丙二醛檢測(cè)試劑盒
使用說明書
僅供體外研究使用，不用于臨床診斷!
第1版（2016年04月修訂）
[ INTENDED USE ]
This kit is improved by many domestic & abroad methods, it is a microscale, convenient, accurate, unharmful measuring method. This assay can reflect lipid peroxide amount of cellular level and subcellular in extracellular fluids such as blood serum (or plasma) and milk, etc., various kinds of animal cells and plant cells such as erythrocytes, leucocytes, cultured cells, etc.
This kit can be used for laboratory research only.
[ REAGENTS AND MATERIALS PROVIDED ]
Reagents | Quantity(100T/50T) | Reagents | Quantity(100T/50T) |
Reagent 1 | 1×20ml/1×10ml | Reagent 3 | 1/1 |
Reagent 2 | 1×12ml/1×6ml | Standard | 1×5ml/1×5ml |
Instruction manual | 1 | 96T plate | 1 |
Note: This kit can be stored by cold preservation for at least 6 months.
[ MATERIALS REQUIRED BUT NOT SUPPLIED ]
1. An spectrophotometer capable of measuring absorbance at 532nm.
2. Thermostatic water bath or air bath capable of controlling temperature at 95℃.
3. Desk centrifuge.
4. Glacial acetic acid (analytical pure, CH3COOH≥99.5%
[ STORAGE OF THE KITS ]
This kit can be stored by cold preservation for at least 6 months.
1. 100T/96S assay kit:
Reagent 1: Solution, can be stored at room temperature. (It may coagulate in cold days, so before use, please warm this bottle in water bath in order to increase dissolving rate. When solution becomes limpid, then you can use it for assay.)
Reagent 2: Solution. When use, add 340ml double distilled water in each bottle. Can be stored at 4℃.
Note: Never drop on your skin.
Reagent 3: Powder. When use, add powder in 60ml 90℃～100℃ hot double distilled water, dissolve sufficiently (you can heat properly during dissolving), add double distilled water until volume reaches to 60ml, add 60ml glacial acetic acid*, mix sufficiently, prepared reagent can be stored by cold preservation away from light.
Standard: 10 nmol/ml tetraethoxypropane, Solution, can be stored at 4℃.
2. 50T/48S assay kit:
Reagent 1: Solution, can be stored at room temperature. (It may coagulate in cold days, so before use, please warm this bottle in water bath in order to increase dissolving rate. When solution becomes limpid, then you can use it for assay.)
Reagent 2: Solution. When use, add 170ml double distilled water in each bottle. Can be stored at 4℃.
Note: Never drop on your skin.
Reagent 3: Powder. When use, add powder in 30ml 90℃～100℃ hot double distilled water, dissolve sufficiently (you can heat properly during dissolving), add double distilled water until volume reaches to 60ml, add 60ml glacial acetic acid*, mix sufficiently, prepared reagent can be stored by cold preservation away from light.
Standard: 10 nmol/ml tetraethoxypropane, Solution, can be stored at 4℃.
[ ASSAY PROCEDURE ]
1. Normal Operation procedure:
(1) Normal operation table:
Blank tube | Standard tube | Sample tube | Contrast tube** | |
10 nmol/ml standard (ml) | a* | |||
Dehydrated alcohol（ml） | a* | |||
Sample to assay（ml） | a* | a* | ||
Reagent 1 (ml) | a* | a* | a* | a* |
Mix sufficiently by shaking test-tube stand | ||||
Reagent 2 (ml) | 3 | 3 | 3 | 3 |
Reagent 3 (ml) | 1 | 1 | 1 | |
50% glacial acetic acid（ml） | 1 | |||
Mix sufficiently by vortex, seal test tube by refreshing film, pierce a small hole by needle, place in 95℃ water bath for 40 minutes, cool in tap water, centrifugate at 3500~4000rpm for 10 minutes.(If you centrifugate at low speed (<3000rpm), then please extend time in order to make sedimentation complete). Take supernatant***, transfer in cuvettes of 25px light path, measure absorbances of all tubes at 532nm (adjust zero by double distilled water). | ||||
a* reflects volumes of sample, standard, dehydrated alcohol, Reagent 1, these four volumes should be equal to each other. For example, if you take 0.1ml sample, then you should also take 0.1ml standard, dehydrated alcohol, Reagent 1; if you take 0.2ml sample, then you should also take 0.2ml standard, dehydrated alcohol, Reagent 1. Absorbance reflects direct proportion with sample volume, so result won’t be disturbed.
