ELISA-Based Assay Development
The Enzyme-Linked Immunosorbent Assay (ELISA) is the most common and widely used immunoassay application. ELISAs are designed for detecting and quantitating substances such as peptides, proteins, antibodies, hormones, cytokines, drugs of abuse and their metabolites. A number of different assay formats are being used, dependent on the system being investigated. The simplest format involves coating of an antigen onto microtiter plates, followed by incubation with a specific antibody. The binding antibody or an appropriate secondary antibody is conjugated to an enzyme that typically catalyzes the formation of a colored product. Color formation is monitored spectrophotometrically and related to concentration of the antigen by calibration to a standard curve. When antigen is limited, a sandwich assay format is used in which the analyte to be measured is bound between two antibodies – the capture antibody and the detection antibody. Competitive assays are often used when the antigen is small and has only one epitope, or antibody binding site. ELISAs are typically performed in 96, 384, and even 1536-well polystyrene plates, and are easily robotized and adapted to high throughput screening.
BIO-SYNTHESIS, INC. has generated many polyclonal antibodies for ELISA and has developed optimized protocols. Most of our catalog antibodies have been tested in ELISA-type assays. They are able to detect antigens reliably, down to concentrations as low as 250 pg/ml. In addition, we have custom-made many polyclonal antibodies for ELISA-based applications required by our customers, including antibodies to phosphoproteins, enzymes, haptens, and drugs of abuse. BIO-SYNTHESIS, INC. offers a wealth of expertise in developing antibodies for ELISA-based applications. Please contact us with your specific application needs.
Showing posts with label STR DNA Typing. Show all posts
Showing posts with label STR DNA Typing. Show all posts
Wednesday, August 4, 2010
Tuesday, August 3, 2010
STR DNA Identification Multiplex
Bio-Synthesis assists client in developing DNA-based test kits for diseases by using custom PCR (Polymerase Chain Reaction) assays which are designed, manufactured, quality controlled, and tested on biological materials at our facilities in Lewisville, Texas. This Molecular Test is also known as an Nucleic Acid Amplification Test. It is based on the amplification of the presence of target DNA in the specimen, rather than the conventional method of bacteria or cell cultures. The group of molecular genetic research scientists at Bio-Synthesis, Inc. has successfully developed the DNA PCR –SSP Fastype™ System Detection Kits for human infectious diseases for clients in need of custom formulation. We also provide fresh prepared DNA-Based Human Infectious Detection kits designed to detect Conjunctivitis, Adenovirus and Chlamydia, useful for molecular testing labs of infectious diseases.
As a one-stop-shop company, we put all of our products to work more efficiently through our Custom Assay Development. Bio-Synthesis has provided industrial and academic scientists with a unique service platform dedicated to the design, development and testing of the DNA-Based assays that you have been waiting for.
Bio-Synthesis can guide you through all steps, starting from your basic needs to finishing with your DNA-based PCR detection kits.
The primary features of the unique Fastype™ System Kits are listed as follows:
All primers, dNTPs and PCR buffer (with red dye as an indicator) are pre-aliquot and lyophilized in high-quality PCR thermal cycle strip tubes or plates. Positive Control Kits are also prepared with the same procedure by just adding genomic DNA template into the wells.
Lyophilized primers, dNTPs and PCR buffer are more stable than in the liquid form under ambient condition, and it is easier to transport.
The final lyophilized kits contain a red dot (reagent) in the bottom of the PCR test tubes, and one can distinguish clearly the kits from the regular PCR tubes. The PCR is performed with the special red color gel loading dye, and you can simply apply the PCR product onto the agarose gel without adding loading buffer.
The Fastype™ PCR-SSP Kits reduce the preparation time to less than 10 minutes. All you have to do is add DNA template, DDH2O and TAQ, and start the PCR program.
We can custom develop testing kits of your research interest, whether you prefer a volume of 10µl or 50µl per PCR experiment. And, we have strong technical support to guide you along the way.
