Anti-Calpastatin Autoantibody Detection ELISA-For in vitro research use only-not for use in diagnostic procedures
Anti-Calpastatin Autoantibody Detection ELISA
-Immunoassay for the quantitative detection of autoantibodies specific for the C-terminus of calpastatin in serum and plasma.
Cat#: RDI-PR59191 $350.00/1 kit $300.00/kit 3 or more
Size: 12 x 8 Determinations
Storage: 2-8'C
For In Vitro Research Use Only-Not For Use in Diagnostics
1. Introduction:
An important role for Ca2+-activated neutral proteases (calpains) was implicated in the pathogeneiss of many diseases with altered Ca2+ homeostasis and altered protein metabolism, cell death, muscle and nerve injury, atrophy, and tissue degeneration. Calpains are important regulators of cellular growth and tissue turnover, cytoskeletal proteins, receptors, and protein kinases. Thus, disturbances of the calpains could cause tissue damage and organ malfunction in manifold ways (1).
Calpstatins are the natural inhibitors of the calpains and important regulators of the calpain activity in cells. Several proteins from cellular extracts exhibit cross-reactivity with 70 kDa calpastatin antigen. At present, the molecular basis of this diersity is not fully explored. How far disturbances in the calpastatin inhibitory activity contribute to disease activity, is not yet known in greater detail (1). Autoantibodies reactive against human calpastatin were detected by screening a cDNA expression library with the serum of a patient with the history of venous thrombosis and suspected antiphospholipid syndrome. By testing further sera it could be shown that >90% of calpastatin autoantibodies, detected by Western blotting against calpastatin, react with the C-terminal peptide of the protein. These results led to the development of an ELISA based on a synthetic peptide containing the C-terminus of calpastatin (3,4).
An association between the occurance of calpastatin autoantibodies and thrombosis was published most recently (5.6). Furthermore an increased incidence of calpastatin autoantibodies was found for a subgroup of patients with rheumatoid arthritis (7). Also observed was the presence of calpastatin autoantibodies in some cases of infertility (8,9).
2. Principle of the Test
The Calpastatin IgG ELISA(5) detects antibodies against calpastatin. A defined human standard allows a quantitative serological determination of IgG antibodies in human serum.
In this indirect solid phase enzyme immunoassay calpastatin peptide antigen is fixed to the wells of a microplate plate. During serum incubation, calpastatin-specific antibodies form immune complexes with the antigen. In a second incubation step, these immune complexes bind to a peroxidase-labelled anti-human IgG antibody. During substrate reaction, a blue stain develops whichturns yellow after adding stop solution. The absorbance measured is proportional to the quantity of bound antibody and is determined photometrically.
3. Precautions
Standards and negative control are sera of human origin and although they have been tested against and confirmed negative for HIV antibody, HBsAg and Treponema sp., these materials must be considered potentially infectious and should be treated as biohazards in use and for disposal.
Some reagents of this test kit contain Thimerosal in a final concentration of 0.01%, i.e. contact with mucous membranes should be avoided!
4. Storage and Shelf Life Store all reagents at 2-8'C and do not use after expiration date on the label.
5. Reagents Supplied
12x8-well microplate strips, coated with calpastatin antigen.
H 6 vials Standard anti-calpastatin IgG antibody (ready-to-use); 2ml, 200 units.
P 1 vial Positive Control (ready-to-use); 1ml
N 1 vial Negative Control (ready-to-use); 1ml
WB 2 bottles Wash Buffer (20x); 20ml
SB 1 bottle Sample Buffer (20x); 20ml
C 1 bottle anti-IgG Peroxidase Conjugate (20x); 750 ul
S 1 vial TMB Substrate (20x); 750 ul(CAUTION: May not be diluted in polystyrol tubes).
SS 1 bottle Stop Solution; 13 ml (contains 1N H2SO4;caustic!)
6. Material Required But Not Supplied
1. Distilled water
2. Test tubes for serum dilution
3. Precision pipettes (20,100,200 and 1000 ul)
4. Adjustable 8-channel pipette
5. Magnetic stirr bar, vortex mixer
6. microplate plate reader with 450 nm filter
7. Specimen Collection and Handling
Collect venous blood specimens and allow to clot completely.After centrifugation, aliquot and store at 2-8'C for max. 48h or deep-freeze for longer storage at -20'C.