**Generally, it only needs to make 1~2 standard tubes, blank tubes, contrast tubes. If there is no haemolysis or lipidemia in samples, then you can skip contrast tube, use blank tube to instead.
***Use micropipet to transfer supernatant in cuvette, please avoid transfer by pouring or sediments may enter cuvette and disturb absorbance.
(2) Referenced sample volume:
● Blood serum (or plasma): 0.1~0.2ml
● Low density lipoprotein: 0.1~0.2ml
● Edible oil: 0.03ml
● Liver tissue, cardiac muscle, muscular tissue, spiral algae: 0.1~0.2ml 5% or 10% homogenate
(3) Normal referenced standard absorbances:
When standard volume is 0.1ml,
ODStandard – ODBlank = 0.065~0.070
When standard volume is 0.2ml,
ODStandard – ODBlank = 0.130~0.140
(4) When you use normal operation procedure or convenient operation procedure, if ODSample is too low, then you can extend water bath period from 40 minutes to 80 minutes. But if you do like this, then please extend all MDA assays’ water bath period to 80 minutes in order to avoid difference between batches.
(5) Formulas:
① Blood serum (or plasma) MDA assay:
② Tissue MDA assay:
* nmol/mgprot means nanomole per protein in milligram
(6) Examples:
① Take 0.1ml blood serum without dilution to measure MDA content, in results, ODSample is 0.036, ODStandard is 0.076, ODContrast is 0.006 (use blank tube to instead contrast tube). Calculate as follows:
② Take 0.1ml yolk, add 0.9ml dehydrated alcohol, mix sufficiently, extract for 3 minutes, centrifugate at 4000rpm for 10 minutes, take 0.2ml supernatant to measure MDA content, in results, ODSample is 0.198, ODStandard is 0.151, ODContrast is 0.009, (use blank tube to instead contrast tube). Calculate as follows:
③ Take 0.1ml 10% rat liver tissue to measure MDA content, in results, ODSample is 0.214, ODStandard is 0.079, ODContrast is 0.009, (use blank tube to instead contrast tube), protein concentration in 10% rat liver tissue homogenate is 8.5mgprot/ml. Calculate as follows:
④ Take 0.1ml 10% carp brain tissue homogenate to measure MDA content, in results, in results, ODSample is 0.314, ODStandard is 0.076, ODContrast is 0.006, (use blank tube to instead contrast tube), protein concentration in 10% carp brain tissue homogenate is 5.0788mgprot/ml. Calculate as follows:
⑤ Take 0.1ml 20% earthworm tissue homogenate to measure MDA content, in results, in results, ODSample is 0.082, ODStandard is 0.074, ODContrast is 0.005, ODBlank is 0.025, protein concentration in 20% earthworm tissue homogenate is 10.3145 mgprot/ml. Calculate as follows:
⑥ Take 0.2ml 10% rice leaf homogenate to measure MDA content, in results, in results, ODSample is 0.077, ODStandard is 0.140, ODContrast is 0.003, (use blank tube to instead contrast tube), protein concentration in 20% earthworm tissue homogenate is 3.2729 mgprot/ml. Calculate as follows:
2. Convenient operation procedure:
If number of samples is huge, then you can use convenient operation procedure.
(1) Mixed reagent preparation (volume ratio):
Working solution 1 preparation:
Reagent 1: Reagent 2: Reagent 3 = a*:3:1, consider solution volume according to you need, this solution should be used at the same day of preparation.
Working solution 2 preparation:
Reagent 1: Reagent 2: 50% glacial acetic acid = a*:3:1, consider solution volume according to you need, this solution should be used at the same day of preparation.