See products on Fastype™ PCR-SSP Detection Kits for Human Infectious Disease
Contact:
Before going into technical steps, your project will be fully analyzed in order to evaluate its feasibility and to build its strategy, providing the highest chance of success. You will be provided with a detailed quotation, listing all steps and associated costs in order to facilitate your decision process.
Please contact us to consult with our specialists and start designing a Custom PCR-SSP Molecular Detection Kit.
As a one-stop-shop company, we put all of our products to work more efficiently through our Custom Assay Development. Bio-Synthesis has provided industrial and academic scientists with a unique service platform dedicated to the design, development and testing of the DNA-Based assays that you have been waiting for.
Bio-Synthesis can guide you through all steps, starting from your basic needs to finishing with your DNA-based PCR detection kits.
The primary features of the unique Fastype™ System Kits are listed as follows:
All primers, dNTPs and PCR buffer (with red dye as an indicator) are pre-aliquot and lyophilized in high-quality PCR thermal cycle strip tubes or plates. Positive Control Kits are also prepared with the same procedure by just adding genomic DNA template into the wells.
Lyophilized primers, dNTPs and PCR buffer are more stable than in the liquid form under ambient condition, and it is easier to transport.
The final lyophilized kits contain a red dot (reagent) in the bottom of the PCR test tubes, and one can distinguish clearly the kits from the regular PCR tubes. The PCR is performed with the special red color gel loading dye, and you can simply apply the PCR product onto the agarose gel without adding loading buffer.
The Fastype™ PCR-SSP Kits reduce the preparation time to less than 10 minutes. All you have to do is add DNA template, DDH2O and TAQ, and start the PCR program.
We can custom develop testing kits of your research interest, whether you prefer a volume of 10µl or 50µl per PCR experiment. And, we have strong technical support to guide you along the way.
See products on Fastype™ PCR-SSP Detection Kits for Human Infectious Disease
Contact:
Before going into technical steps, your project will be fully analyzed in order to evaluate its feasibility and to build its strategy, providing the highest chance of success. You will be provided with a detailed quotation, listing all steps and associated costs in order to facilitate your decision process.
Please contact us to consult with our specialists and start designing a Custom PCR-SSP Molecular Detection Kit.
Sunday, July 25, 2010
Custom DNA Peptide-Based Assay Development
Custom DNA Peptide-Based Assay Development
As researchers approach the developing field of genomics with custom DNA/peptide based assays, the need for microarray services or productions that maximize flexibility and precision, without compromising speed and cost, has become critical. In order to meet the demands for custom microarraying, BSI offers a complete suite of microarray services, which includes:
To download more specific instructions and an appropriate order form, please click here.
Quality Control
Replicated spots and slides are used to monitor technical variations. In addition, pre-scanning with fast dye staining is conducted to make sure the spot size and morphology meet expectations. Sometimes a test hybridization is conducted, if needed. Using similar high-precision printing technology, the peptides or proteins can be spotted on membrane-coated glass slides. Using different detection technologies, recombinant proteins/antibodies, cell lysate or serum samples can be quantitatively assayed. This has wide applications in protein-protein interaction studies, including antigen-antibody recognitions, kinase-substrate bindings, pathway analysis, biomarker screenings, and much more.
Services highlight
As researchers approach the developing field of genomics with custom DNA/peptide based assays, the need for microarray services or productions that maximize flexibility and precision, without compromising speed and cost, has become critical. In order to meet the demands for custom microarraying, BSI offers a complete suite of microarray services, which includes:
- Experimental design consultation, provide expert support with DNA/peptide sequence selection, microarray chip layout design, and quality control.
- Synthesis chemistry of custom DNA, RNA, peptide, BNA or hybrid oligos with backbone modifications such as phosphorothioate for antisense study, or nucleotide modifications
- Flexible chip fabrication, which allow fine-tune client's probe sets for high-density custom microarrays.
- Target sample labeling and hybridization
- Other related services DNA/RNA Extraction, RNA quality check, Real-Time PCR, Western blot.
- BioInformatic data analysis
To download more specific instructions and an appropriate order form, please click here.
