RAPID IDENTIFICATION OF BURKHOLDERIA PSEUDOMALLEI IN BLOOD CULTURES BY LATEX AGGLUTINATION USING LIPOPOLYSACCHARIDE-SPECIFIC MONOCLONAL ANTIBODY

Size: px
Start display at page:

Download "RAPID IDENTIFICATION OF BURKHOLDERIA PSEUDOMALLEI IN BLOOD CULTURES BY LATEX AGGLUTINATION USING LIPOPOLYSACCHARIDE-SPECIFIC MONOCLONAL ANTIBODY"

Transcription

1 Am. J. Trop. Med. Hyg., 61(4), 1999, pp Copyright 1999 by The American Society of Tropical Medicine and Hygiene RAPID IDENTIFICATION OF BURKHOLDERIA PSEUDOMALLEI IN BLOOD CULTURES BY LATEX AGGLUTINATION USING LIPOPOLYSACCHARIDE-SPECIFIC MONOCLONAL ANTIBODY TARARAJ DHARAKUL, SIRIRURG SONGSIVILAI, SAIJAI SMITHIKARN, CHARIN THEPTHAI, AND AMORNRAT LEELAPORN Laboratory of Cellular and Molecular Immunology, Department of Immunology, and Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand Abstract. Melioidosis, an infection caused by Burkholderia pseudomallei, is endemic in Southeast Asia. The septicemic form of melioidosis is the leading cause of death due to community-acquired bacteremia in the northeastern part of Thailand. The delay in isolation and identification of the causative organism is a major contributing factor to the high mortality. The present study describes the evaluation of a latex agglutination test for rapid identification of the bacteria directly from blood cultures. The Bps-L1 monoclonal antibody recognized the lipopolysaccharide antigen of 96.8% of B. pseudomallei clinical isolates and was highly specific for B. pseudomallei. The diagnostic value of the latex agglutination test based on Bps-L1 monoclonal antibody was prospectively evaluated in an area endemic for melioidosis. The agglutination test kit was evaluated in 88 blood cultures with gram-negative bacteria identified with Gram staining. The sensitivity and specificity of the test kit were both 100%. These results indicated that the detection of B. pseudomallei lipopolysaccharide by specific monoclonal antibody in a latex agglutination format is clinically useful for the rapid identification of the bacteria in blood cultures in areas endemic for melioidosis. Melioidosis is an important infectious disease known to be endemic in Southeast Asia and the northern part of Australia. The causative agent, Burkholderia pseudomallei, is one of the most important causes of fatality in communityacquired septicemia in northeastern Thailand. 1 The highest mortality occurs in patients with the septicemic form of melioidosis, which is characterized by dissemination of the bacteria in the circulation and isolation of the bacteria from blood and various organs. The clinical course of septicemic melioidosis often deteriorates rapidly and death occurs within the first few days after hospitalization. Rapid diagnosis and prompt treatment with appropriate antibiotics can reduce the mortality at least by half. 2 Current clinical practice in areas endemic for melioidosis requires a combination of high clinical suspicion and bacterial isolation by culture. The gold standard method for laboratory diagnosis of melioidosis is the isolation of the bacteria, which generally takes 3 7 days. However, in many cases the results are obtained after the patient has died. 1 Blood is the main clinical specimen subjected to bacterial isolation in septicemic melioidosis. Therefore, methods that can shorten the time required for the bacterial isolation, as well as for bacterial identification, are urgently needed. Our previous study demonstrated that the time required for growth of B. pseudomallei that was sufficient for isolation from blood culture was within 48 hr in 93% of the patients. 3 The present study was therefore undertaken to evaluate the applicability of a method for rapid identification of B. pseudomallei in blood culture. A latex agglutination test kit based on a specific monoclonal antibody to the bacterial lipopolysaccharide was developed and the performance of the test was prospectively evaluated in an area endemic for melioidosis. MATERIALS AND METHODS Bacterial strains. A total of 126 clinical isolates of B. pseudomallei, including 65 isolates from blood and 61 isolates from other sites, were obtained from the Department 658 of Microbiology, Faculty of Medicine, Siriraj Hospital (Bangkok) and the Department of Clinical Pathology, Khonkaen Regional Hospital (Khonkaen). All of these isolates had the arabinose-negative phenotype. Fifty isolates of other medically important bacteria were used as controls. These included 1 clinical isolate each of Acinetobacter anitratus, Burkholderia cepacia, Escherichia coli, Enterobacter cloacae, Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas putida, Serratia marcescens, Staphylococcus aureus, Streptococcus pneumoniae, group A, group B, and group D streptococci, as well as 17 isolates of Aeromonas hydrophila and 3 isolates of Aeromonas veronii obtained from the Department of Microbiology, Faculty of Medicine, Siriraj Hospital (Bangkok). In addition, 14 isolates of B. cepacia, and 1 isolate each of Stenotrophomonas maltophilia (DMS904/NCTC10257) and Pseudomonas fluorescens (DMS2589) type strains were provided by the National Institute of Health of Thailand. Stocks of the bacteria in 20% glycerol were aliquoted and kept at 20 C until use. Burkholderia pseudomallei specific monoclonal antibody. The monoclonal antibody was produced using a standard hybridoma technique 4 by fusion of the splenocytes with the P3X63-Ag8/653 mouse myeloma cell line using polyethylene glycol (PEG-4000; Accurate Chemical and Scientific Co., Westbury, NY). The antigen used for immunizing BALB/c mice was prepared from sonicated B. pseudomallei. Several hybridoma clones that recognized the lipopolysaccharide (LPS) of B. pseudomallei were produced, all of which produced antibodies of IgM isotype. One clone was selected for further characterization (Bps-L1). This monoclonal antibody recognized the LPS of B. pseudomallei as demonstrated by a characteristic ladder pattern of staining by Western blot analysis (Figure 1). The monoclonal antibody was produced in the CELLMAX artificial capillary cell culture system (Cellco, Inc., Germantown, MD), according to the manufacturer s recommendation. The culture supernatant fluid was concentrated by 50% ammonium sul-