(2) Convenient operation table:
Blank tube | Standard tube | Sample tube | Contrast tube** | |
10 nmol/m standard（ml） | a* | |||
Dehydrated alcohol（ml） | a* | |||
Sample to assay（ml） | a * | a* | ||
Working solution I （ml） | 4ml | 4ml | 4ml | |
Working solution II (ml) | 4ml | |||
Mix sufficiently by vortex, seal test tube by refreshing film, pierce a small hole by needle, place in 95℃ water bath for 40 minutes, cool in tap water, centrifugate at 3500~4000rpm for 10 minutes.(If you centrifugate at low speed (<3000rpm), then please extend time in order to make sedimentation complete). Take supernatant***, transfer in cuvettes of 25px light path, measure absorbances of all tubes at 532nm (adjust zero by double distilled water). | ||||
a* reflects volumes of sample, standard, dehydrated alcohol, Reagent 1, these four volumes should be equal to each other. For example, if you take 0.1ml sample, then you should also take 0.1ml standard, dehydrated alcohol, Reagent 1; if you take 0.2ml sample, then you should also take 0.2ml standard, dehydrated alcohol, Reagent 1. Absorbance reflects direct proportion with sample volume, so result won’t be disturbed.
** You can skip contrast tube, use blank tube to instead.
***Use micropipet to transfer supernatant in cuvette, please avoid transfer by pouring or sediments may enter cuvette and disturb absorbance.
Note1: Normal method and convenient method apply to samples of human, aninmals & plants (include serum, animal & plant tissue, body fluid, cells & cell culture fluid, etc.).
Note2: When you use normal operation procedure or convenient operation procedure, if ODSample is too low, then you can extend water bath period from 40 minutes to 80 minutes. But if you do like this, then please extend all MDA assays’ water bath period to 80 minutes in order to avoid difference between batches.
(3) Formulas:
① Blood serum (or plasma) MDA assay:
② Tissue MDA assay:
(4) Examples:
① Take 0.1ml undiluted blood serum to measure MDA content, in results, ODSample is 0.041, ODStandard is 0.080, ODContrast is 0.006 (use blank tube to instead contrast tube). Calculate as follows:
② Take 0.1ml 10% rat liver tissue homogenate to measure MDA content, in results, ODSample is 0.204, ODStandard is 0.082, ODContrast is 0.013 (use blank tube to instead contrast tube), protein concentration in 10% rat liver tissue homogenate is 8.1mgprot/ml. Calculate as follows:
2. Microscale operation procedure:
You can use microscale operation method for low MDA containing or small scale samples such as mouse blood serum (or plasma), child blood serum (or plasma), mouse lung tissue, skin tissue, kidney tissue, retina, cells and cell culture fluid, etc:
(1) Sample pretreatment follows tissue homogenate treatment in Experimental Methodology, make 10% or 5% homogenate. But please remember that it is better NOT to centrifugate because sample (such as lyzed cultured cells) is small scaled. Please take sample immediately after shaking.
(2) Reagent composition & preparation:
① 100T/96S assay kit:
Reagent 1: Solution, 20ml×1 bottle, can be stored at room temperature. (It may coagulate in cold days, so before use, please warm this bottle in water bath in order to increasing dissolving rate. When solution becomes limpid, then you can use it for assay.)
Reagent 2: Solution, 12ml×1 bottle. When use, add 340ml double distilled water in each bottle. Can be stored at 4℃. Note: Never drop on your skin.
Reagent 3: Powder×1 vial, can be stored at 4℃.
Standard: 10 nmol/ml tetraethoxypropane, 5ml×1 bottle, can be stored at 4℃.
Microscale Reagent 3 preparation in microscale operation procedure:
(a) Prepare MDA Reagent 3 according to normal operation procedure: Pour 1 vial of MDA Reagent 3 in flask, add 64ml* 90℃～100℃ hot double distilled water, dissolve completely (you can heat solution properly during dissolving). Add 60ml glacial acetic acid after cooling, mix sufficiently.
(b) Dilute prepared MDA Reagent 3 with 50%** glacial acetic acid at ratio of 2:1, consider solution volume according to you need.
For example, if you need 15ml Reagent 3, then you can prepare 10ml Reagent 3 according to procedure above, then add 5ml 50% glacial acetic acid, mix sufficiently. Prepared can be stored at 4℃ in fridge away from light (you should get glacial acetic acid***).
Note: * Double distilled water will bulge when it becomes hot, and evaporation can not be ignored during heating process. So you need to add 64ml hot double distilled water to make sure 60ml double distilled water (after cooling) is add in reaction system .
