Name
Hycult Dx Human MBL ELISA kit
Catalog nr
DH001
(lot number and expiry date are indicated on the label)
Short description
Mannose Binding Lectin (MBL) is a C-type lectin and an important element in innate immunity. MBL belongs to the collectin family of proteins that consists of a collagen-like domain and a carbohydrate recognition domain (CRD). Through this, MBL recognizes carbohydrates (e.g. mannose and N-acetylglucosamine) on pathogens. MBL forms several sizes of oligomers that are composed of subunits of thr...
Size
1 x 96 det.
Application
Manual
Description
Mannose Binding Lectin (MBL) is a C-type lectin and an important element in innate immunity. MBL belongs to the collectin family of proteins that consists of a collagen-like domain and a carbohydrate recognition domain (CRD). Through this, MBL recognizes carbohydrates (e.g. mannose and N-acetylglucosamine) on pathogens. MBL forms several sizes of oligomers that are composed of subunits of three identical 32 kDa polypeptides. MBL is a pattern recognition receptor, and upon recognition of the infectious agent, MBL triggers the activation of the lectin-complement pathway through attached C1r/C1s-like serine proteases, designated MASP. The MBL-MASP complex proteolytically cleaves C4, C2 and C3. The lectin pathway is antibody and C1q-independent and is, therefore, independent of the classical and alternative complement activation pathways. MBL is synthesized by hepatocytes and has been isolated from the liver or serum of several vertebrate species.
Normal human plasma contains MBL concentrations ranging from 10 to 5,000 ng/ml. Up to 12% of healthy Caucasian blood donors have MBL concentrations below 100 ng/ml. Low plasma concentrations have been associated with an inherited defect in opsonization. The MBL concentration is enhanced in infectious diseases. Measuring of MBL is indicated in recurrent infections (especially in children), primary/secondary immunodeficiencies, artherosclerosis/coronary heart disease, cystic fibrosis, transplantation, autoimmune diseases (SLE/Rheumatoid arthritis) and habitual abortion. MBL measurement is not affected by the presence of antibodies against mannan. In the human MBL assay, any influence of the classical pathway has been eliminated by using a special MBL-binding buffer, which inhibits the binding of C1q to immunocomplexes and disrupts the C1 complex, while leaving the function of the MBL complex intact.
Cross Reactivity
The Hycult Dx human MBL ELISA has been developed for the quantitative measurement of natural human MBL.
No potential cross-reacting proteins have been detected in the Hycult Dx human MBL ELISA.
Features
Working time of 4 hours.
Minimum concentration which can be measured is 0.41 ng/ml.
Measurable concentration range of 0.41 to 100 ng/ml.
Working volume of 100 µl/well.
Minimum concentration which can be measured is 0.41 ng/ml.
Measurable concentration range of 0.41 to 100 ng/ml.
Working volume of 100 µl/well.
Typical standard curve
Principle
The Hycult Dx human MBL ELISA is a ready-to-use ligand-lectin solid-phase enzyme-linked immuno assay with a working time of 4 hours.
The efficient format of 1 plate with twelve disposable 8-well strips allows free choice of batch size for the assay.
Samples, standards and controls are incubated in microtiter wells coated with mannan.
During this incubation the mannan binding property of functional human MBL is used to capture human MBL by solid bound mannan.
Biotinylated tracer antibody to human MBL is added to the wells.
If functional, mannan binding human MBL is present in the sample, the tracer antibodies will bind to the mannan bound human MBL.
Streptavidin-peroxidase will bind to the biotinylated tracer antibody.
Streptavidin-peroxidase will react with the substrate, tetramethylbenzidine (TMB).
The enzyme reaction is stopped by the addition of oxalic acid.
The absorbance at 450 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorbance (linear) versus the corresponding concentrations of the human MBL standards (log).
The human MBL concentration of samples and controls, which are run concurrently with the standards, can be determined from the standard curve.
The efficient format of 1 plate with twelve disposable 8-well strips allows free choice of batch size for the assay.
Samples, standards and controls are incubated in microtiter wells coated with mannan.
During this incubation the mannan binding property of functional human MBL is used to capture human MBL by solid bound mannan.
Biotinylated tracer antibody to human MBL is added to the wells.
If functional, mannan binding human MBL is present in the sample, the tracer antibodies will bind to the mannan bound human MBL.