Do not use hyperlipemic or contaminated sera or heat-inactivated serum samples (30 min, 56'C). Precipitated or ciscous samples can give false results.
8. Preparation of Reagents
Allow reagents to reach room temperature (22+2'C). Buffer concentrates may contain salt cristals which can be quickly dissolved at 37'C.
Dilute required reagents immediately before use!
Example for 1 x 8-well microplate strip:
SB 1 ml + 19 ml bidistilled water
WB 1 ml + 19 ml bidistilled water
C 50 ul + 0.95 ml ready-to-use wash buffer
S 50 ul + 0.95 ml bidistilled water
A standard curve is obtained by consecutive dilutions of equal volumes of H and ready-to-use sample buffer.
200 units: H is provided ready-to-use
100 units: 500 ul H + 500 ul sample buffer
50 units: 500 ul 100 units + 500 ul sample buffer
25 units: 500 ul 50 units + 500 ul sample buffer
12 units: 500 ul 25 units + 500 ul sample buffer
Serum dilution is 1:201:
1 ml ready-to-use sample buffer + 5 ul serum, mix well.
9. Test Procedure
1. Pipette 100 ul each of standards, negative and positive con trol as well as diluted sample into the respective wells.
2. Cover strips with adhesive foil and incubate 45 min at 37'C.
3. Aspirate microplate strips and fill with 200 ul ready-to-use wash buffer. Aspirate after approx. 15 sec, invert and tap to remove excess liquid. Repeat wash step 3x.
4. Pipette 100 ul conjugate solution into each well; cover strips with adhesive foil and incubate 45 min at 37'C.
5. Repeat wash step as described in 3.
6. Pipette 100 ul ready-to-use substrate solution into each well and incubate 10 min at RT.
7. Stop color reaction by addition of 100 ul stop solution into each well.
8. Measure absorbance within 30 min. at 450 nm with a MTPL reader blank against air.
10. Quality ControlThe following criteria have to be met for a valid test run:
H(200 U/ml) Absorbance: > 0.800
N(negative control) Absorbance: < 0.150
P(positive control) Absorbance: 50 -70 U/ml
If these criteria are not met, the test is invalid and should be repeated.
11. Calculation of Results
For the calculation of results a standard curve is constructed on semilogarithmic paper by plotting the measured absorbance values of the standarfs (Y-axis) versus the corresponding concentration on the x-axis. The values of the patient samples are then read from the standard curve. Alternatively, a software program can used for calculation. An example of a standard curve is shown below.
12. Interpretation of Results
The results should be interpreted as shown below:
< 30 units Negative: no IgG antibodies specific to Calpastatin detected.
> 30 units Positive: specific IgG antibodies to Calpastatin detected.
References:
1. Coroall DE, Demartino GN (1991) Physiol Rev. 71:813-847.
2. Despres N, Talbot G, Plouffe B, Boire G. Menard HA (1995) J.Clin Invest 95: 1891-96.
3. Schlosser U, Lackner KJ, Schreckenhofer C, and Schmitz G. Calpastatin autoantibodies: detection, development of a specific peptide ELISA, and epitope mapping. Clin Chem 42: 1250-1256 (1996).
4. German Patent No. 19518488 .
5. Schlosser et al. 3rd Dresden Symposium on Autoantibodies; September 1996, Abstract.
6. Schlosser U, Lackner KJ, Scheckenhofer C, Spannagl M, Spengel FA, Hahn G, Lang B, Schmitz G. Autoantibodies Against the Protease Inhibitor Calpastating: A New Risk Factor for Venous Thrombosis. (1997) Thromb Haemost 77(1), in press.
7. Mimori T, Suganuma K, Tanamo Y; Mojima t, Matsumura M, Fujii T, Yoshizawa St, Suzuki K, and Akizuki M. (1995) Proc Natl Acad Sci 92:7267-7271
8. Wang LF, Wei SG, Miao SY, Lin QY, Koide SS (1995) Biochem Mol Biol Int 33:245-51.
9. Liang ZG, O'Hern PA, Yaretz B, Yretz H, Goldberg E (1995) Reprod Fertil Dev 6:297-305.