2 LATEX AGGLUTINATION KIT FOR DIAGNOSIS OF MELIOIDOSIS 659 TABLE 1 Reactivities of the monoclonal antibody Bps-L1 with 126 clinical isolates of Burkholderia pseudomallei from blood and other sites Source of B. pseudomallei clinical isolates Number tested Number of isolates tested positive with the Bps-L1 coated latex particles (%) Blood (100) Sputum (94.1) Urine (90.9) Pus (93.9) Total (96.8) FIGURE 1. Western blot analysis of the Burkholderia pseudomallei crude lipopolysaccharide with Bps-L1 monoclonal antibody. Lane 1, Escherichia coli; lanes 2 5, non-agglutinable B. pseudomallei isolates; lane 6, agglutinable B. pseudomallei; lane 7, molecular weight markers. kd kilodaltons. fate precipitation and the IgM antibodies were further purified by Sephadex G-200 gel filtration chromatography. Western blot analysis. The supernatant fluid of the bacterial culture was treated with 100 g/ml of proteinase K (Sigma Chemical Co., St. Louis, MO) at 56 C for 1 hr and the enzyme was inactivated at 95 C for 10 min. The proteinase K-treated bacterial lysate was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) and blotted onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). The blotted nitrocellulose membrane was blocked with skim milk and was subsequently incubated with the culture supernatant fluid of Bps-L1 monoclonal antibody. The reaction was detected using alkaline phosphatase conjugated goat anti-mouse IgM (Kirkegaard and Perry Laboratories, Gaithersburg, MD) o-dianisidine and -naphthyl phosphate as substrates. 4 Silver staining of the LPS. The crude bacterial lysate containing LPS was prepared by treatment with proteinase K and subjected to SDS-PAGE as above. The gel was incubated in 30% ethanol and 10% acetic acid overnight, followed by 30% ethanol for 30 min twice and 3 changes of deionized water. The gel was then incubated with 0.1% fresh silver nitrate for 30 min in the dark. The reaction was developed with 2.5% sodium carbonate and 0.02% formaldehyde until bands began to appear. 4 Passive adsorption of the IgM monoclonal antibody onto latex particles. Purified IgM monoclonal antibody (200 g/ml) was passively adsorbed onto m polystyrene latex particles (Interfacial Dynamics Corp., Portland, OR) for 2 hr at room temperature. The adsorbed latex particles were washed 3 times in glycine-buffered saline. The remaining protein binding sites on the surface of the latex were blocked with a 1% (w/v) solution of bovine serum albumin (BSA). The coated latex particles were then washed and resuspended in glycine-buffered saline containing 0.1% BSA and 0.1% sodium azide to give a final concentration of 0.5% latex suspension. The latex suspension was stored at 4 C for up to 1 year and brought to room temperature before use. Latex agglutination assay. One drop of the bacterial culture was mixed with an equal volume of a diluent buffer containing 2% n-octyl -D-glucopyranoside ( -OG) on a clean glass slide using a wooden applicator. A drop of the latex reagent was added to the mixture and the slide was rotated gently. Agglutination could be seen in the dark background within 2 min. The agglutination reaction was scored from 1 to 4. Typically, a 4 reaction was observed as large floccules formed instantly with clear background fluid. A negative reaction was characterized by a uniform milky fluid with no visible floccules. Field evaluation of the Bps-L1 latex agglutination test kit for the detection of B. pseudomallei antigen in blood cultures. The Bps-L1-based latex agglutination test kit was prospectively evaluated in an area endemic for melioidosis. The evaluation was performed at the clinical microbiology laboratory at Burirum General Hospital (Burirum, Thailand) by the local laboratory personnel who routinely processed the blood culture specimens. This laboratory used a manual blood culture system that was widely used in Thailand. Briefly, blood from patients with suspected bacteremia was inoculated into brain heart infusion broth and cultured at 37 C. The cultures were examined daily by the laboratory personnel for turbidity and the specimens with turbid culture fluid were subjected to Gram staining. All specimens that showed gram-negative staining were then tested using the latex agglutination test kit and the results were recorded. The result of standard bacterial identification by biochemical reactions, which were usually obtained 1 2 days later, were also independently recorded. At the end of the study period, the results of both tests were analyzed. RESULTS Burkholderia pseudomallei LPS-specific monoclonal antibody. The reactivity of the monoclonal antibody Bps- L1 was evaluated using 126 B. pseudomallei clinical isolates (Table 1). This antibody reacted with 122 (96.8%) of 126 B. pseudomallei isolates, and reacted with all isolates from blood (100%), but not with 1, 1, and 2 specimens from sputum, urine, and pus, respectively. The 4 B. pseudomallei clinical isolates that were not agglutinated by the Bps-L1 monoclonal antibody were further characterized. The identity of these isolates was confirmed by polymerase chain reaction amplification of the 16S ribosomal RNA gene 5 and by standard biochemical identification. The reactivities of the monoclonal antibody was con-

3 660 DHARAKUL AND OTHERS FIGURE 2. Silver staining of the Burkholderia pseudomallei lipopolysaccharide (LPS), demonstrating the distinct LPS pattern of the 4 nonagglutinable B. pseudomallei isolates. Lane 1, molecular weight markers; lanes 2, 3, and 8, agglutinable B. pseudomallei isolates; lanes 4 7, non-agglutinable B. pseudomallei isolates. kd kilodaltons. firmed in Western blot analysis. The Bps-L1 antibody reacted with the LPS of 122 B. pseudomallei isolates, seen as a ladder of bands approximately kd (Figure 1), but did not react with these 4 isolates. Silver staining of the LPS of these 4 isolates showed the pattern that was distinctively different from that of other B. pseudomallei isolates recognized by Bps-L1 (Figure 2). The specificity of the Bps-L1 monoclonal antibody was evaluated by latex agglutination using 50 isolates of other medically important bacteria. This antibody demonstrated no cross-reactivity with any of the other bacteria tested (Table 2). TABLE 2 Reactivities Burkholderia pseudomallei and other baterial species with the Bps-L1 coated latex particles Organisms identified No. tested No. positive (%) Burkholderia pseudomallei (96.8%) Other bacteria Acinetobacter anitratus 1 0 Aeromonas hydrophila 17 0 Aeromonas veronii 3 0 Burkholderia cepacia 15 0 Enterobacter cloacae 1 0 Escherichia coli 1 0 Klebsiella pneumoniae 1 0 Pseudomonas aeruginosa 1 0 Pseudomonas fluorescens (DMS2589) 1 0 Pseudomonas putida 1 0 Stenotrophomonas maltophilia (DMS904/NCTC10257) 1 0 Haemophilus influenzae 1 0 Serratia marcescens 1 0 Staphylococcus aureus 1 0 Streptococcus pneumoniae 1 0 Group A streptococcus 1 0 Group B streptococcus 1 0 Group D streptococcus 1 0 Total 50 0 (0%) Increased sensitivity of the latex agglutination test by treatment with detergents. Approximately 10 8 colonyforming units (CFU)/ml of B. pseudomallei was required for agglutination by Bps-L1-coated latex particles. This amount of bacteria was obtained after 20 hr of culture with an initial bacterial count of 10 CFU/ml. The sensitivity of the latex agglutination test can be enhanced by inducing the release of B. pseudomallei LPS by treatment with heat or detergents. Two anionic detergents, SDS and sarkosyl, and 5 nonionic detergents, 3-[(3-cholamidopropyl) dimethylammonio]-1- propane sulfonate (CHAPS), Triton X-114, Triton X-100, Tween 20, and -OG, all at a final concentration of 1% were evaluated for their ability to release LPS. CHAPS, Triton X- 114, and -OG enhanced the agglutination activity from to 4, similar to that observed after boiling the bacterial culture for 5 min. However, Triton X-114 caused autoagglutination of the coated latex in the absence of bacteria. Further evaluation of CHAPS and -OG indicated that 2.0% CHAPS or 1.0% -OG was optimal in significantly enhancing the release of LPS and increasing the sensitivity of the latex agglutination from negative to 4. When 2% -OG was used, the detection limit of the latex agglutination test kit was approximately 10 7 CFU/ml of B. pseudomallei. Development of the latex agglutination test kit. The latex agglutination test kit was developed based on Bps-L1 monoclonal antibody. The kit contained 100 tests each of the latex reagent and diluent buffer (2% -OG in normal saline), and the positive and negative control samples. The test kit was stable and maintained its activity for at least 12 months. The test was performed by mixing 1 drop (30 50 l) of blood culture fluid with 30 l of diluent buffer containing detergent for 1 min on a clean glass slide, followed by adding 30 l of the latex reagent and mixing; the agglutination reaction was clearly visible within 2 min. Evaluation of the Bps-L1 latex agglutination test kit for the detection of B. pseudomallei antigen in blood cultures in clinical microbiology laboratory in an area endemic for melioidosis. During the study period, all positive