** Mix 50ml double distilled water & 50ml glacial acetic acid together in order to prepare 50% glacial acetic acid.
*** Glacial acetic acid is also named as ethanoic acid, you can buy it from pharmaceuticals company or medicament company generally. It is better to buy analytical pure (AR) ethanoic acid, CH3COOH>99%.
This kit can be stored by cold preservation for at least 1 year.
② 50T/48S assay kit:
Reagent 1: Solution, 10ml×1 bottle, can be stored at room temperature. (It may coagulate in cold days, so before use, please warm this bottle in water bath in order to increasing dissolving rate. When solution becomes limpid, then you can use it for assay.)
Reagent 2: Solution, 6ml×1 bottle. When use, add 340ml double distilled water in each bottle. Can be stored at 4℃. Note: Never drop on your skin.
Reagent 3: Powder×1 vial, can be stored at 4℃.
Standard: 10 nmol/ml tetraethoxypropane, 5ml×1 bottle, can be stored at 4℃.
Microscale Reagent 3 preparation in microscale operation procedure:
(a) Prepare MDA Reagent 3 according to normal operation procedure: Pour 1 vial of MDA Reagent 3 in flask, add 32ml* 90℃～100℃ hot double distilled water, dissolve completely (you can heat solution properly during dissolving). Add 30ml glacial acetic acid after cooling, mix sufficiently.
(b) Dilute prepared MDA Reagent 3 with 50%** glacial acetic acid at ratio of 2:1, how much you use, how much you make.
For example, if you need 15ml Reagent 3, then you can prepare 10ml Reagent 3 according to procedure above, then add 5ml 50% glacial acetic acid, mix sufficiently. Prepared can be stored at 4℃ in fridge away from light (you should get glacial acetic acid***).
Note: * Double distilled water will bulge when it becomes hot, and evaporation can not be ignored during heating process. So you need to add 32ml hot double distilled water to make sure 30ml double distilled water (after cooling) is add in reaction system .
** Mix 50ml double distilled water & 50ml glacial acetic acid together in order to prepare 50% glacial acetic acid.
*** Glacial acetic acid is also named as ethanoic acid, you can buy it from pharmaceuticals company or medicament company generally. It is better to buy analytical pure (AR) ethanoic acid, CH3COOH>99%.
This kit can be stored by cold preservation for at least 1 year.
(3) Microscale operation table:
Standard tube | Blank tube | Sample tube | Contrast tube** | |
10 nmol/ml standard (ml) | a* | |||
Dehydrated alcohol（ml） | a* | |||
Sample to assay（ml） | a* | a* | ||
Reagent 1 (ml) | a* | a* | a* | a* |
Mix sufficiently by shaking test-tube stand | ||||
Reagent 2 (ml) | 1.5 | 1.5 | 1.5 | 1.5 |
Reagent 3 (ml) | 1.5 | 1.5 | 1.5 | |
50% glacial acetic acid（ml） | 1.5 | |||
Mix sufficiently by vortex, seal test tube by refreshing film, pierce a small hole by needle, place in 95℃ water bath for 40 minutes, cool in tap water, centrifugate at 3500~4000rpm for 10 minutes.(If you centrifugate at lower speed (<3000rpm), then please extend time in order to make sedimentation complete). Take supernatant***, transfer in cuvettes of 25px light path, measure absorbances of all tubes at 532nm (adjust zero by double distilled water). | ||||
a* reflects volumes of sample, standard, dehydrated alcohol, Reagent 1, these four volumes should be equal to each other. For example, if you take 0.1ml sample, then you should also take 0.1ml standard, dehydrated alcohol, Reagent 1; if you take 0.2ml sample, then you should also take 0.2ml standard, dehydrated alcohol, Reagent 1. Absorbance reflects direct proportion with sample volume, so result won’t be disturbed.
**Generally, it only need to make 1~2 standard tubes, blank tubes, contrast tubes. If there is no haemolysis or lipidemia in samples, then you can skip contrast tube, use blank tube to instead.
***Use micropipet to transfer supernatant in cuvette, please avoid transfer by pouring or sediments may enter cuvette and disturb absorbance.
Note 1: OD values measured by microscale method are higher than use normal method, but final results won’t be disturbed.
Note 2: For 5% homogenate, a*=0.1~0.2ml.
Note 3: If ODSample is too low, then you can extend water bath period from 40 minutes to 80 minutes. But if you do like this, then please extend all MDA assays’ water bath period to 80 minutes in order to avoid difference between batches.
