Suparnostic
Encyclopedia
suPARnostic is a simplified double monoclonal antibody sandwich enzyme-linked immunosorbent assay (ELISA
) that measures the amount of soluble urokinase plasminogen activator receptor (suPAR
) in blood. Elevated plasma suPAR levels have been observed in various infectious
, inflammatory
and autoimmune diseases. suPAR concentration positively correlates to the activation level of the immune system. suPARnostic can be used as a prognostic tool to determine the severity of a disease within a patient, but is not used as a reliable diagnostic tool, as it can detect the severity of the immune response in a patient, but does not reveal the specific disease from which the patient may be suffering. Recently, increase suPAR levels were shown to be associated with increased risk of systemic inflammatory response syndrome (SIRS)/sepsis
, cardiovascular disease
, type 2 diabetes, infectious diseases, HIV
, cancer
tuberculosis
, malaria
, bacterial and viral CNS infections, rheumatoid arthritis
, multiple sclerosis
and mortality
in the general population.
requires two antibodies with high specificity for suPAR
. The blood plasma sample from the patient that contains an unknown amount of suPAR is immobilized on the microwells on the clear microtiter plate
and a detection antibody forms a complex with suPAR.
Between each step the plate is rinsed with a wash buffer to dispose of any proteins that do not specifically bind to any of the wells on the plate. After the final wash step, the plate is developed by adding the TMB
substrate to produce a visible signal, which indicates the quantity of suPAR in the sample. The measured absorbance can, based on the values from the standard curve, be converted to the concentration (ng/mL) of suPAR in the sample. This level can then suggest whether or not the patient is experiencing challenges to their immune system.
is a simplified double monoclonal antibody sandwich assay that measures the level of suPAR
and suPARII-III in the body . The suPARnostic ELISA utilizes monoclonal mouse and rat antibodies against human suPAR.
The advantages of using monoclonal antibodies
compared to using polyclonal antibodies includes: High homogeneity
, absence of nonspecific antibodies and no batch-to-batch or lot-to-lot variability. This results in a very robust and reliable assay.
A ‘sandwich’ is formed of solid-phase antibody
, suPAR and peroxidase-conjugated antibody. The concentration (ng/mL plasma) of suPAR in the patient sample is determined via interpolation, based on a calibration curve
prepared from seven suPAR standards. Recombinant suPAR standards are calibrated against healthy human blood donor samples. Absorbance is measured using a microtiter plate
reader, at 450 nm with a 650 nm reference filter. Measurement of suPAR levels from blood samples provides greater accuracy and precision than measurement from urine
or cerebral spinal fluid. suPAR level is not changed by transient illness such as cold. It also remains stable after a blood sample is taken despite storage.
suPARnostic measurements between 0.1 to 4.0 ng/mL suggest that a patient is healthy, with no challenges to their immune system
and no signs or symptoms of an opportunistic infection
or inflammation
; the average level among the population is 3.4 ng/mL. However, a patient’s immune system can be considered ‘negatively activated’ at suPAR levels above 4.0 and up to 6.0 ng/mL, indicating a potential infection or high level of inflammation. In this case, a patient’s health is likely to worsen and he or she should be referred for further testing. suPARnostic measurements from 6.0 ng/mL to double digit levels can indicate a serious illness that is progressing rapidly to a critical situation. Patients in the intensive care unit average a level of 10.0 ng/mL. There is no difference in suPAR levels intrinsic to various races; however, the scale varies for male and female.
There are two suPARnostic tests available. The suPARnostic Standard ELISA (Code No. A001) is for research use and large trials, one batch consisting of 41 samples in doublets. The suPARnostic Flex ELISA (Code No. A002) has been developed for clinical applications consisting of 93 samples, is modular and flexible, and gives fully quantitative results in 2 hours.
of several years and when frozen, may be kept for longer. The kit should sit at room temperature for half an hour before use but it may be held at room temperature for as long as three to four hours. The suPARnostic Flex ELISA (Code No. A002) is able to provide fully quantitative results in 2 hours. suPARnostic is run as large, batch test with up to 41 samples in doublets for research purposes or 93 samples for clinical use at one time.
Although suPARnostic currently does not have FDA approval, it is CE/IVD marked for distribution throughout Europe. suPAR is a prognostic test to indicate general health, and it cannot be used as a diagnostic tool to suggest a particular illness. suPAR cannot be used in the detection of brain tumors because the suPAR molecule cannot migrate through the blood brain barrier.
ELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
) that measures the amount of soluble urokinase plasminogen activator receptor (suPAR
SuPAR
suPAR, soluble urokinase-type plasminogen activator receptor, is the soluble form of uPAR. uPAR is a membrane bound receptor for uPA, otherwise known as urokinase. suPAR results from the cleavage and release of membrane-bound uPAR...
) in blood. Elevated plasma suPAR levels have been observed in various infectious
Infectious disease
Infectious diseases, also known as communicable diseases, contagious diseases or transmissible diseases comprise clinically evident illness resulting from the infection, presence and growth of pathogenic biological agents in an individual host organism...
, inflammatory
Inflammation
Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process...
and autoimmune diseases. suPAR concentration positively correlates to the activation level of the immune system. suPARnostic can be used as a prognostic tool to determine the severity of a disease within a patient, but is not used as a reliable diagnostic tool, as it can detect the severity of the immune response in a patient, but does not reveal the specific disease from which the patient may be suffering. Recently, increase suPAR levels were shown to be associated with increased risk of systemic inflammatory response syndrome (SIRS)/sepsis
Sepsis
Sepsis is a potentially deadly medical condition that is characterized by a whole-body inflammatory state and the presence of a known or suspected infection. The body may develop this inflammatory response by the immune system to microbes in the blood, urine, lungs, skin, or other tissues...
, cardiovascular disease
Cardiovascular disease
Heart disease or cardiovascular disease are the class of diseases that involve the heart or blood vessels . While the term technically refers to any disease that affects the cardiovascular system , it is usually used to refer to those related to atherosclerosis...
, type 2 diabetes, infectious diseases, HIV
HIV
Human immunodeficiency virus is a lentivirus that causes acquired immunodeficiency syndrome , a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive...
, cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
tuberculosis
Tuberculosis
Tuberculosis, MTB, or TB is a common, and in many cases lethal, infectious disease caused by various strains of mycobacteria, usually Mycobacterium tuberculosis. Tuberculosis usually attacks the lungs but can also affect other parts of the body...
, malaria
Malaria
Malaria is a mosquito-borne infectious disease of humans and other animals caused by eukaryotic protists of the genus Plasmodium. The disease results from the multiplication of Plasmodium parasites within red blood cells, causing symptoms that typically include fever and headache, in severe cases...
, bacterial and viral CNS infections, rheumatoid arthritis
Rheumatoid arthritis
Rheumatoid arthritis is a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks synovial joints. The process produces an inflammatory response of the synovium secondary to hyperplasia of synovial cells, excess synovial fluid, and the development...
, multiple sclerosis
Multiple sclerosis
Multiple sclerosis is an inflammatory disease in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, leading to demyelination and scarring as well as a broad spectrum of signs and symptoms...
and mortality
Mortality rate
Mortality rate is a measure of the number of deaths in a population, scaled to the size of that population, per unit time...
in the general population.
Performing the suPARnostic ELISA
Performing the suPARnostic ELISAELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
requires two antibodies with high specificity for suPAR
SuPAR
suPAR, soluble urokinase-type plasminogen activator receptor, is the soluble form of uPAR. uPAR is a membrane bound receptor for uPA, otherwise known as urokinase. suPAR results from the cleavage and release of membrane-bound uPAR...
. The blood plasma sample from the patient that contains an unknown amount of suPAR is immobilized on the microwells on the clear microtiter plate
Microtiter plate
A Microtiter plate or microplate or microwell plate, is a flat plate with multiple "wells" used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories...
and a detection antibody forms a complex with suPAR.
Between each step the plate is rinsed with a wash buffer to dispose of any proteins that do not specifically bind to any of the wells on the plate. After the final wash step, the plate is developed by adding the TMB
3,3’,5,5’-Tetramethylbenzidine
3,3’,5,5’-Tetramethylbenzidine or TMB is a chromogenic substrate used in staining procedures in immunohistochemistry as well as being a visualising reagent used in enzyme-linked immunosorbent assays...
substrate to produce a visible signal, which indicates the quantity of suPAR in the sample. The measured absorbance can, based on the values from the standard curve, be converted to the concentration (ng/mL) of suPAR in the sample. This level can then suggest whether or not the patient is experiencing challenges to their immune system.