4 LATEX AGGLUTINATION KIT FOR DIAGNOSIS OF MELIOIDOSIS 661 TABLE 3 Field evaluation of the latex agglutination kit for rapid identification of Burkholderia pseudomallei in blood culture Organisms identified No. tested No. positive (%) Burkholderia pseudomallei (100%) Other gram-negative bacteria Burkholderia cepacia 1 0 Acinetobacter anitratus 3 0 Enterobacter cloacae 1 0 Escherichia coli 35 0 Klebsiella pneumoniae 8 0 Providencia rettgeri 1 0 Pseudomonas aeruginosa 4 0 Salmonella group E 1 0 Salmonella paratyphi 2 0 Total 56 0 (0%) blood cultures were subjected to Gram staining. A total of 88 blood culture specimens gave gram-negative bacterial staining. Thirty-two of these were confirmed to be B. pseudomallei by a standard microbiologic technique, and the other 56 were other gram-negative bacteria. The latex agglutination test was performed on all samples before the biochemical identification. The result of the agglutination test is summarized in Table 3. All B. pseudomallei cultures (100%) tested positive by the latex agglutination test kit. All but 1 of the specimens showed 3 agglutination, and 1 specimen showed a 1 agglutination reaction. None of other gram-negative bacteria showed a positive agglutination reaction. Therefore, the field evaluation of the test kit in the microbiology laboratory in the endemic area indicated that the sensitivity and specificity of the test kit were both 100%. DISCUSSION The latex agglutination test using polyclonal anti-b. pseudomallei antiserum was previously reported for the detection of bacteria in blood culture and in clinical specimens. 6,7 Although the polyclonal antibody contained high titer anti-lps activity, it also contained antibodies to other components of the bacteria that may contribute to the cross-reactivity unless pre-absorption was performed. The purpose of the present study was to develop and evaluate the test kit based on a specific monoclonal antibody to B. pseudomallei. This antibody would detect bacterial antigen in the blood after initial cultures as a supplement to the standard bacterial identification method and hasten the diagnosis of melioidosis. The use of the monoclonal antibody provided several advantages, including high specificity with no cross-reactivity that may be present when polyclonal antibody to whole bacteria was used, the minimal batch-to-batch variation, and the ability to scale up production of the test kit. Recently, a latex agglutination test based on a monoclonal antibody against an exopolysaccharide of B. pseudomallei was also shown to react with B. pseudomallei isolated from several countries, including Thailand, but its application in an actual clinical situation was not evaluated. 8 In the present study, the monoclonal antibody directed against LPS, which is a well-known major structural antigenic component of the bacteria, was used. The LPS of B. pseudomallei has been extensively studied and it is generally accepted that it represents a highly species-specific antigen that can be used for diagnostic purposes The test kit based on this LPS-specific monoclonal antibody was developed and its performance was independently evaluated in a clinical microbiology laboratory in an area endemic for melioidosis. The monoclonal antibody described in this study recognizes the LPS of approximately 97% of the B. pseudomallei isolates. This finding supported the general conclusion that B. pseudomallei circulating in Thailand have only a single major serotype. The homogeneity of B. pseudomallei LPS was demonstrated in 2 previous studies. 11,12 However, another study suggested the presence of 2 bacterial serotypes based on the O antigen. 13 Interestingly, the 4 isolates that demonstrated the distinct ladder pattern of LPS and were not recognized by the monoclonal antibody in this study may represent another population of B. pseudomallei. Burkholderia pseudomallei with a distinct LPS pattern was also reported in 4 of 123 clinical isolates from Thailand, 12 although the immunoreactivity was not described. In addition, an atypical B. pseudomallei isolate that contained only O-polysaccharide (O-PS) I and was not recognized by the polyclonal and monoclonal antibodies to O-PS II was also reported. 9 Further study is required to elucidate the identity of these isolates. In addition, it should be noted that the test was evaluated in a clinical setting in Thailand where melioidosis is endemic. Although B. pseudomallei isolates are rather homogeneous and little differences are found among isolates from different geographic regions, the performance of this latex agglutination test kit in other countries remains to be investigated. Field evaluation of the Bps-L1 latex agglutination test kit showed a sensitivity and specificity of 100% after initial Gram staining; only gram-negative bacterial cultures were subjected to the agglutination test. This procedure appeared to be economical and effective in detecting B. pseudomallei in cultures. In addition, a minimal extra work load was required in incorporating the latex agglutination test in routine laboratory procedures. Identification of B. pseudomallei by the latex agglutination test can be done in less than 5 min, and diagnosis of melioidosis can be provided at least 1 day earlier. The earlier diagnosis will result in earlier treatment with appropriate antibiotics that may be able to save the lives of the critically ill patients. A previous study has shown that appropriate antibiotic therapy in septicemic melioidosis saved lives after the first 48 hr. 2 The use of ceftazidime as a first-line drug before culture results are known appears attractive in a highly endemic area such as the northeastern part of Thailand; however, the high cost of such treatment prevent its wide use. The availability of this latex agglutination test kit may help hasten the diagnosis of septicemic melioidosis and appropriate treatment can therefore be provided earlier. In summary, the present study demonstrated the clinical applicability of the latex agglutination test kit that uses a monoclonal antibody (Bps-L1) to B. pseudomallei LPS. This test kit was evaluated for the detection of B. pseudomallei antigen in blood culture; the test was highly sensitive and specific. Studies are underway to evaluate this test kit on a larger scale in several laboratories in areas endemic for melioidosis. In addition, more sensitive assays based on this

5 662 DHARAKUL AND OTHERS monoclonal antibody are being developed for the earlier detection of B. pseudomallei antigen in blood cultures, as well as in other specimens, such as urine or pus, which contain less of the bacterial antigen. Acknowledgments: We thank Nuanchan Piyasangthong, Stitaya Sirisinha, Surang Dejsirilert, and Somporn Srifuangfung for providing bacterial isolates; Kavi Rattanabannangkoon and Prakaithip Thongkum for technical assistance; and Somchai Jitrathai and Chaiya Rengpimai for help in the field evaluation of the latex agglutination test kit. Financial support: This work was supported by a grant from the National Science and Technology Development Agency of Thailand. Authors addresses: Tararaj Dharakul, Sirirurg Songsivilai, Saijai Smithikarn, and Charin Thepthai, Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkok 10700, Thailand. Amornrat Leelaporn, Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkok 10700, Thailand. Reprint requests: Tararaj Dharakul, Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkok 10700, Thailand. REFERENCES 1. Chaowagul W, White NJ, Dance DAB, Wattanagoon Y, Naigowit P, Davis TME, Looareesuwan S, Pitakwatchara N, Melioidosis: a major cause of community-acquired septicemia in northeastern Thailand. J Infect Dis 159: White NJ, Dance DAB, Chaowagul W, Wattanagoon Y, Wuthiekanun V, Pitakwatchara N, Halving of mortality of severe melioidosis by ceftazidime. Lancet ii: Tiangpitayakorn C, Songsivilai S, Piyasangthong N, Dharakul T, Speed of detection of Burkholderia pseudomallei in blood cultures and its correlation with the clinical outcome. Am J Trop Med Hyg 57: Harrow E, Lane D, Antibodies. A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. 5. Dharakul T, Songsivilai S, Viriyachitra S, Luangwedchakarn V, Tassaneetritap B, Chaowagul W, Detection of Burkholderia pseudomallei DNA in patients with septicemic melioidosis. J Clin Microbiol 34: Smith MD, Wuthieckanun V, Walsh AL, Pitt TL, Latex agglutination test for identification of Pseudomonas pseudomallei. J Clin Pathol 46: Smith MD, Wuthieckanun V, Walsh AL, Teerawattanasook N, Desakorn V, Suputtamongkol Y, Pitt TL, White NJ, Latex agglutination for rapid detection of Pseudomonas pseudomallei antigen in urine of patients with melioidosis. J Clin Pathol 48: Steinmetz I, Reganzerowski A, Brenneke B, Haussler S, Simpson A, White NJ, Rapid identification of Burkholderia pseudomallei by latex agglutination based on an exopolysaccharide-specific monoclonal antibody. J Clin Microbiol 37: Perry MB, MacLean LL, Schollaardt T, Bryan LE, Ho M, Structural characterization of the lipopolysaccharide O antigens of Burkholderia pseudomallei. Infect Immun 63: Ho M, Schollaardt T, Smith MD, Perry M, Brett PJ, Chaowagul W, Bryan LE, Specificity and functional activity of anti- Burkholderia pseudomallei polysaccharide antibodies. Infect Immun 65: Pitt TL, Aucken H, Dance DAB, Homogeneity of lipopolysaccharide antigens in Pseudomonas pseudomallei. J Infect 25: Anuntagool N, Intachote P, Wuthiekanun V, White NJ, Sirisinha S, Lipopolysaccharide from nonvirulent Ara Burkholderia pseudomallei isolates is immunologically indistinguishable from lipopolysaccharide from virulent Ara- clinical isolates. Clin Diag Lab Immunol 5: Dodin A, Fournier J, Antigenes precipitants et antigenes agglutinants de Pseudomonas pseudomallei (B. de Whitmore). Ann Inst Pasteur 119:

Short Report: Failure of Burkholderia pseudomallei to Grow in an Automated Blood Culture System

Short Report: Failure of Burkholderia pseudomallei to Grow in an Automated Blood Culture System Accepted for Publication, Published online October 13, 2014; doi:10.4269/ajtmh.14-0018. The latest version is at http://ajtmh.org/cgi/doi/10.4269/ajtmh.14-0018 In order to provide our readers with timely

More information

Basic Immunologic Procedures. Complex Serological Tests

Basic Immunologic Procedures. Complex Serological Tests Basic Immunologic Procedures Complex Serological Tests Amal Alghamdi 2014-2015 1 Classification of antigen-antibody interactions: 1. Primary serological tests: (Marker techniques) e.g. Enzyme linked immuonosorben

More information

Chapter 18: Applications of Immunology

Chapter 18: Applications of Immunology Chapter 18: Applications of Immunology 1. Vaccinations 2. Monoclonal vs Polyclonal Ab 3. Diagnostic Immunology 1. Vaccinations What is Vaccination? A method of inducing artificial immunity by exposing

More information

Chapter 2 Antibodies. Contents. Introduction

Chapter 2 Antibodies. Contents. Introduction Chapter 2 Antibodies Keywords Immunohistochemistry Antibody labeling Fluorescence microscopy Fluorescent immunocytochemistry Fluorescent immunohistochemistry Indirect immunocytochemistry Immunostaining

More information

Pure-IP Western Blot Detection Kit

Pure-IP Western Blot Detection Kit Product Manual Pure-IP Western Blot Detection Kit Catalog Number PRB-5002 20 blots FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction The technique of immunoprecipitation (IP) is used

More information

Chapter 3 Contd. Western blotting & SDS PAGE

Chapter 3 Contd. Western blotting & SDS PAGE Chapter 3 Contd. Western blotting & SDS PAGE Western Blot Western blots allow investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different

More information

Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a

Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a Ryan S. Stowers, 1 Jacqueline A. Callihan, 2 James D. Bryers 2 1 Department of Bioengineering, Clemson University, Clemson,

More information

Protein immunoblotting

Protein immunoblotting Protein immunoblotting (Western blotting) Dr. Serageldeen A. A. Sultan Lecturer of virology Dept. of Microbiology SVU, Qena, Egypt seaas@lycos.com Western blotting -It is an analytical technique used to

More information

LABORATORY PROCEDURE BBL Pneumoslide Test for Streptococcus pneumoniae

LABORATORY PROCEDURE BBL Pneumoslide Test for Streptococcus pneumoniae I. INTENDED USE LABORATORY PROCEDURE BBL Pneumoslide Test for Streptococcus pneumoniae The BBL Pneumoslide Test is a serologic latex slide agglutination method for the qualitative detection of capsular

More information

Pharmaceutical Biotechnology. Recombinant DNA technology Western blotting and SDS-PAGE

Pharmaceutical Biotechnology. Recombinant DNA technology Western blotting and SDS-PAGE Pharmaceutical Biotechnology Recombinant DNA technology Western blotting and SDS-PAGE Recombinant DNA Technology Protein Synthesis Western Blot Western blots allow investigators to determine the molecular

More information

Mouse Typer Sub-Isotyping Kit Instruction Manual

Mouse Typer Sub-Isotyping Kit Instruction Manual Mouse Typer Sub-Isotyping Kit Instruction Manual Protocol for Mouse Typer Sub-Isotyping Kit (Catalog Number 172-2051) and Mouse Typer Sub Isotyping Panel (Catalog Number 172-2055) For Technical Service

More information

Topic: Serological reactions: the purpose and a principle of reactions. Agglutination test. Precipitation test. CFT, IFT, ELISA, RIA.

Topic: Serological reactions: the purpose and a principle of reactions. Agglutination test. Precipitation test. CFT, IFT, ELISA, RIA. Topic: Serological reactions: the purpose and a principle of reactions. Agglutination test. Precipitation test. CFT, IFT, ELISA, RIA. Serology is the study and use of immunological tests to diagnose and

More information

Dot Blot Analysis. Teacher s Guidebook. (Cat. # BE 502) think proteins! think G-Biosciences www.gbiosciences.com

Dot Blot Analysis. Teacher s Guidebook. (Cat. # BE 502) think proteins! think G-Biosciences www.gbiosciences.com PR110 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Dot Blot Analysis Teacher s Guidebook (Cat. # BE 502) think proteins! think G-Biosciences

More information

Molecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION

Molecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION Molecular Biology Techniques: A Classroom Laboratory Manual THIRD EDITION Susan Carson Heather B. Miller D.Scott Witherow ELSEVIER AMSTERDAM BOSTON HEIDELBERG LONDON NEW YORK OXFORD PARIS SAN DIEGO SAN

More information

WESTERN BLOTTING TIPS AND TROUBLESHOOTING GUIDE TROUBLESHOOTING GUIDE

WESTERN BLOTTING TIPS AND TROUBLESHOOTING GUIDE TROUBLESHOOTING GUIDE WESTERN BLOTTING TIPS AND TROUBLESHOOTING GUIDE TIPS FOR SUCCESSFUL WESTERB BLOTS TROUBLESHOOTING GUIDE 1. Suboptimal protein transfer. This is the most common complaint with western blotting and could

More information

KMS-Specialist & Customized Biosimilar Service

KMS-Specialist & Customized Biosimilar Service KMS-Specialist & Customized Biosimilar Service 1. Polyclonal Antibody Development Service KMS offering a variety of Polyclonal Antibody Services to fit your research and production needs. we develop polyclonal

More information

Antigens & Antibodies II. Polyclonal antibodies vs Monoclonal antibodies

Antigens & Antibodies II. Polyclonal antibodies vs Monoclonal antibodies A Brief Review of Antibody Structure A Brief Review of Antibody Structure The basic antibody is a dimer of dimer (2 heavy chain-light chain pairs) composed of repeats of a single structural unit known

More information

Lyme (IgG and IgM) Antibody Confirmation

Lyme (IgG and IgM) Antibody Confirmation Pathology & Laboratory Medicine Lyme (IgG and IgM) Antibody Confirmation TEST UPDATE: New Test Notification Date: 1/9/2013 Effective Date: 1/7/2013 CONTACT INFO Call 802-847-5121 800-991-2799 email labmarketing@vtmednet.org

More information

Standard Operating Procedure (SOP)

Standard Operating Procedure (SOP) Standard Operating Procedure (SOP) Latex Agglutination for the detection of Burkholderia pseudomallei Compiled by: Ms Vanaporn Wuthiekanun (VW), Dr Narisara Chantratita (NC), Dr David Dance (DD), Dr Direk

More information

ELISA BIO 110 Lab 1. Immunity and Disease

ELISA BIO 110 Lab 1. Immunity and Disease ELISA BIO 110 Lab 1 Immunity and Disease Introduction The principal role of the mammalian immune response is to contain infectious disease agents. This response is mediated by several cellular and molecular

More information

Some Immunological Test. Presented by Alaa Faeiz Ashwaaq Dyaa Aseel Abd AL-Razaq Supervised by D.Feras

Some Immunological Test. Presented by Alaa Faeiz Ashwaaq Dyaa Aseel Abd AL-Razaq Supervised by D.Feras Some Immunological Test Presented by Alaa Faeiz Ashwaaq Dyaa Aseel Abd AL-Razaq Supervised by D.Feras Alaa Faeiz Antigen -Antibody Reactions. Antigen antibody reactions are performed to determine the presence

More information

Chapter 6. Antigen-Antibody Properties 10/3/2012. Antigen-Antibody Interactions: Principles and Applications. Precipitin reactions

Chapter 6. Antigen-Antibody Properties 10/3/2012. Antigen-Antibody Interactions: Principles and Applications. Precipitin reactions Chapter 6 Antigen-Antibody Interactions: Principles and Applications Antigen-Antibody Properties You must remember antibody affinity (single) VS avidity (multiple) High affinity: bound tightly and longer!