Streptavidin-peroxidase will bind to the biotinylated tracer antibody.
Streptavidin-peroxidase will react with the substrate, tetramethylbenzidine (TMB).
The enzyme reaction is stopped by the addition of oxalic acid.
The absorbance at 450 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorbance (linear) versus the corresponding concentrations of the human MBL standards (log).
The human MBL concentration of samples and controls, which are run concurrently with the standards, can be determined from the standard curve.
Use
The Hycult Dx human MBL ELISA kit is to be used for the in vitro quantitative determination of human MBL in serum and plasma samples.
The results of the kit are to be used in combination with other laboratory and clinical findings for the purpose of providing information about a pathological state. The analysis should be performed by trained laboratory professionals.
Storage and stability
- Upon receipt, store individual components at 2 - 8°C, do not freeze.
- Do not use components beyond the expiration date printed on the kit label.
- Once reconstituted, , and are stable for 12 hours, if stored at 2 – 8 °C. For longer stability we recommend storage of aliquots at –20°C. Stored at -20°C the standard and controls will be stable for one month.
- The exact concentration of the standard is indicated on the label of the vial and the certificate of quality control.
- Once reconstituted, and are stable for 1 month if stored at 2 - 8°C.
- Upon receipt, foil pouch around the should be vacuum-sealed and unpunctured. Any irregularities to aforementioned conditions may influence plate performance in the assay.
- Return unused strips immediately to the foil pouch containing the desiccant pack and reseal along the entire edge of the zip-seal. Store until expiration date at 2 - 8°C.
Recovery
Normal human blood samples (plasma) containing baseline levels of human MBL, were spiked with human MBL in concentrations of 5 and 50 ng/ml. Samples with and without human MBL were incubated for 1 hour at room temperature. Samples were measured using the ELISA. Values for human MBL ranged between 90.6% and 101.5% (mean 93.9%).
Linearity
Precision and reproducibility
The intra-assay precision and reproducibility was tested with samples of three different, known concentrations in 6 replicates, performed by 3 different operators.
The inter-assay precision and reproducibility was tested with samples of three different, known concentrations, assayed in duplicate in 6 separate runs. These were performed by 3 different operators: single run per operator on 2 different days.
|
Parameter |
CV acceptance limit |
CV of current batch |
|
Intra-assay |
≤ 20% |
12.7% |
|
Inter-assay |
≤ 25% |
15.2% |
The linearity of the assay was determined by serially diluting a sample containing 250 ng/ml human MBL. The diluted samples were measured in the assay. The line obtained a slope of 1.124 and a correlation coefficient of 0.997.
References
- Kirkpatrick, B et al; Serum mannose-binding lectin deficiency is associated with cryptosporidiosis in young Haitian children. Clin Infect Dis 2006, 43: 289
- Druszczynska, M et al; Tuberculosis bacilli still posing a threat. Polymorphism of genes regulating anti-mycobacterial properties of macrophages. Pol J Microbiol 2006, 55: 7
- Wang, X et al; Mannose-binding lectin gene polymorphisms and the development of coal workers pneumoconiosis in Japan. Am J Ind Med 2008, 51: 548
- Ribeiro, L et al; Serum mannose-binding lectin levels are linked with respiratory syncytial virus (RSV) disease. J clin immunol 2008, 28: 166
- Koutsounaki, E et al; Mannose-binding lectin MBL2 gene polymorphisms and the outcome of hepatitis C virus-infected patients. J Clin Immunol 2008, 28: 495
References
- Kirkpatrick, B et al; Serum mannose-binding lectin deficiency is associated with cryptosporidiosis in young Haitian children. Clin Infect Dis 2006, 43: 289
- Druszczynska, M et al; Tuberculosis bacilli still posing a threat. Polymorphism of genes regulating anti-mycobacterial properties of macrophages. Pol J Microbiol 2006, 55: 7
- Wang, X et al; Mannose-binding lectin gene polymorphisms and the development of coal workers pneumoconiosis in Japan. Am J Ind Med 2008, 51: 548
- Ribeiro, L et al; Serum mannose-binding lectin levels are linked with respiratory syncytial virus (RSV) disease. J clin immunol 2008, 28: 166
- Koutsounaki, E et al; Mannose-binding lectin MBL2 gene polymorphisms and the outcome of hepatitis C virus-infected patients. J Clin Immunol 2008, 28: 495
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