Quality Control
Replicated spots and slides are used to monitor technical variations. In addition, pre-scanning with fast dye staining is conducted to make sure the spot size and morphology meet expectations. Sometimes a test hybridization is conducted, if needed. Using similar high-precision printing technology, the peptides or proteins can be spotted on membrane-coated glass slides. Using different detection technologies, recombinant proteins/antibodies, cell lysate or serum samples can be quantitatively assayed. This has wide applications in protein-protein interaction studies, including antigen-antibody recognitions, kinase-substrate bindings, pathway analysis, biomarker screenings, and much more.
Services highlight
- DNA/Peptide selection and modification
- sample to data full service
Wednesday, July 14, 2010
Human Cell Line Authentication Services
Bio-Synthesis, Inc. (BSI) offers fast and reliable human cell line authentication services, using STR DNA typing, to assist researchers in confirming species identity and detection of possible intra-species contamination or misidentification. This technology is/a provides:
* Highly Informative
Using multiplex STR technology which contains 15 loci and 1 sex determination marker (Amelogenin) for high resolution screening to detect intra-species cross contamination.
* Highly Sensitive
Capable of detecting as low as 500 picograms of genomic DNA (~ 15 diploid cells).
* High Throughput
Based on 5-dye chemistry which allows higher density multiplexing with better spectral resolution to minimize false positive calls due to fluorescent artifacts..
* Integrated Workflow
Using Applied Biosystems Genetic Analysyzer, GeneScane, GeneMapper ® ID software
* Compatible Pricing
as low as $120/sample and up
* Fast Turnaround
3-5 business days
Please contact us directly to discuss options that may be applicable to your project.
For decades, inadvertent cell line contamination has been a major issue for biomedical researchers. Cell line integrity is of utmost importance especially in stem cell, cancer tumor research and other new, developing research. In the culture of mammalian cells, laboratory personnel usually are more concerned about microbial (bacteria, yeast, mycoplasma) contamination, while intraspecific1, 2 and interspecific3 cross-contamination between different cell lines may be overlooked. The contamination and overgrowth of cell lines is a well documented, but often neglected phenomenon. Contamination by HeLa cells is only the best known example, and shows that any rapidly growing cell line can overtake both tumor cell cultures and normal cell lines. Nelson-Rees et al4 were among the first to document the extent of this problem. The implications are far reaching, including invalidation of published data and lost time, money and effort. As a result, NIH has recommended that all cell lines used in research undergo authentication to verify their identity (NIH Notice Number OT-OD-08-17) and stated that "(g)rant applications that fail to employ such practices would not be considered of the highest quality and such manuscripts would not fare well in the journal review process." See detailed lists of misidentified malignant hematopoietic cell lines.
About STR Technology
Most DNA present in the genomes of higher animals does not actually code for protein, as well as gene introns, regulatory and structurally important sequences, RNA genes, pseudogenes and sequences of questionable function are present. A significant part of non-coding DNA is comprised of repetitive sequences. The "mini- or microsatellites" are highly polymorphic zones inside these non-coding regions and characterized by reiterated DNA blocks, each containing individual sets of repeats5. The single-locus short tandem repeats (STR) are located at specific loci, while multi-locus STRs are spread throughout the entire genome6, 7. Repeat-number-variability polymorphisms define the length of the individual specific "alleles", which can be analyzed by Southern blot or polymerase chain reaction (PCR) techniques. The analysis of STR at each single-locus site results in the detection of two DNA fragments differing in length unless the paternal and maternal alleles comigrate. In contrast, the analysis of the genome using a multi-locus probe, detects a large and complex pattern of DNA fragments (see figure). A similarly high level of information can be obtained by STR DNA typing when several highly polymorphic regions are analyzed simultaneously to facilitate a more reliable typing of individual cell lines4
Solution by Using STR DNA Typing
Molecular profiling by short tandem repeats (STR or microsatellite markers) is a fast, affordable and highly discriminating method to authenticate a cell line. Due to the highly polymorphic nature of each STR loci, every cell line will have a unique digital bar code that can distinguish it from other sources. A common set of STR loci are widely used for forensic applications and can be used for intra-species authentication.