More information

Guide to Purification of Polyclonal Antibodies

Guide to Purification of Polyclonal Antibodies Guide to Purification of Polyclonal Antibodies When choosing a polyclonal antibody, either as a primary or secondary antibody in an immunoassay, researchers are often inundated with an array of antibody

More information

THE His Tag Antibody, mab, Mouse

THE His Tag Antibody, mab, Mouse THE His Tag Antibody, mab, Mouse Cat. No. A00186 Technical Manual No. TM0243 Update date 01052011 I Description.... 1 II Key Features. 2 III Storage 2 IV Applications.... 2 V Examples - ELISA..... 2 VI

More information

Mouse IFN-gamma ELISpot Kit

Mouse IFN-gamma ELISpot Kit Page 1 of 8 Mouse IFN-gamma ELISpot Kit Without Plates With Plates With Sterile Plates Quantity Catalog Nos. 862.031.001 862.031.001P 862.031.001S 1 x 96 tests 862.031.005 862.031.005P 862.031.005S 5 x

More information

TECHNICAL BULLETIN. HIS-Select Nickel Affinity Gel. Catalog Number P6611 Storage Temperature 2 8 C

TECHNICAL BULLETIN. HIS-Select Nickel Affinity Gel. Catalog Number P6611 Storage Temperature 2 8 C HIS-Select Nickel Affinity Gel Catalog Number P6611 Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description HIS-Select Nickel Affinity Gel is an immobilized metalion affinity chromatography (IMAC)

More information

Serotyping Techniques

Serotyping Techniques Serotyping Techniques Thomas A. Kruzel, M.T., N. D. Southwest College of Naturopathic Medicine & Health Sciences ABO Blood Groups Blood Group RBC Antigens Serum Antibodies Percentage O none Anti A & B

More information

竞 争 性 分 析 Epitope Mapping 实 验 方 法

竞 争 性 分 析 Epitope Mapping 实 验 方 法 竞 争 性 分 析 Epitope Mapping 实 验 方 法 ABSTRACT The simplest way to determine whether two monoclonal antibodies bind to distinct sites on a protein antigen is to carry out a competition assay. The assay can

More information

6 Characterization of Casein and Bovine Serum Albumin

6 Characterization of Casein and Bovine Serum Albumin 6 Characterization of Casein and Bovine Serum Albumin (BSA) Objectives: A) To separate a mixture of casein and bovine serum albumin B) to characterize these proteins based on their solubilities as a function

More information

Classic Immunoprecipitation

Classic Immunoprecipitation 292PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Classic Immunoprecipitation Utilizes Protein A/G Agarose for Antibody Binding (Cat.

More information

Western Blot Analysis with Cell Samples Grown in Channel-µ-Slides

Western Blot Analysis with Cell Samples Grown in Channel-µ-Slides Western Blot Analysis with Cell Samples Grown in Channel-µ-Slides Polyacrylamide gel electrophoresis (PAGE) and subsequent analyses are common tools in biochemistry and molecular biology. This Application

More information

Geniron. Custom Antibody Services. Serum services Antibody Monoclonal. Purification Antibody Mono Y Genetic Immunization Genbody Polyclonal Antibody

Geniron. Custom Antibody Services. Serum services Antibody Monoclonal. Purification Antibody Mono Y Genetic Immunization Genbody Polyclonal Antibody Geniron Custom Antibody Services Serum services Antibody Monoclonal Purification Antibody Mono Y Genetic Immunization Genbody Polyclonal Antibody Geniron Poly Y WE PROVIDE OUR SERVICES TO With Expertise

More information

Direct Testing Systems and Serology

Direct Testing Systems and Serology Direct Testing Systems and Serology Rapid Manual Tests 6-2 Serology Diagnostics 6-6 BD Diagnostics Diagnostic Systems Catalog 2005/2006 6-1 Rapid Manual Tests Meningitis Test Systems 252360 Directigen

More information

QuickTiter FeLV Core Antigen ELISA Kit (FeLV p27)

QuickTiter FeLV Core Antigen ELISA Kit (FeLV p27) Product Manual QuickTiter FeLV Core Antigen ELISA Kit (FeLV p27) Catalog Numbers VPK-155 96 wells FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Feline leukemia virus (FeLV) is

More information

Anti-ATF6 α antibody, mouse monoclonal (1-7)

Anti-ATF6 α antibody, mouse monoclonal (1-7) Anti-ATF6 α antibody, mouse monoclonal (1-7) 73-500 50 ug ATF6 (activating transcription factor 6) is an endoplasmic reticulum (ER) membrane-bound transcription factor activated in response to ER stress.

More information

Aviva Systems Biology

Aviva Systems Biology Aviva Custom Antibody Service and Price Mouse Monoclonal Antibody Service Package Number Description Package Contents Time Price Customer provides antigen protein $6,174 Monoclonal package1 (From protein

More information

DNA Isolation Kit for Cells and Tissues

DNA Isolation Kit for Cells and Tissues DNA Isolation Kit for Cells and Tissues for 10 isolations of 00 mg each for tissue or 5 x 10 7 cultured cells Cat. No. 11 81 770 001 Principle Starting material Application Time required Results Benefits

More information

LAB 14 ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

LAB 14 ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) STUDENT GUIDE LAB 14 ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) GOAL The goal of this laboratory lesson is to explain the concepts and technique of enzyme linked immunosorbent assay (ELISA). OBJECTIVES

More information

STANDARD OPERATING PROCEDURE

STANDARD OPERATING PROCEDURE STANDARD OPERATING PROCEDURE Title: Antibody Production at Strategic Diagnostics Inc. SOP#: M-119 Version #: 1 Date Approved: August 6, 2009 Author: Strategic Diagnostic Inc. Date Modified: 1. PURPOSE

More information

Protocol for Western Blotting

Protocol for Western Blotting Protocol for Western Blotting Materials Materials used on Day 3 Protease inhibitor stock: 1 μg/μl pepstatin A in DMSO 200 μm leupeptin in OG Buffer 200 mm PMSF: Freshly made. Ex) 34.8 mg PMSF in 1 ml isopropanol

More information

QuickTiter Hepatitis B Surface Antigen (HBsAg) ELISA Kit

QuickTiter Hepatitis B Surface Antigen (HBsAg) ELISA Kit Product Manual QuickTiter Hepatitis B Surface Antigen (HBsAg) ELISA Kit Catalog Numbers VPK-5004 VPK-5004-5 96 assays 5 x 96 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction

More information

Covalent Conjugation to Cytodiagnostics Carboxylated Gold Nanoparticles Tech Note #105

Covalent Conjugation to Cytodiagnostics Carboxylated Gold Nanoparticles Tech Note #105 Covalent Conjugation to Cytodiagnostics Carboxylated Gold Nanoparticles Tech Note #105 Background Gold nanoparticle conjugates have been widely used in biological research and biosensing applications.

More information

ArC Amine Reactive Compensation Bead Kit

ArC Amine Reactive Compensation Bead Kit ArC Amine Reactive Compensation Bead Kit Catalog no. A1346 Table 1. Contents and storage information. Material Amount Composition Storage Stability ArC reactive beads (Component A) ArC negative beads (Component

More information

50 g 650 L. *Average yields will vary depending upon a number of factors including type of phage, growth conditions used and developmental stage.

