Hybridoma clone C3A8, which is a fusion product between splenic lymphocytes of Balb/c mice immunized with MCF7 breast carcinoma cells and SP2/0 myelomas, was produced and characterized. A stable clone that secreted IgM monoclonal antibody (MAb) with kappa light chain was obtained through limiting dilutions. Cell-ELISA screening, flow cytometry analysis, and immunofluorescence staining revealed that the MAb C3A8 had bound specifically and strongly to MCF7 and HT29 but cross reacted weakly or not on HeLa cell line. The MAb C3A8 reacted positively with paraffin-embedded tissues of human breast and colon cancers but there were no positive reactions on normal tissues. Western blot analysis showed the MAb recognized a 55 kDa protein, which was present in the extract of MCF7 and HT29 cell lines. Our results demonstrated that MAb C3A8 could be used for basic and clinical research of breast and colon cancers.
The Candida species are the most common fungal pathogens of systemic candidiasis. The diagnosis of invasive candidiasis remains a laboratory and clinical challenge. Thus, development of diagnostic assays to detect systemic candidiasis and to identify Candida virulence factors and associated pathogenesis through immunohistochemistry using specific monoclonals and polyclonals will be useful. Inbred Balb/c mice were immunized with C. albicans antigens, and blood was checked for the presence of reactive antibodies using ELISA. Fusion was performed using the harvested spleen cells and NS1 myeloma cells, and the clones were screened for the presence of antibody producing hybrid cells by dot-blot. Western blot analysis showed that the L2D10 monoclonal antibody was reactive against the antigens with molecular weight of 20 kDa. Experimental systemic candidiasis in mice was induced through intravenous injection of C. albicans and all the vital organs were collected for immunohistochemistry study. The monoclonal antibody reacted to surface epitopes on the yeast cells, germ tubes, and hyphae, and to immune complexes. It was used with the polyclonal antibody in a sandwich ELISA for the detection of circulating antigens in experimental candiadiasis in mice. Antibody levels were also determined using the ELISA method, and the antibody levels of C. albicans infected mice were increased compared with uninfected animals. The monoclonal antibody was used in immunoperoxidase and immunofluorescence techniques for the detection of fungal infection in tissue sections and was found to be more sensitive than conventional periodic acid Schiff or silver staining techniques. This monoclonal antibody may serve as potential primary capture antibodies for the development of a rapid diagnostic test for human systemic fungal infection.
Prostate-specific antigen (PSA) is widely used as a diagnostic marker for the detection of prostate cancer in men. We have generated stable hybridomas producing specific monoclonal antibodies (MAbs) of the IgG class against PSA from fusions of splenocytes from immunized mice with myeloma cells. The hybridomas were adapted into serum-free media and cultured in CELLine CL-1000 bioreactors to produce milligram quantities of MAbs. Cross-reactivity study demonstrated that all the MAbs reacted did not cross-react with several other types of tumor antigens. Two of the MAbs were successfully radiolabeled by the iodogen method. The (125)I-labeled MAbs demonstrated strong binding to PSA on the surface of the LNCaP cells (Kd of 1.16 x 10(-9) M and 1.4 x 10(-9) M). Thus the (125)I-labeled MAbs retained their immunoreactivity and possessed high affinity and is potentially useful for binding to tumor cells. In conclusion, the MAbs can be used to develop radioimmunodiagnostic, radioimaging, and immunohistochemistry techniques for the early detection and treatment of prostate cancer.
Burkholderia pseudomallei, the causative agent of melioidosis, is endemic to Southeast Asia and northern Australia. Clinical manifestations of the disease are diverse, ranging from chronic localized infection to acute septicaemia, with death occurring within 24-48 h after the onset of symptoms. Definitive diagnosis of melioidosis involves bacterial culture and identification, with results obtained within 3-4 days. This delayed diagnosis is a major contributing factor to high mortality rates. Rapid diagnosis is vital for successful management of the disease. This study describes the purification and evaluation of three recombinant antigenic proteins, BPSL0972, BipD and OmpA from B. pseudomallei 08, for their potential in the serodiagnosis of melioidosis using an indirect enzyme-linked immunosorbent assay (ELISA) method. The recombinant proteins were evaluated using 74 serum samples from culture-confirmed melioidosis patients from Malaysia, Thailand and Australia. In addition, 62 nonmelioidosis controls consisting of serum samples from clinically suspected melioidosis patients (n=20) and from healthy blood donors from an endemic region (n=18) and a nonendemic region (n=24) were included. The indirect ELISAs using BipD and BPSL0972 as antigens demonstrated poor to moderate sensitivities (42% and 51%, respectively) but good specificity (both 100%). In contrast, the indirect ELISA using OmpA as an antigen achieved 95% sensitivity and 98% specificity. These results highlight the potential for OmpA to be used in the serodiagnosis of melioidosis in an endemic area.