The DNA typing process used at Bio-Synthesis involves simultaneously amplifying fifteen STR loci and the amelogenin gene in a multiplex PCR reaction (from Applied Biosystems AmpFℓSTR® Identifiler) capable of detecting as low as 500 picograms of genomic DNA (~ 15 diploid cells). The amplicons are separated by capillary electrophoresis and analyzed using Applied Biosystem Identifiler kit and GeneMapper software. The pattern of repeats results in a unique STR identity profile for each cell line analyzed. The profile can be used as a baseline for comparison with future tests. On discovering an untoward match between newly accessioned cell lines and an existing DNA profile, further independent approaches should be used to confirm cross-contamination, including independent DNA typing systems, such as VNTR or multi locus STR typing, as well as immunological and cytogenetic analyses.
See sample preparation and delivery
Cell line validation request form
References
1. MacLeod RAF, Dirks WG, Matsuo Y, Kaufmann M, Milch H, Drexler HG (1999). Widespread intraspecies cross-contamination of human tumor cell lines arising at source. Int. J. Cancer 83: 555-563.
2. Dirks W, MacLeod RAF, Jäger K, Milch H, Drexler HG (1999). First searchable database for DNA profiles of human cell lines: Sequential use of fingerprint techniques for authentication. Cell. Mol. Biol. 45: 841-853
3. Parodi B, Arescu O, Bini D, Lorenzini R, Schena F, Visconti P, Cesaro M, Ferrera D, Andreotti V, Ruzzon T (2002). Species identification and confirmation of human and animal cell lines: A PCR method. Biotechniques 32: 432-440
4. Nelson-Rees, WA, Daniels D, Flandermeyer RR (1981). Cross-contamination of cells in culture. Science 212: 446-452.
5. Jeffreys AJ, Wilson V, Thein SL: Hypervariable 'minisatellite' regions in human DNA. Nature 314: 67-73 (1985).
6. Zischler H, Nanda I, Schäfer R, Schmid M, Epplen JT: Digoxigenated oligonucleotide probes specific for simple repeats in DNA fingerprinting and hybridization in situ. Hum Genet 82: 227- 233 (1989).
7. Häne B, Tümmler M, Jäger K, Schleithoff L, Janssen JWG, Drexler HG: Differences in DNA fingerprints of continuous leukemia-lymphoma cell lines from different sources. Leukemia 6: 1129-1133 (1992).
* Highly Informative
Using multiplex STR technology which contains 15 loci and 1 sex determination marker (Amelogenin) for high resolution screening to detect intra-species cross contamination.
* Highly Sensitive
Capable of detecting as low as 500 picograms of genomic DNA (~ 15 diploid cells).
* High Throughput
Based on 5-dye chemistry which allows higher density multiplexing with better spectral resolution to minimize false positive calls due to fluorescent artifacts..
* Integrated Workflow
Using Applied Biosystems Genetic Analysyzer, GeneScane, GeneMapper ® ID software
* Compatible Pricing
as low as $120/sample and up
* Fast Turnaround
3-5 business days
Please contact us directly to discuss options that may be applicable to your project.
For decades, inadvertent cell line contamination has been a major issue for biomedical researchers. Cell line integrity is of utmost importance especially in stem cell, cancer tumor research and other new, developing research. In the culture of mammalian cells, laboratory personnel usually are more concerned about microbial (bacteria, yeast, mycoplasma) contamination, while intraspecific1, 2 and interspecific3 cross-contamination between different cell lines may be overlooked. The contamination and overgrowth of cell lines is a well documented, but often neglected phenomenon. Contamination by HeLa cells is only the best known example, and shows that any rapidly growing cell line can overtake both tumor cell cultures and normal cell lines. Nelson-Rees et al4 were among the first to document the extent of this problem. The implications are far reaching, including invalidation of published data and lost time, money and effort. As a result, NIH has recommended that all cell lines used in research undergo authentication to verify their identity (NIH Notice Number OT-OD-08-17) and stated that "(g)rant applications that fail to employ such practices would not be considered of the highest quality and such manuscripts would not fare well in the journal review process." See detailed lists of misidentified malignant hematopoietic cell lines.
