-immunized (group I) or immunized with heat-killed A. actinomycetemcomitans (groups II-VII). Colchicine was administered intraperitoneally before (group III), on the same day of (group IV), or after (group V) the primary immunization and on the same day of (group VI) or after (group VII) the secondary immunization. All groups were challenged with viable A. actinomycetemcomitans. The levels of serum-specific IgG subclasses and both IFN-gamma and IL-4 before and after bacterial challenge were assessed. The diameter of skin lesions was assessed. The results showed that colchicine augmented splenic-specific IgG1 and IL-4 as well as cell proliferation but suppressed specific IgG2a and IFN-gamma levels. Enhancement of serum-specific IgG1 and IL-4 levels, suppression of specific IgG2a and IFN-gamma levels as well as DTH response, and delayed healing of the lesions were observed in groups IV and VI, but not in the remaining groups of animals. Therefore, these results suggest that colchicine may induce a T helper 2 (Th2)-like immunity specific to A. actinomycetemcomitans in vitro and that colchicine administered on the same day as the immunization may stimulate a non-protective Th2-like immunity in A. actinomycetemcomitans-induced infections in mice.

Matched MeSH terms: Antibodies/immunology
Fulltext Chronic hepatitis C infection associated with neuromyelitis optica and antinuclear antibodies

Earnest BS, Men LC, Sukvinder Kaur G, Alias RB, Sunita Devi H

J Assoc Physicians India, 2010 Feb;58(2):118-20.
PMID: 20653157

The neurological manifestations of chronic hepatitis C is most often a peripheral sensory neuropathy characterised by numbness, burning and sensation of "pins and needles". Peripheral motor neuropathy, mononeuropathy, mononeuropathy multiplex and transverse myelitis also occur. Ischemic stroke and transient cerebral ischemia have also been reported. Anterior ischemic optic neuropathy is seen, often following interferon therapy. We report an exceptional case of neuromyelitis optica in chronic hepatitis C infection in the absence of interferon therapy.

Matched MeSH terms: Hepatitis C Antibodies/immunology*
Fulltext Highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms

Too WC, Wong MT, Few LL, Konrad M

PLoS One, 2010;5(9):e12999.
PMID: 20886003 DOI: 10.1371/journal.pone.0012999

Choline kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKα1, α2, and β). Specific inhibition of CKα has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CKα used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the α1 or the α2 isoform.

Matched MeSH terms: Antibodies/immunology
Fulltext Anti-hepatitis A seroprevalence among chronic viral hepatitis patients in Kelantan, Malaysia

Ahmad F, Hamzah NA, Mustaffa N, Gan SH

World J Gastroenterol, 2011 Sep 28;17(36):4130-4.
PMID: 22039329 DOI: 10.3748/wjg.v17.i36.4130

To determine the seroprevalence of anti-hepatitis A virus (HAV) antibodies in patients with chronic liver disease (CLD) and to justify the need for hepatitis A vaccination.

Matched MeSH terms: Hepatitis A Antibodies/immunology
Fulltext Evaluation of the dried blood spot (DBS) collection method as a tool for detection of HIV Ag/Ab, HBsAg, anti-HBs and anti-HCV in a Malaysian tertiary referral hospital

Lee CE, Sri Ponnampalavanar S, Syed Omar SF, Mahadeva S, Ong LY, Kamarulzaman A

Ann Acad Med Singap, 2011 Oct;40(10):448-53.
PMID: 22206053 DOI: 10.47102/annals-acadmedsg.V40N10p448

INTRODUCTION: Dried blood spot (DBS) collection is an appealing alternative to whole blood or plasma sampling, as it has technical and economic advantages over the latter.

MATERIALS AND METHODS: A prospective cross-sectional study was conducted at a Malaysian tertiary referral hospital from November 2009 to March 2010. One hundred and fifty paired specimens of DBS and plasma were analysed by the standard assays for HIV Ag/Ab, HBsAg, anti-HBS and anti-HCV, separately (total 600 paired specimens). DBS sample titres were then compared to the results of plasma testing, which was used as the gold standard.

RESULTS: For the HIV Ag/Ab assay with a cut-off point of 0.35 Relative Light Units (RLUs), the sensitivity and specificity were both 100%. For the HBsAg assay, the sensitivity was 96.5% and the specificity was 97.8%, with a cut-off point of 1.72 RLUs. Sensitivity for the anti-HBs test was 74.2% and the specificity was 86.9%, using a cut-off point of 0.635 RLUs. For the anti-HCV assay, the sensitivity was 97.3% and the specificity was 100%, with a cut-off point of 0.10 RLUs.