Principles
The suPARnostic ELISAELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
is a simplified double monoclonal antibody sandwich assay that measures the level of suPAR
SuPAR
suPAR, soluble urokinase-type plasminogen activator receptor, is the soluble form of uPAR. uPAR is a membrane bound receptor for uPA, otherwise known as urokinase. suPAR results from the cleavage and release of membrane-bound uPAR...
and suPARII-III in the body . The suPARnostic ELISA utilizes monoclonal mouse and rat antibodies against human suPAR.
The advantages of using monoclonal antibodies
Monoclonal antibodies
Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....
compared to using polyclonal antibodies includes: High homogeneity
Homogeneity and heterogeneity
Homogeneity and heterogeneity are concepts relating to the uniformity or lack thereof in a substance. A material that is homogeneous is uniform in composition or character; one that is heterogeneous lacks uniformity in one of these qualities....
, absence of nonspecific antibodies and no batch-to-batch or lot-to-lot variability. This results in a very robust and reliable assay.
A ‘sandwich’ is formed of solid-phase antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
, suPAR and peroxidase-conjugated antibody. The concentration (ng/mL plasma) of suPAR in the patient sample is determined via interpolation, based on a calibration curve
Calibration curve
In analytical chemistry, a calibration curve is a general method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard samples of known concentration...
prepared from seven suPAR standards. Recombinant suPAR standards are calibrated against healthy human blood donor samples. Absorbance is measured using a microtiter plate
Microtiter plate
A Microtiter plate or microplate or microwell plate, is a flat plate with multiple "wells" used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories...
reader, at 450 nm with a 650 nm reference filter. Measurement of suPAR levels from blood samples provides greater accuracy and precision than measurement from urine
Urine
Urine is a typically sterile liquid by-product of the body that is secreted by the kidneys through a process called urination and excreted through the urethra. Cellular metabolism generates numerous by-products, many rich in nitrogen, that require elimination from the bloodstream...
or cerebral spinal fluid. suPAR level is not changed by transient illness such as cold. It also remains stable after a blood sample is taken despite storage.
suPARnostic measurements between 0.1 to 4.0 ng/mL suggest that a patient is healthy, with no challenges to their immune system
Immune system
An immune system is a system of biological structures and processes within an organism that protects against disease by identifying and killing pathogens and tumor cells. It detects a wide variety of agents, from viruses to parasitic worms, and needs to distinguish them from the organism's own...
and no signs or symptoms of an opportunistic infection
Opportunistic infection
An opportunistic infection is an infection caused by pathogens, particularly opportunistic pathogens—those that take advantage of certain situations—such as bacterial, viral, fungal or protozoan infections that usually do not cause disease in a healthy host, one with a healthy immune system...
or inflammation
Inflammation
Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process...
; the average level among the population is 3.4 ng/mL. However, a patient’s immune system can be considered ‘negatively activated’ at suPAR levels above 4.0 and up to 6.0 ng/mL, indicating a potential infection or high level of inflammation. In this case, a patient’s health is likely to worsen and he or she should be referred for further testing. suPARnostic measurements from 6.0 ng/mL to double digit levels can indicate a serious illness that is progressing rapidly to a critical situation. Patients in the intensive care unit average a level of 10.0 ng/mL. There is no difference in suPAR levels intrinsic to various races; however, the scale varies for male and female.
There are two suPARnostic tests available. The suPARnostic Standard ELISA (Code No. A001) is for research use and large trials, one batch consisting of 41 samples in doublets. The suPARnostic Flex ELISA (Code No. A002) has been developed for clinical applications consisting of 93 samples, is modular and flexible, and gives fully quantitative results in 2 hours.
Practical Considerations
The suPARnostic kit has a refrigerated shelf lifeShelf life
Shelf life is the length of time that food, drink, medicine, chemicals, and many other perishable items are given before they are considered unsuitable for sale, use, or consumption...
of several years and when frozen, may be kept for longer. The kit should sit at room temperature for half an hour before use but it may be held at room temperature for as long as three to four hours. The suPARnostic Flex ELISA (Code No. A002) is able to provide fully quantitative results in 2 hours. suPARnostic is run as large, batch test with up to 41 samples in doublets for research purposes or 93 samples for clinical use at one time.
Although suPARnostic currently does not have FDA approval, it is CE/IVD marked for distribution throughout Europe. suPAR is a prognostic test to indicate general health, and it cannot be used as a diagnostic tool to suggest a particular illness. suPAR cannot be used in the detection of brain tumors because the suPAR molecule cannot migrate through the blood brain barrier.