About STR Technology
Most DNA present in the genomes of higher animals does not actually code for protein, as well as gene introns, regulatory and structurally important sequences, RNA genes, pseudogenes and sequences of questionable function are present. A significant part of non-coding DNA is comprised of repetitive sequences. The "mini- or microsatellites" are highly polymorphic zones inside these non-coding regions and characterized by reiterated DNA blocks, each containing individual sets of repeats5. The single-locus short tandem repeats (STR) are located at specific loci, while multi-locus STRs are spread throughout the entire genome6, 7. Repeat-number-variability polymorphisms define the length of the individual specific "alleles", which can be analyzed by Southern blot or polymerase chain reaction (PCR) techniques. The analysis of STR at each single-locus site results in the detection of two DNA fragments differing in length unless the paternal and maternal alleles comigrate. In contrast, the analysis of the genome using a multi-locus probe, detects a large and complex pattern of DNA fragments (see figure). A similarly high level of information can be obtained by STR DNA typing when several highly polymorphic regions are analyzed simultaneously to facilitate a more reliable typing of individual cell lines4
Solution by Using STR DNA Typing
Molecular profiling by short tandem repeats (STR or microsatellite markers) is a fast, affordable and highly discriminating method to authenticate a cell line. Due to the highly polymorphic nature of each STR loci, every cell line will have a unique digital bar code that can distinguish it from other sources. A common set of STR loci are widely used for forensic applications and can be used for intra-species authentication.
The DNA typing process used at Bio-Synthesis involves simultaneously amplifying fifteen STR loci and the amelogenin gene in a multiplex PCR reaction (from Applied Biosystems AmpFℓSTR® Identifiler) capable of detecting as low as 500 picograms of genomic DNA (~ 15 diploid cells). The amplicons are separated by capillary electrophoresis and analyzed using Applied Biosystem Identifiler kit and GeneMapper software. The pattern of repeats results in a unique STR identity profile for each cell line analyzed. The profile can be used as a baseline for comparison with future tests. On discovering an untoward match between newly accessioned cell lines and an existing DNA profile, further independent approaches should be used to confirm cross-contamination, including independent DNA typing systems, such as VNTR or multi locus STR typing, as well as immunological and cytogenetic analyses.
See sample preparation and delivery
Cell line validation request form
References
1. MacLeod RAF, Dirks WG, Matsuo Y, Kaufmann M, Milch H, Drexler HG (1999). Widespread intraspecies cross-contamination of human tumor cell lines arising at source. Int. J. Cancer 83: 555-563.
2. Dirks W, MacLeod RAF, Jäger K, Milch H, Drexler HG (1999). First searchable database for DNA profiles of human cell lines: Sequential use of fingerprint techniques for authentication. Cell. Mol. Biol. 45: 841-853
3. Parodi B, Arescu O, Bini D, Lorenzini R, Schena F, Visconti P, Cesaro M, Ferrera D, Andreotti V, Ruzzon T (2002). Species identification and confirmation of human and animal cell lines: A PCR method. Biotechniques 32: 432-440
4. Nelson-Rees, WA, Daniels D, Flandermeyer RR (1981). Cross-contamination of cells in culture. Science 212: 446-452.
5. Jeffreys AJ, Wilson V, Thein SL: Hypervariable 'minisatellite' regions in human DNA. Nature 314: 67-73 (1985).
6. Zischler H, Nanda I, Schäfer R, Schmid M, Epplen JT: Digoxigenated oligonucleotide probes specific for simple repeats in DNA fingerprinting and hybridization in situ. Hum Genet 82: 227- 233 (1989).
7. Häne B, Tümmler M, Jäger K, Schleithoff L, Janssen JWG, Drexler HG: Differences in DNA fingerprints of continuous leukemia-lymphoma cell lines from different sources. Leukemia 6: 1129-1133 (1992).
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