50 g 650 L. *Average yields will vary depending upon a number of factors including type of phage, growth conditions used and developmental stage. 3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com Phage DNA Isolation Kit Product # 46800, 46850 Product Insert

More information

2.1.2 Characterization of antiviral effect of cytokine expression on HBV replication in transduced mouse hepatocytes line

2.1.2 Characterization of antiviral effect of cytokine expression on HBV replication in transduced mouse hepatocytes line i 1 INTRODUCTION 1.1 Human Hepatitis B virus (HBV) 1 1.1.1 Pathogenesis of Hepatitis B 1 1.1.2 Genome organization of HBV 3 1.1.3 Structure of HBV virion 5 1.1.4 HBV life cycle 5 1.1.5 Experimental models

More information

CUSTOM ANTIBODIES. Fully customised services: rat and murine monoclonals, rat and rabbit polyclonals, antibody characterisation, antigen preparation

CUSTOM ANTIBODIES. Fully customised services: rat and murine monoclonals, rat and rabbit polyclonals, antibody characterisation, antigen preparation CUSTOM ANTIBODIES Highly competitive pricing without compromising quality. Rat monoclonal antibodies for the study of gene expression and proteomics in mice and in mouse models of human diseases available.

More information

Your partner in immunology

Your partner in immunology Your partner in immunology Expertise Expertise Reactivity Reactivity Quality Quality Advice Advice Who are we? Specialist of antibody engineering Covalab is a French biotechnology company, specialised

More information

Guidance for Industry. Monoclonal Antibodies Used as Reagents in Drug Manufacturing

Guidance for Industry. Monoclonal Antibodies Used as Reagents in Drug Manufacturing Guidance for Industry Monoclonal Antibodies Used as Reagents in Drug Manufacturing U.S. Department of Health and Human Services Food and Drug Administration enter for Drug Evaluation and Research (DER)

More information

Running protein gels and detection of proteins

Running protein gels and detection of proteins Running protein gels and detection of proteins 1. Protein concentration determination using the BIO RAD reagent This assay uses a colour change reaction to give a direct measurement of protein concentration.

More information

SSI STREPTOCOCCUS LATEX GROUP KIT

SSI STREPTOCOCCUS LATEX GROUP KIT SSI STREPTOCOCCUS LATEX GROUP KIT SSI STREPTOCOCCUS LATEX GROUP KIT Latex particles coated with streptococcal antiserum raised in rabbits Application The Streptococcus Latex Group Kit is a ready-to-use

More information

About Our Products. Blood Products. Purified Infectious/Inactivated Agents. Native & Recombinant Viral Proteins. DNA Controls and Primers for PCR

About Our Products. Blood Products. Purified Infectious/Inactivated Agents. Native & Recombinant Viral Proteins. DNA Controls and Primers for PCR About Our Products Purified Infectious/Inactivated Agents ABI produces a variety of specialized reagents, allowing researchers to choose the best preparations for their studies. Available reagents include

More information

Blood-Based Cancer Diagnostics

Blood-Based Cancer Diagnostics The Biotechnology Education Company Blood-Based Cancer Diagnostics EDVO-Kit 141 Store entire experiment at room temperature. EXPERIMENT OBJECTIVE: The objective of this experiment is to learn and understand

More information

Chromatin Immunoprecipitation (ChIP)

Chromatin Immunoprecipitation (ChIP) Chromatin Immunoprecipitation (ChIP) Day 1 A) DNA shearing 1. Samples Dissect tissue (One Mouse OBs) of interest and transfer to an eppendorf containing 0.5 ml of dissecting media (on ice) or PBS but without

More information

Serology: Fluorescent antibody tests and other tests employing conjugated antibodies

Serology: Fluorescent antibody tests and other tests employing conjugated antibodies Serology: Fluorescent antibody tests and other tests employing conjugated antibodies Authors: Adapted by Prof M van Vuuren. Originally compiled by Dr RW Worthington. (Retired) Licensed under a Creative

More information

Custom Antibody Services

Custom Antibody Services prosci-inc.com Custom Antibody Services High Performance Antibodies and More Broad Antibody Catalog Extensive Antibody Services CUSTOM ANTIBODY SERVICES Established in 1998, ProSci Incorporated is a leading

More information

4A. Types of Laboratory Tests Available and Specimens Required. Three main types of laboratory tests are used for diagnosing CHIK: virus

4A. Types of Laboratory Tests Available and Specimens Required. Three main types of laboratory tests are used for diagnosing CHIK: virus 4. LABORATORY 4A. Types of Laboratory Tests Available and Specimens Required Three main types of laboratory tests are used for diagnosing CHIK: virus isolation, reverse transcriptase-polymerase chain reaction

More information

Bovine Mastitis. 062612tr

Bovine Mastitis. 062612tr Bovine Mastitis 062612tr Hardy Diagnostics has everything for your laboratory! Mastitis Microbiology Made Easy! Our products are designed to aid in the rapid identification of bovine mastitis organisms

More information

Methionine Sulfoxide Immunoblotting Kit

Methionine Sulfoxide Immunoblotting Kit Methionine Sulfoxide Immunoblotting Kit Item No. 600160 Customer Service 800.364.9897 * Technical Support 888.526.5351 www.caymanchem.com TABLE OF CONTENTS GENERAL INFORMATION 3 Materials Supplied 4 Precautions

More information

PRODUCTION OF MONOCLONAL ANTIBODIES FOR USE IN IMMUNOASSAYS BASED ON THE MAGNETIZABLE SOLID PHASE SEPARATION TECHNIQUE

PRODUCTION OF MONOCLONAL ANTIBODIES FOR USE IN IMMUNOASSAYS BASED ON THE MAGNETIZABLE SOLID PHASE SEPARATION TECHNIQUE PRODUCTION OF MONOCLONAL ANTIBODIES FOR USE IN IMMUNOASSAYS BASED ON THE MAGNETIZABLE SOLID PHASE SEPARATION TECHNIQUE W. CHAROENSIRIWATANA, N. JANEJAI, P.KRASAO XA9643133 Department of Medical Sciences,

More information

WHO Prequalification of Diagnostics Programme PUBLIC REPORT. Product: Genscreen ULTRA HIV Ag-Ab Number: PQDx 0096-031-00. Abstract

WHO Prequalification of Diagnostics Programme PUBLIC REPORT. Product: Genscreen ULTRA HIV Ag-Ab Number: PQDx 0096-031-00. Abstract WHO Prequalification of Diagnostics Programme PUBLIC REPORT Product: Genscreen ULTRA HIV Ag-Ab Number: PQDx 0096-031-00 Abstract Genscreen ULTRA HIV Ag-Ab with product codes 72386 and 72388, manufactured

More information

EVALUATION OF DARK FIELD MICROSCOPY, ISOLATION AND MICROSCOPIC AGGLUTINATION TEST FOR THE DIAGNOSIS OF CANINE LEPTOSPIROSIS

EVALUATION OF DARK FIELD MICROSCOPY, ISOLATION AND MICROSCOPIC AGGLUTINATION TEST FOR THE DIAGNOSIS OF CANINE LEPTOSPIROSIS Page85 Research Article Biological Sciences EVALUATION OF DARK FIELD MICROSCOPY, ISOLATION AND MICROSCOPIC AGGLUTINATION TEST FOR THE DIAGNOSIS OF CANINE LEPTOSPIROSIS S. Vamshi Krishna *, Siju Joseph,

More information

serology Agglutination Techniques and Blood Cell Identification

serology Agglutination Techniques and Blood Cell Identification Serology: Agglutination Techniques and Blood Cell Identification S erology is a branch of immunology dealing with techniques to identify and measure antigens, and to detect serum antibodies. Agglutination

More information

Interim Progress Report R&D Project 348. Development of a Field Test Kit for Detection of Blue-Green Algal Toxins

Interim Progress Report R&D Project 348. Development of a Field Test Kit for Detection of Blue-Green Algal Toxins Interim Progress Report R&D Project 348 Development of a Field Test Kit for Detection of Blue-Green Algal Toxins Biocode Limited November 1992 R&D 348/04/A ENVIRONMENT AGENCY 135357 CONTENTS SUMMARY KEYWORDS