CONCLUSION: DBS is an ideal choice to be used as a screening tool for the detection of HIV, Hepatitis B and Hepatitis C virus infections. However, different cut-off values need to be used for the validation of test positivity in DBS samples because the small amount of blood in the DBS specimens leads to lower assay titres.

Matched MeSH terms: Hepatitis B Antibodies/immunology; HIV Antibodies/immunology; Hepatitis C Antibodies/immunology
Display of the antigenic region of Nipah virus nucleocapsid protein on hepatitis B virus capsid

Yap WB, Tey BT, Alitheen NB, Tan WS

J Biosci Bioeng, 2012 Jan;113(1):26-9.
PMID: 22024533 DOI: 10.1016/j.jbiosc.2011.09.007

The C-terminal domain of Nipah virus (NiV) nucleocapsid protein (NP₄₀₁₋₅₃₂) was inserted at the N-terminus and the immunodominant loop of hepatitis B core antigen (HBc). The stability of NP₄₀₁₋₅₃₂ increased tremendously when displayed on the HBc particles. These particles reacted specifically with the swine anti-NiV and the human anti-HBc antisera.

Matched MeSH terms: Hepatitis B Antibodies/immunology
Fulltext Construction of single-chain variable fragment antibodies against MCF-7 breast cancer cells

Zuhaida AA, Ali AM, Tamilselvan S, Alitheen NB, Hamid M, Noor AM, et al.

Genet. Mol. Res., 2013;12(4):5547-59.
PMID: 24301925 DOI: 10.4238/2013.November.18.5

A phage display library of single chain variable fragment (scFv) against MCF-7 breast cancer cells was constructed from C3A8 hybridoma cells. RNA from the C3A8 was isolated, cDNA was constructed, and variable heavy and light immunoglobulin chain gene region were amplified using PCR. The variable heavy and light chain gene regions were combined with flexible linker, linked to a pCANTAB 5E phagemid vector and electrophoresed into supE strain of Escherichia coli TG1 cells. Forty-eight clones demonstrated positive binding activity to MCF-7 breast cancer cell membrane fragments and the strongest of 48 clones was selected for analysis. The anti-MCF-7 library evaluated by SfiI and NotI digests demonstrated that anti-MCF-7 scFv antibodies possess individual patterns that should be able to recognize distinct human breast cancer cells. The C3A8 scFv, with an apparent molecular weight of 32 kDa, showed high homology (99%) with single chain antibody against rice stripe virus protein P20. In summary, the anti MCF-7 scFv antibody can be used for pretargeting breast cancer for clinical diagnosis of patients; it also has potential for therapeutic applications.

Matched MeSH terms: Single-Chain Antibodies/immunology*
Current aspects in immunosensors

Gopinath SC, Tang TH, Citartan M, Chen Y, Lakshmipriya T

Biosens Bioelectron, 2014 Jul 15;57:292-302.
PMID: 24607580 DOI: 10.1016/j.bios.2014.02.029

Sensing applications can be used to report biomolecular interactions in order to elucidate the functions of molecules. The use of an analyte and a ligand is a common set-up in sensor development. For several decades, antibodies have been considered to be potential analytes or ligands for development of so-called "immunosensors." In an immunosensor, formation of the complex between antibody and antigen transduces the signal, which is measurable in various ways (e.g., both labeled and label-free based detection). Success of an immunosensor depends on various factors, including surface functionalization, antibody orientation, density of the antibody on the sensor platform, and configuration of the immunosensor. Careful optimization of these factors can generate clear-cut results for any immunosensor. Herein, current aspects, involved in the generated immunosensors, are discussed.

Matched MeSH terms: Antibodies/immunology
Delineation of BmSXP antibody V-gene usage from a lymphatic filariasis based immune scFv antibody library

Rahumatullah A, Ahmad A, Noordin R, Lim TS

Mol Immunol, 2015 Oct;67(2 Pt B):512-23.
PMID: 26277276 DOI: 10.1016/j.molimm.2015.07.040

Phage display technology is an important tool for antibody generation or selection. This study describes the development of a scFv library and the subsequent analysis of identified monoclonal antibodies against BmSXP, a recombinant antigen for lymphatic filariasis. The immune library was generated from blood of lymphatic filariasis infected individuals. A TA based intermediary cloning approach was used to increase cloning efficiency for the library construction process. A diverse immune scFv library of 10(8) was generated. Six unique monoclonal antibodies were identified from the 50 isolated clones against BmSXP. Analysis of the clones showed a bias for the IgHV3 and Vκ1 (45.5%) and IgHV2 and Vκ3 (27.3%) gene family. The most favored J segment for light chain is IgKJ1 (45.5%). The most favored D and J segment for heavy chain are IgHD6-13 (75%) and IgHJ3 (47.7%). The information may suggest a predisposition of certain V genes in antibody responses against lymphatic filariasis.