Nipah encephalitis is a particular dangerous disease that affects animals and man. Fatal cases of the disease have been identified in the persons looking after pigs in the villages of Malaysia. The causative agent is presumably referred to as morbilliviruses of the Paramixoviridae family. Two hundred persons died among the ill patients with the signs of encephalitis. The principal hosts of the virus were fox-bats (Megaschiroptera) inhabiting in the surrounding forests. The present paper descries the epidemiological features of the disease, its clinical manifestations, abnormal anatomic changes, diagnosis, and implemented controlling measures.
Helicobacter pylori is one of the few remaining major pathogens that accompanied humans on their travels from Africa. A recently published study reports the unexpected finding of a low H. pylori prevalence among pregnant women in Zanzibar (Farag, T.H., Stolzfus, R.J., Khalfan, S.S., Tielsch, J.M., 2007. Unexpectedly low prevalence of Helicobacter pylori infection among pregnant women on Pemba Island, Zanzibar. Trans. R. Soc. Trop. Med. Hyg. 101). The apparent epidemiology of higher prevalence with higher socioeconomic status and decrease with age are unprecedented. As with many 'unexpected' events, a search of the literature reveals evidence of low prevalence populations in Java and Malaysia, with clues dating back to the mid-twentieth century. Why some populations apparently lost H. pylori infection remains an open question. However, the tools needed to resolve the dilemma are readily available and we hope investigators will soon rise to the challenge.
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus which causes epidemic fever, rash and polyarthralgia in Africa and Asia. Two outbreaks have been reported in Malaysia, in Klang, Selangor (1998) and Bagan Panchor, Perak (2006). It is not known if the outbreaks were caused by the recent introduction of CHIKV, or if the virus was already circulating in Malaysia. Seroprevalence studies from the 1960s suggested previous disease activity in certain parts of the country. In Asia, CHIKV is thought to be transmitted by the same mosquitoes as dengue, Aedes aegypti and Ae. albopictus. Due to similarities in clinical presentation with dengue, limited awareness, and a lack of laboratory diagnostic capability, CHIKV is probably often underdiagnosed or misdiagnosed as dengue. Treatment is supportive. The prognosis is generally good, although some patients experience chronic arthritis. With no vaccine or antiviral available, prevention and control depends on surveillance, early identification of outbreaks, and vector control. CHIKV should be borne in mind in sporadic cases, and in patients epidemiologically linked to ongoing local or international outbreaks or endemic areas.
An effective live attenuated rubella vaccine was available since 1969 and congenital rubella syndrome can be prevented with appropriate vaccination. We report a baby with congenital rubella syndrome born in Klang valley to indicate that the Universal Rubella Vaccination Programme adopted by the Ministry of Health Malaysia since 2002 has yet to achieve its effect of eliminating transmission of rubella and preventing congenital rubella infection in the community. To our knowledge, the virus isolate represents the first successful isolation of rubella virus in this country and will serve as the reference strain for future comparison in molecular epidemiological tracking of rubella virus activity this country.
A 55 year old female presented with fever, skin rash and subconjunctival hemorrhage. She also developed hepatitis. Fever and skin rash lasted for more than three weeks. This patient was diagnosed to have rubella, highlighting the fact that rubella can present with atypical features like prolonged fever and rash, subconjunctival hemorrhage and hepatitis, especially in adults.
Systemic Lupus Erythematosus (SLE) is a disease with multiorgan involvement and multiple autoantibody production including antineutrophil cytoplasmic antibodies (ANCA). Despite its reported prevalence in more than one third of SLE patients, the role of ANCA in the pathogenesis or otherwise in SLE remains unresolved. 131 SLE patients had been previously studied for various serologic parameters of disease activity. Their cumulative organ involvement in the course of their disease had also been determined and the Lupus Activity Index (LAI) calculated. Their stored sera were then screened for the presence of ANCA by two methods viz Indirect immunofluorescence (IIF) and also enzyme-linked immunosorbent assay (ELISA). ANCA was present in 24.8% of these SLE patients. The atypical ANCA pattern was predominant and accounted for an overall of 20.6%. Anti-MPO and anti-PR3 were detected in 1.5% of patients respectively. No association was found between ANCA positivity and disease activity. There was also no association of ANCA with specific organ involvement. Despite the high prevalence of ANCA especially the atypical variant in SLE, they probably represent only one of the wide repertoire of autoantibodies found in this disease. Routine testing for ANCA in lupus patients is therefore not recommended.