More information

SDS-PAGE Protocol Mutated from the SDS-PAGE protocol written by the Lord of the Flies

SDS-PAGE Protocol Mutated from the SDS-PAGE protocol written by the Lord of the Flies SDS-PAGE Protocol Mutated from the SDS-PAGE protocol written by the Lord of the Flies Pouring the resolving gel 1. Clean glass plates with soap and water, then with ethanol. Assemble the glass plates and

More information

CHARACTERIZATION OF PSEUDOMONAS PSEUDOMALLEI ANTIGENS BY SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS AND WESTERN BLOT

CHARACTERIZATION OF PSEUDOMONAS PSEUDOMALLEI ANTIGENS BY SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS AND WESTERN BLOT CHARACTERIZATION OF PSEUDOMONAS PSEUDOMALLEI ANTIGENS BY SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS AND WESTERN BLOT Surasakdi ~on~ratanacheewin', Unchalee ~attawasart', Viraphong ~ulitanond', Suwin Wongwajanal,

More information

Western Blotting: Mini-gels

Western Blotting: Mini-gels Western Blotting: Mini-gels Materials a Protein Extraction Buffer (for callus or kernel), Solution Stock Final Volume Tris-HCl ph 80 1 M 200 mm 20 ml NaCl 4 M 100 mm 25 ml Sucrose 2 M 400 mm 20 ml EDTA

More information

(From the Department of Bacteriology, Preventive Medicine and Public Health, School o] Medicine, Howard University, Washington, D. C.

(From the Department of Bacteriology, Preventive Medicine and Public Health, School o] Medicine, Howard University, Washington, D. C. A THERMOPRECIPITATION EQUIPERDUM INFECTION REACTION IN TRYPANOSOMA IN LABORATORY ANIMALS BY HILDRUS A. POINDEXTER, M.D. (From the Department of Bacteriology, Preventive Medicine and Public Health, School

More information

Chromatin Immunoprecipitation

Chromatin Immunoprecipitation Chromatin Immunoprecipitation A) Prepare a yeast culture (see the Galactose Induction Protocol for details). 1) Start a small culture (e.g. 2 ml) in YEPD or selective media from a single colony. 2) Spin

More information

Basics of Immunology

Basics of Immunology Basics of Immunology 2 Basics of Immunology What is the immune system? Biological mechanism for identifying and destroying pathogens within a larger organism. Pathogens: agents that cause disease Bacteria,

More information

EZ-Run Protein Gel Solution. EZ-Run Protein Standards. EZ-Run Gel Staining Solution. Traditional SDS-Page Reagents. Protein Electrophoresis

EZ-Run Protein Gel Solution. EZ-Run Protein Standards. EZ-Run Gel Staining Solution. Traditional SDS-Page Reagents. Protein Electrophoresis EZ-Run Protein Gel Solution EZ-Run Protein Standards EZ-Run Gel Staining Solution Traditional SDS-Page Reagents Protein Electrophoresis protein electrophoresis Introduction Sodium dodecyl sulfate polyacrylamide

More information

WESTERN BLOT PROTOCOL FOR LICOR ODYSSEY SCANNER (HAKE S LAB)

WESTERN BLOT PROTOCOL FOR LICOR ODYSSEY SCANNER (HAKE S LAB) WESTERN BLOT PROTOCOL FOR LICOR ODYSSEY SCANNER (HAKE S LAB) WESTERN BLOT FOR ANALYSIS ON LICOR ODYSSEY SCANNER. 1) The Licor Odyssey protein marker is optimal as it is visible on channel 700 (2ul is enough

More information

Anti-V5 Antibody Anti-V5-HRP Antibody

Anti-V5 Antibody Anti-V5-HRP Antibody Anti-V5 Antibody Anti-V5-HRP Antibody Catalog nos. R960-25, R961-25 Version F 073001 28-0140 www.invitrogen.com tech_service@invitrogen.com ii Table of Contents Table of Contents...iii Overview...1 Western

More information

WESTERN BLOT DETECTION KIT Buffers and detection reagents for up to ten 10 x 10 cm 2 blots. Fluorescent detection via: Goat anti-mouse SureLight P3

WESTERN BLOT DETECTION KIT Buffers and detection reagents for up to ten 10 x 10 cm 2 blots. Fluorescent detection via: Goat anti-mouse SureLight P3 WESTERN BLOT DETECTION KIT Buffers and detection reagents for up to ten 10 x 10 cm 2 blots Fluorescent detection via: Goat anti-mouse SureLight P3 Cat. #: WK-P112 6440 Dobbin Road, Suite D Phone (443)

More information

Chapter 6: Antigen-Antibody Interactions

Chapter 6: Antigen-Antibody Interactions Chapter 6: Antigen-Antibody Interactions I. Strength of Ag-Ab interactions A. Antibody Affinity - strength of total noncovalent interactions between single Ag-binding site on an Ab and a single epitope

More information

APPLICATION FOCUS. Application Solutions for Western Blotting

APPLICATION FOCUS. Application Solutions for Western Blotting APPLICATION FOCUS Application Solutions for Western Blotting WESTERN BLOTTING Companion Products Solutions for consistently better blotting. Companion products from Cell Signaling Technology (CST) are

More information

Compromise Elsewhere Protocols. Western Blotting Methods. 800-656-ROCK www.rockland-inc.com info@rockland-inc.com 1 of 11

Compromise Elsewhere Protocols. Western Blotting Methods. 800-656-ROCK www.rockland-inc.com info@rockland-inc.com 1 of 11 Compromise Elsewhere Protocols Western Blotting Methods info@rockland-inc.com 1 of 11 Odyssey Western Blotting Protocols Odyssey reagents: Additional reagents needed: IR-labeled secondary antibodies Odyssey

More information

Hypoxyprobe -1 Plus Kit Kit contents:

Hypoxyprobe -1 Plus Kit Kit contents: Updated 2015 1 PRODUCT INSERT Hypoxyprobe, Inc 121 Middlesex Turnpike Burlington, MA 01803 USA www.hypoxyprobe.com Hypoxyprobe -1 Plus Kit Kit contents: Solid pimonidazole HCl (Hypoxyprobe -1) FITC conjugated

More information

The Use of Antibodies in Immunoassays

The Use of Antibodies in Immunoassays TECHNICAL NOTE The Use of Antibodies in Immunoassays Introduction Structure of an IgG Antibody Immunological reagents are the backbone of every immunoassay system. Immunoassays can be utilized to quantitatively

More information

Nitrotyrosine Western blot starter pack

Nitrotyrosine Western blot starter pack Nitrotyrosine Western blot starter pack Cat no. A010-513 A convenient reagent pack containing Badrilla s high integrity nitrotyrosine monoclonal antibody (clone 2E11) and associated positive and negative

More information

an antirubella antibody labelled with iodine-125 not require purified rubella antigen and, in general, are resistant to false positive results due to

an antirubella antibody labelled with iodine-125 not require purified rubella antigen and, in general, are resistant to false positive results due to J Clin Pathol 1985;38:1150-1154 Public Health Laboratory Service IgM antibody capture enzyme linked immunosorbent assay for detecting rubella specific IgM KATHRYN BELLAMY,* J HODGSON,t PS GARDNER,* P MORGAN-CAPNERt

More information

CONTENT. Chapter 1 Review of Literature. List of figures. List of tables

CONTENT. Chapter 1 Review of Literature. List of figures. List of tables Abstract Abbreviations List of figures CONTENT I-VI VII-VIII IX-XII List of tables XIII Chapter 1 Review of Literature 1. Vaccination against intracellular pathogens 1-34 1.1 Role of different immune responses

More information

Direct Antiglobulin Test (DAT)