Matched MeSH terms: Single-Chain Antibodies/immunology*
Fulltext Selection of phage-displayed human antibody fragments specific for CD1b presenting the Mycobacterium tuberculosis glycolipid Ac2SGL

Camacho F, Sarmiento ME, Reyes F, Kim L, Huggett J, Lepore M, et al.

Int J Mycobacteriol, 2016 06;5(2):120-7.
PMID: 27242221 DOI: 10.1016/j.ijmyco.2015.12.002

OBJECTIVE/BACKGROUND: The development of new tools capable of targeting Mycobacterium tuberculosis (Mtb)-infected cells have potential applications in diagnosis, treatment, and prevention of tuberculosis. In Mtb-infected cells, CD1b molecules present Mtb lipids to the immune system (Mtb lipid-CD1b complexes). Because of the lack of CD1b polymorphism, specific Mtb lipid-CD1b complexes could be considered as universal Mtb infection markers. 2-Stearoyl-3-hydroxyphthioceranoyl-2'-sulfate-α-α'-d-trehalose (Ac2SGL) is specific for Mtb, and is not present in other mycobacterial species. The CD1b-Ac2SGL complexes are expressed on the surface of human cells infected with Mtb. The aim of this study was to generate ligands capable of binding these CD1b-Ac2SGL complexes.
METHODS: A synthetic human scFv phage antibody library was used to select phage-displayed antibody fragments that recognized CD1b-Ac2SGL using CD1b-transfected THP-1 cells loaded with Ac2SGL.
RESULTS: One clone, D11-a single, light-variable domain (kappa) antibody (dAbκ11)-showed high relative binding to the Ac2SGL-CD1b complex.
CONCLUSION: A ligand recognizing the Ac2SGL-CD1b complex was obtained, which is a potential candidate to be further tested for diagnostic and therapeutic applications.

Matched MeSH terms: Single-Chain Antibodies/immunology
Generation of a naïve human single chain variable fragment (scFv) library for the identification of monoclonal scFv against Salmonella Typhi Hemolysin E antigen

Lim BN, Chin CF, Choong YS, Ismail A, Lim TS

Toxicon, 2016 Jul;117:94-101.
PMID: 27090555 DOI: 10.1016/j.toxicon.2016.04.032

Antibody phage display is a useful tool for the isolation and identification of monoclonal antibodies. Naive antibody libraries are able to overcome the limitations associated with the traditional hybridoma method for monoclonal antibody generation. Antibody phage display is also a preferred method for antibody generation against toxins as it does not suffer from toxicity mediated complications. Here, we describe a naïve multi ethnic scFv antibody library generated via two-step cloning with an estimated diversity of 2 × 10(9). The antibody library was used to screen for monoclonal antibodies against Hemolysin E antigen, a pore forming toxin produced by Salmonella enterica serovar Typhi. A soluble monoclonal scFv antibody against the HlyE toxin (IgM scFv D7 anti-hlyE) was isolated from the library. This shows the value of the naïve library to generate antibodies against toxin targets in addition to the potential use of the library to isolate antibodies against other immunogenic targets.

Matched MeSH terms: Single-Chain Antibodies/immunology*
Surface Antigen Profiling of Helicobacter pylori-Infected and -Uninfected Gastric Cancer Cells Using Antibody Microarray

Sukri A, Hanafiah A, Kosai NR, Mohamed Taher M, Mohamed Rose I

Helicobacter, 2016 Oct;21(5):417-27.
PMID: 26807555 DOI: 10.1111/hel.12295

Comprehensive immunophenotyping cluster of differentiation (CD) antigens in gastric adenocarcinoma, specifically between Helicobacter pylori-infected and -uninfected gastric cancer patients by using DotScan(™) antibody microarray has not been conducted. Current immunophenotyping techniques include flow cytometry and immunohistochemistry are limited to the use of few antibodies for parallel examination. We used DotScan(™) antibody microarray consisting 144 CD antibodies to determine the distribution of CD antigens in gastric adenocarcinoma cells and to elucidate the effect of H. pylori infection toward CD antigen expression in gastric cancer.

