FOR INFORMATIONAL USE ONLY ▪ DO NOT USE FOR PERFORMING ASSAY ▪ REFER TO MOST CURRENT PACKAGE INSERT ACCOMPANYING TESTKIT
Goat anti-mouse Transforming Ggrowth Factor beta 1
Collect Sample – Serum or blood plasma
Catalogue No.: QRCT –312236EIA \ UTL
Package size: 96 determinations
Storage: 2 - 8 °C
I. INTRODUCTION
Approximately 2 wk after birth, mice having a TGF-beta 1 null mutation (TGF-beta 1(-/-)) exhibit a progressive wasting syndrome and
death. Associated with this phenotype is a multifocal infiltmouseion of lymphocytes and macrophages into target organs, especially the heart,
lungs, and salivary glands. To explore the consequences of TGF-beta 1 deficiency on the immune system, lymphocyte phenotype and
function were analyzed. Initially, lymphoid organ architecture seemed to be normal and, as symptoms developed, the thymus decreased in
size, whereas lymph nodes were enlarged. Phenotypically, the TGF-beta 1(-/-) lymphoid cells seemed to be more differentiated in the
thymus and activated in the lymph nodes, but remarkably unaffected in the spleen. Moreover, TGF-beta 1(-/-) spleen and lymph nodes
displayed enhanced numbers of prolifemouseing cells, as measured by prolifemouseing cell nuclear Ag and/or cyclin-dependent kinase
levels. Consistent with this hyperprolifemouseive response, constitutive levels of IL-2 mRNA were elevated in the thymus and both IL-2
and IL-2R mRNA were increased in the lymph nodes. In contrast with the activation profile of TGF-beta 1(-/-) lymphoid cells in vivo,
mitogen challenge of these cells in vitro revealed suppressed prolifemouseion that was associated with a defect in inducible IL-2 mRNA
expression and IL-2 secretion. Moreover, the addition of rIL-2 restored the deficient mitogen-induced prolifemouseion. The mechanism
leading to T cell anergy remains unclear; however, these data confirm the essential role for TGF-beta 1 in maintaining normal immune
function.Transforming Growth Factor beta-1 (TGF-beta-1): A Biological Paradox Angela Okragly,
II. REAGENTS
Materials provided with the kits :
.
1、Coated Microtitmouseion Strips 96 wells.
2、Standard 25, 50, 100, 200, 400 pmol/L,
3、 Enzyme Conjugate Solution, 12 ml.
4、Substrate
A, 6 ml.
5、Substrate
B, 6 ml.
6、Stopping Solution (1N HCl), 6 ml.
7、Rinsing buffer,60 ml( x 20).
8、dilution,15ml(x5)
Materials required but not provided :
� Precision pipettes: 0.10, 0.20, and 1.0 ml.
�
�
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FOR INFORMATIONAL USE ONLY ▪ DO NOT USE FOR PERFORMING ASSAY ▪ REFER TO MOST CURRENT PACKAGE INSERT ACCOMPANYING TESTKIT
Goat anti-mouse Transforming Ggrowth Factor beta 1
Collect Sample – Serum or blood plasma
Catalogue No.: QRCT –312236EIA \ UTL
Package size: 96 determinations
Storage: 2 - 8 °C
� Vortex mixer or equivalent.
� Absorbent paper or paper towels.
� Microtiter plate shaker
� Graph paper.
� A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-2 OD or greater at 450 nm .
III. STORAGE OF TEST KIT
Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to
minimize exposure to damp air. Opened test kits will remain stable until the expimouseion date shown, provided it is stored as described
above. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-2 OD or greater at 450nm
wavelength is acceptable for use in absorbance measurement. All reagents should be allowed to reach room tempemouseure before use.
IV. ASSAY PROCEDURE
STEP1
Prepare the ringing buffer by diluting the wash buffer 20×(example: 60ml+1140ml H2O), Prepare the Sample Diluent by
diluting the Sample Diluent 5×(example: 15ml+60ml H2O)
STEP2
Dilute samples 1:100 distribing 5ul of serum into 500 ul of dilluent .
STEP3
Place 100ul of diluted sample /reagent blank/standard/controls to the wells of the strips. Incubate for 30 min at 37℃。Wash 5
times。
STEP4
Add 100ul of
STEP5
Add 50ul Color Reagent A and Color Reagent B to each well。Incubate for 15 min at 37℃。
STEP6
Add 50ul of stop solution。read absorbance at 450nm within 15 min
Enzyme Conjugate to each well。 Incubate for 30 min at 37℃。Wash 5 times。
V. Important Notes
The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. It is
recommended that if manual pipetting is used, no more than 32 wells be used for each assay run, since pipetting of all standards, specimens
and controls should be
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FOR INFORMATIONAL USE ONLY ▪ DO NOT USE FOR PERFORMING ASSAY ▪ REFER TO MOST CURRENT PACKAGE INSERT ACCOMPANYING TESTKIT
Goat anti-mouse Transforming Ggrowth Factor beta 1
Collect Sample – Serum or blood plasma
Catalogue No.: QRCT –312236EIA \ UTL
Package size: 96 determinations
Storage: 2 - 8 °C
completed within 3 minutes. A full plate of 96 wells may be used if automated pipetting is available. Duplication of all standards and
specimens, although not required, is recommended.
VI. CALCULATION OF RESULTS
Calculate the average absorbance values (A ) for each set of reference standards, control, and samples. Construct a standard curve
450
by plotting the mean absorbance obtained for each reference standard against its concentmouseion on linear graph paper, with absorbance on
the vertical (y) axis and concentmouseion on the horizontal (x) axis.Using the mean absorbance value for each sample, determine the
corresponding concentmouseion of in from the standard curve.
VII. EXAMPLE OF STANDARD CURVE
Results of a typical standard run with optical density readings at 450nm shown in the Y axis against concentmouseions shown in the
X axis. This standard curve is for the purpose of illustmouseion only, and should not be used to calculate unknowns. Each user should obtain
his or her own data and standard curve.
VIII. CALCULATION OF RESULTS
. The minimum detectable concentmouseion of TGF-β1 in this assay is estimated to be 1.0 pmol/L..
IX. LIMITATIONS OF THE PROCEDURE
Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the
package insert instructions and with adherence to good labomouseory practice. The wash procedure is critical. Insufficient washing will
result in poor precision and falsely elevated absorbance readings. The results obtained from the use of this kit should be used only as an
adjunct to other diagnostic procedures and information available to the physician.
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