Direct Antiglobulin Test (DAT) Exercise 8 Exercise 9 Direct Antiglobulin Test (DAT) Elution Study Task Aim Introduction To perform the DAT and elution procedure with correct interpretation of results. To perform with 100% accuracy the

More information

SCANTIBODIES Laboratory, Inc. Contract Monoclonal Antibody Production

SCANTIBODIES Laboratory, Inc. Contract Monoclonal Antibody Production A Technical Publication of SCANTIBODIES Laboratory, Inc. Volume 1 Number 4 9336 Abraham Way Santee, CA 92071 USA (619) 258-9300 fax (619) 258-9366 www.scantibodies.com SCANTIBODIES Laboratory, Inc. Contract

More information

Antibody Production Price List

Antibody Production Price List Antibody Production Price List Presenting Insight Biotechnology s price list for custom polyclonal and monoclonal antibody production services. We are happy to tailor individual packages towards the specific

More information

Western Blotting. USA: proteintech@ptglab.com UK & Europe: europe@ptglab.com China: service@ptglab.com. www.ptglab.com

Western Blotting. USA: proteintech@ptglab.com UK & Europe: europe@ptglab.com China: service@ptglab.com. www.ptglab.com Western Blotting All steps are carried out at room temperature unless otherwise indicated. Recipes for all solutions highlighted bold are included at the end of the protocol. SDS-PAGE 1. Construct an SDS-PAGE

More information

Protein transfer from SDS-PAGE to nitrocellulose membrane using the Trans-Blot SD cell (Western).

Protein transfer from SDS-PAGE to nitrocellulose membrane using the Trans-Blot SD cell (Western). Western Blot SOP Protein transfer from SDS-PAGE to nitrocellulose membrane using the Trans-Blot SD cell (Western). Date: 8/16/05, 10/31/05, 2/6/06 Author: N.Oganesyan, R. Kim Edited by: R. Kim Summary:

More information

TABLE OF CONTENT. Page ACKNOWLEDGEMENTS. iii ENGLISH ABSTRACT THAI ABSTRACT. vii LIST OF TABLES LIST OF FIGURES. xvi ABBREVIATIONS.

TABLE OF CONTENT. Page ACKNOWLEDGEMENTS. iii ENGLISH ABSTRACT THAI ABSTRACT. vii LIST OF TABLES LIST OF FIGURES. xvi ABBREVIATIONS. x TABLE OF CONTENT ACKNOWLEDGEMENTS ENGLISH ABSTRACT THAI ABSTRACT LIST OF TABLES LIST OF FIGURES ABBREVIATIONS iii iv vii xv xvi xviii CHAPTER I: INTRODUCTION 1.1 Statement of problems 1 1.2 Literature

More information

PURIFICATION AND CHARACTERIZATION OF CELL WALL MANNOPROTEINS OF CANDIDA ALBICANS USING INTACT CELL METHOD

PURIFICATION AND CHARACTERIZATION OF CELL WALL MANNOPROTEINS OF CANDIDA ALBICANS USING INTACT CELL METHOD Medical Journal of the Islamic Republic of Iran Z. Farahnejad, et al. Volume 18 Number 2 Summer 1383 August 2004 PURIFICATION AND CHARACTERIZATION OF CELL WALL MANNOPROTEINS OF CANDIDA ALBICANS USING INTACT

More information

MagExtractor -Genome-

MagExtractor -Genome- Instruction manual MagExtractor-Genome-0810 F0981K MagExtractor -Genome- NPK-101 100 preparations Store at 4 C Contents [1] Introduction [2] Components [3] Materials required [4] Protocol 1. Purification

More information

Marmara Üniversitesi Fen-Edebiyat Fakültesi Kimya Bölümü / Biyokimya Anabilim Dalı PURIFICATION AND CHARACTERIZATION OF PROTEINS

Marmara Üniversitesi Fen-Edebiyat Fakültesi Kimya Bölümü / Biyokimya Anabilim Dalı PURIFICATION AND CHARACTERIZATION OF PROTEINS EXPERIMENT VI PURIFICATION AND CHARACTERIZATION OF PROTEINS I- Protein isolation and dialysis In order to investigate its structure and properties a protein must be obtained in pure form. Since proteins

More information

Custom Antibodies Services. GeneCust Europe. GeneCust Europe

Custom Antibodies Services. GeneCust Europe. GeneCust Europe GeneCust Europe Laboratoire de Biotechnologie du Luxembourg S.A. 6 rue Dominique Lang L-3505 Dudelange Luxembourg Tél. : +352 27620411 Fax : +352 27620412 Email : info@genecust.com Web : www.genecust.com

More information

Monoclonal Antibodies Production for Quantification of Ochratoxin A in Feedstuffs by Enzyme-Linked Immunosorbent Assay

Monoclonal Antibodies Production for Quantification of Ochratoxin A in Feedstuffs by Enzyme-Linked Immunosorbent Assay Kasetsart J. (Nat. Sci.) 37 : 137-144 (2003) Monoclonal Antibodies Production for Quantification of Ochratoxin A in Feedstuffs by Enzyme-Linked Immunosorbent Assay Montri Punyatong, Puntipa Pongpiachan

More information

Laboratory Protocols Level 2 Training Course Serotypning of Salmonella enterica O and H antigen.

Laboratory Protocols Level 2 Training Course Serotypning of Salmonella enterica O and H antigen. Global Salm-Surv A global Salmonella surveillance and laboratory support project of the World Health Organization Laboratory Protocols Level 2 Training Course Serotypning of Salmonella enterica O and H

More information

STANDING Tall CUSTOM PRODUCTS AND SERVICES

STANDING Tall CUSTOM PRODUCTS AND SERVICES STANDING Tall CUSTOM PRODUCTS AND SERVICES COME GROW WITH US! Contact KPL and let us know how we can support your OEM needs. Quality antibodies, substrates, and immunoassay support reagents Large scale

More information

How To Make A Tri Reagent

How To Make A Tri Reagent TRI Reagent For processing tissues, cells cultured in monolayer or cell pellets Catalog Number T9424 Store at room temperature. TECHNICAL BULLETIN Product Description TRI Reagent is a quick and convenient

More information

ELITE Custom Antibody Services

ELITE Custom Antibody Services ELITE Custom Antibody Services ELITE Custom Antibody Services Experience, confidence, and understanding As a manufacturer and service provider, we have the experience, confidence, and understanding to

More information

Standard Operating Procedure (SOP)

Standard Operating Procedure (SOP) Standard Operating Procedure (SOP) Title: Direct Fluorescent Antibody Test (DFAT) for the detection of Renibacterium salmoninarum in tissues Number: BACT-3 Version: 02 Created December 14, 2011 Approval:

More information

Diagnosis of HIV-1 Infection. Estelle Piwowar-Manning HPTN Central Laboratory The Johns Hopkins University

Diagnosis of HIV-1 Infection. Estelle Piwowar-Manning HPTN Central Laboratory The Johns Hopkins University Diagnosis of HIV-1 Infection Estelle Piwowar-Manning HPTN Central Laboratory The Johns Hopkins University Tests Used to Diagnose HIV-1 Infection HIV antibody (today s topic) HIV p24 antigen HIV DNA HIV

More information

Detection of a Species-specific Antigen of Gardnerella vaginalis by Western Blot Analysis

Detection of a Species-specific Antigen of Gardnerella vaginalis by Western Blot Analysis Journal of General Microbiology (1986), 132, 1969-1973. Printed in Great Britain 1969 Detection of a Species-specific Antigen of Gardnerella vaginalis by Western Blot Analysis By Y. L. BOUSTOULLER,* A.

More information

Protein extraction from Tissues and Cultured Cells using Bioruptor Standard & Plus

Protein extraction from Tissues and Cultured Cells using Bioruptor Standard & Plus Protein extraction from Tissues and Cultured Cells using Bioruptor Standard & Plus Introduction Protein extraction from tissues and cultured cells is the first step for many biochemical and analytical

More information