Note 4: Referenced ODStandard of this method:
When standard volume is 0.1ml,
ODStandard – ODBlank = 0.103~0.112
When standard volume is 0.2ml,
ODStandard – ODBlank = 0.206~0.224
(4) Formulas:
① Blood serum (or plasma) MDA assay:
② Tissue MDA assay:
* nmol/mgprot means nanomole per protein in milligram
(5) Examples:
① Take 0.1ml human blood serum without dilution to measure MDA content, in results, ODSample is 0.030, ODStandard is 0.104, ODBlank is 0.009(use blank tube to instead contrast tube). Calculate as follows:
② Take 0.1ml 5% rat lung tissue homogenate to measure MDA content, in results, ODSample is 0.062, ODStandard is 0.104, ODBlank is 0.009 (use blank tube to instead contrast tube), protein concentration in 5% rat lung tissue homogenate is 2.45mgprot/ml. Calculate as follows:
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4. Hyperlipidemia sample MDA assay procedure:
This method can be used to measure MDA content in hyperlipidemia blood or lipids. For example, you can use this method to measure MDA content in soybean oil, salad oil or rubsen seed oil, etc.
(1) Slight hyperlipidemia blood MDA assay:
Slight hyperlipidmia blood serum (or plasma) appears less limpid, you can also measure MDA according to common operation method or midrange hyperlipidemia operation method.
① Operation table:
Standard tube | Blank tube | Sample tube | Contrast tube** | |
10 nmol/ml standard (ml) | a* | |||
Dehydrated alcohol（ml） | a* | |||
lipid sample or slight hyperlipidemiablood serum(or plasma)（ml） | a* | a* | ||
Reagent 1 (ml) | a* | a* | a* | a* |
Mix sufficiently by shaking test-tube stand | ||||
Reagent 2 (ml) | 3.0 | 3.0 | 3.0 | 3.0 |
Reagent 3 (ml) | 1.0 | 1.0 | 1.0 | |
50% glacial acetic acid（ml） | 1.0 | |||
Mix sufficiently by vortex, seal test tube by refreshing film, pierce a small hole by needle, place in 95℃ water bath for 40 minutes, cool in tap water, centrifugate at 3500~4000rpm for 10 minutes.(If you centrifugate at lower speed (<3000rpm), then please extend time in order to make sedimentation complete). Take supernatant***, transfer in cuvettes of 25px light path, measure absorbances of all tubes at 532nm (adjust zero by double distilled water). | ||||
a* is sample volume, hyperlipidemia blood serum (or plasma) sample volume is 100μl, lipid sample volume is 30~50μl, volumes of standard, sample & Reagent 1 should be equal to each other.
**Generally, it only need to make 1~2 standard tubes, blank tubes, contrast tubes. If there is no haemolysis or lipidemia in samples, then you can skip contrast tube, use blank tube to instead.
***Use micropipet to transfer supernatant in cuvette, please avoid transfer by pouring or sediments may enter cuvette and disturb absorbance.
② Referenced ODStandard:
When standard volume is 0.1ml,
ODStandard – ODBlank = 0.065~0.070
When standard volume is 0.2ml,
ODStandard – ODBlank = 0.130~0.140
③ Formula:
④ Example:
Take slight hyperlipidemia rabbit blood serum without dilution to measure MDA content, in results, ODSample is 0.080, ODStandard is 0.046, ODBlank is 0.010(use blank tube to instead contrast tube). Calculate as follows:
(2) Midrange hyperlipidemia blood MDA assay:
Midrange hyperlipidmia blood serum (or plasma) appears quite turbid.
① Sample pretreatment:
Dilute midrange hyperlipidmia sample with physiological saline at ratio of 1:2 or 1:3.
② Operation table A:
<td wi
Standard tube | Blank tube | Sample tube | Contrast tube** | |
10 nmol/ml standard (ml) | a* | |||
Dehydrated alcohol（ml） | a* | |||
Diluted midrange hyperlipidemia blood serum(or plasma（ml） | a* | a* | ||
Reagent 1 (ml) | a* | a* | a* | a* |
Mix sufficiently by shaking test-tube stand | ||||
Reagent 2 (ml) | 1.0 | 1.0 | 1.0 | |