Matched MeSH terms: Antibodies/immunology
Immune Antibody Libraries: Manipulating The Diverse Immune Repertoire for Antibody Discovery

Lim TS, Chan SK

Curr Pharm Des, 2016;22(43):6480-6489.
PMID: 27669969 DOI: 10.2174/1381612822666160923111924

BACKGROUND: Antibody phage display is highly dependent on the availability of antibody libraries. There are several forms of libraries depending mainly on the origin of the source materials. There are three major classes of libraries, mainly the naïve, immune and synthetic libraries.
METHODS: Immune antibody libraries are designed to isolate specific and high affinity antibodies against disease antigens. The pre-exposure of the host to an infection results in the production of a skewed population of antibodies against the particular infection.
RESULTS: This characteristic takes advantage of the in vivo editing machinery to generate bias and specific immune repertoire. The skewed but diverse repertoire of immune libraries has been adapted successfully in the generation of antibodies against a wide range of diseases.
CONCLUSION: We envisage immune antibody libraries to play a greater role in the discovery of antibodies for diseases in the near future.

Matched MeSH terms: Antibodies/immunology*
Aptahistochemistry in diagnostic pathology: technical scrutiny and feasibility

Bukari BA, Citartan M, Ch'ng ES, Bilibana MP, Rozhdestvensky T, Tang TH

Histochem Cell Biol, 2017 May;147(5):545-553.
PMID: 28321500 DOI: 10.1007/s00418-017-1561-9

Antibodies have been the workhorse for diagnostic immunohistochemistry to specifically interrogate the expression of certain protein to aid in histopathological diagnosis. This review introduces another dimension of histochemistry that employs aptamers as the core tool, the so-called aptahistochemistry. Aptamers are an emerging class of molecular recognition elements that could recapitulate the roles of antibodies. The many advantageous properties of aptamers suited for this diagnostic platform are scrutinized. An in-depth discussion on the technical aspects of aptahistochemistry is provided with close step-by-step comparison to the more familiarized immunohistochemical procedures, namely functionalization of the aptamer as a probe, antigen retrieval, optimization with emphasis on incubation parameters and visualization methods. This review offers rationales to overcome the anticipated challenges in transition from immunohistochemistry to aptahistochemistry, which is deemed feasible for an average diagnostic pathology laboratory.

Matched MeSH terms: Antibodies/immunology
Fulltext Virus-like Particles Identify an HIV V1V2 Apex-Binding Neutralizing Antibody that Lacks a Protruding Loop

Cale EM, Gorman J, Radakovich NA, Crooks ET, Osawa K, Tong T, et al.

Immunity, 2017 05 16;46(5):777-791.e10.
PMID: 28514685 DOI: 10.1016/j.immuni.2017.04.011

Most HIV-1-specific neutralizing antibodies isolated to date exhibit unusual characteristics that complicate their elicitation. Neutralizing antibodies that target the V1V2 apex of the HIV-1 envelope (Env) trimer feature unusually long protruding loops, which enable them to penetrate the HIV-1 glycan shield. As antibodies with loops of requisite length are created through uncommon recombination events, an alternative mode of apex binding has been sought. Here, we isolated a lineage of Env apex-directed neutralizing antibodies, N90-VRC38.01-11, by using virus-like particles and conformationally stabilized Env trimers as B cell probes. A crystal structure of N90-VRC38.01 with a scaffolded V1V2 revealed a binding mode involving side-chain-to-side-chain interactions that reduced the distance the antibody loop must traverse the glycan shield, thereby facilitating V1V2 binding via a non-protruding loop. The N90-VRC38 lineage thus identifies a solution for V1V2-apex binding that provides a more conventional B cell pathway for vaccine design.

Matched MeSH terms: HIV Antibodies/immunology*
The design of target specific antibodies (scFv) by applying de novo workflow: Case study on BmR1 antigen from Brugia malayi

Khor BY, Lim TS, Noordin R, Choong YS

J Mol Graph Model, 2017 09;76:543-550.
PMID: 28811153 DOI: 10.1016/j.jmgm.2017.07.004

De novo approach was applied to design single chain fragment variable (scFv) for BmR1, a recombinant antigen from Bm17DIII gene which is the primary antigen used for the detection of anti-BmR1 IgG4 antibodies in the diagnostic of lymphatic filariasis. Three epitopes of the BmR1 was previously predicted form an ab initio derived three-dimensional structure. A collection of energetically favourable conformations was generated via hot-spot-centric approach. This resulted in a set of three different scFv scaffolds used to compute the high shape complementary conformations via dock-and-design approach with the predicted epitopes of BmR1. A total of 4227 scFv designs were generated where 200 scFv designs produced binding energies of less than -20 R.E.U with shape complementarity higher than 0.5. We further selected the design with at least one hydrogen bond and one salt bridge with the epitope, thus resulted in a total of 10, 1 and 19 sFv designs for epitope 1, 2 and 3, respectively. The results thus showed that de novo design can be an alternative approach to yield high affinity in silico scFv designs as a starting point for antibody or specific binder discovery processes.

Matched MeSH terms: Single-Chain Antibodies/immunology

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