Diagnosis of human toxocariasis, caused by Toxocara canis or Toxocara cati, normally relies on a combination of the presence of clinical signs and symptoms backed by positive serology. The use of Toxocara excretory-secretory antigen (TES) in ELISA assays increases the test specificity. However, in tropical countries where soil-transmitted helminths are endemic, cross-reactivity from antibodies to these intestinal parasites poses a significant limitation for Toxocara serodiagnosis. To increase the specificity of serodiagnosis, we compared the use of IgG-ELISA to the use of IgG4-ELISA using commercially manufactured TES-coated plates. The sensitivity of the IgG-ELISA was 97.1%, while that of the IgG4-ELISA was 45.7%; the specificities were 36.0 and 78.6%, respectively. The study shows that employing both assays can improve the serodiagnosis of toxocariasis. An IgG4 immunoassay would also be useful in the secondary screening of antigen clones in the effort to develop improved serological tests for toxocariasis.
A 39-year-old patient developed a disseminated rash with scattered petechiae, fever, malaise and arthralgia after a trip to Malaysia. The patient displayed increasing dengue IgG titers and borderline dengue IgM titers. Dengue fever with a hemorrhagic course is a rare condition in adult patients. Patients who have previously had dengue fever and retained non-neutralizing heterotypic antibodies are more likely to develop this complication via the phenomenon of antibody-dependent enhancement.
An in-house prepared M. furfur antigen was used to carry out a seroprevalence study in an urban population in Malaysia by indirect immunofluorescence assay. Of the 800 serum samples from all ages screened, 738 samples were positive for M. furfur specific IgG, giving an overall seropositive rate of 92.3%. There was no significant difference in the seropositive rates among the different gender group and races. However, there was a statistical significant difference in the seropositive rate among different age groups with a lower rate (73%) for the age group 5 years old and below, which increased rapidly to 99% for the 16 to 20 years old age group but declined slightly for the oldest age group. The degree of seropositivity, which semi-quantitatively reflect the anti-M. furfur specific IgG titre, did not show any significant difference among the gender and racial groups. On the other hand, there was a significant difference in the degree of seropositivity among the various age groups, with the 16 to 20 years old age group having the highest antibody titre and the extreme of age groups having the lower antibody titre.
Balamuthia mandrillaris is a protist pathogen that can cause encephalitis with a mortality rate of more than 95%. Early diagnosis followed by aggressive treatment is a pre-requisite for successful prognosis. Current methods for identifying this organism rely on culture and microscopy, antibody-based methods using animals, or involve the use of molecular tools that are expensive. Here, we describe the isolation of antibody fragments that can be used for the unequivocal identification of B. mandrillaris. B. mandrillaris-specific antibody fragments were isolated from a bacteriophage antibody display library. Individual clones were studied by enzyme-linked immunosorbent assay, and immunofluorescence. Four antibody clones showed specific binding to B. mandrillaris. The usefulness of phage antibody display technology as a diagnostic tool for isolating antibody fragments against B. mandrillaris antigens and studying their biological role(s) is discussed further.
Immunohistochemistry is a histological technique that allows detection of one or more proteins of interest within a cell using specific antibody binding, followed by microscopic visualization of a chromogenic substrate catalyzed by peroxidase and/or alkaline phosphatase. Here, we describe a method to localize Chikungunya virus (CHIKV) antigens in formalin-fixed and paraffin-embedded infected mouse brain.
Chikungunya virus (CHIKV) is a mosquito-borne arbovirus which poses a major threat to global public health. Definitive CHIKV diagnosis is crucial, especially in distinguishing the disease from dengue virus, which co-circulates in endemic areas and shares the same mosquito vectors. Laboratory diagnosis is mainly based on serological or molecular approaches. The E2 glycoprotein is a good candidate for serological diagnosis since it is the immunodominant antigen during the course of infection, and reacts with seropositive CHIKV sera. In this chapter, we describe the generation of stable clone Sf9 (Spodoptera frugiperda) cells expressing secreted, soluble, and native recombinant CHIKV E2 glycoprotein. We use direct plasmid expression in insect cells, rather than the traditional technique of generating recombinant baculovirus. This recombinant protein is useful for serological diagnosis of CHIKV infection.
Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by a persistently high titer of antiphospholipid antibodies (aPLs). In addition to pregnancy morbidity, arterial and/or venous thrombosis is another clinical feature of APS. Regardless of the type of APS, the thrombi formed by the induction of aPLs can lead to deep vein thrombosis, pulmonary embolism, myocardial infarction, stroke and gangrene. Although the concept of APS was introduced approximately 32 years ago, its thrombogenic pathophysiology is still unclear. Therefore, patients are treated with anticoagulant and/or antiplatelet regimens just as in other thrombotic disorders even though the thrombotic pathophysiology is mainly aPLs-mediated. In this review, we provided an update of the cellular, auto-immune and genetic factors known to play important roles in the generation of thrombi. Current successful regimens are also outlined along with potential emerging treatment strategies that may lead to the optimum management of thrombotic APS patients.
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.