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Pathophysiology of Delirium

UNSW Cell Biology Delirium_Catalogue


Cystatin C ELISA Protocol

Test Principle

In the CST3 (Human) ELISA Kit (Cat # KA0022), standards, quality controls and samples are incubated in microtitrate plate wells pre-coated with polyclonal anti-human cystatin C antibody. After 30 minutes incubation and washing, polyclonal anti-human cystatin C antibody, conjugated with horseradish peroxidase (HRP) is added to the wells and incubated for 30 minutes with captured cystatin C. Following another washing step, the remaining HRP conjugate is allowed to react with the substrate solution (TMB). The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured. The absorbance is proportional to the concentration of cystatin C. A standard curve is constructed by plotting absorbance values against concentrations of cystatin C standards, and concentrations of unknown samples are determined using this standard curve.

Assay reagents supplied concentrated:

Dilution Buffer Conc. (10x)

Dilution Buffer Concentrate (10x) ten-fold is diluted in 90 ml distilled water to prepare a 1x working solution,

e.g.10 ml of Dilution Buffer Concentrate (10x) + 90 ml of distilled water for use of all 96-wells. . Set of Standards

Each concentration of Standard 400x is diluted with the Dilution Buffer just prior to the assay in two steps : Dilution A (10x): 10 μl of Standard is diluted into 90 μl of Dilution Buffer and mixed well. Dilution B (40x): 10 μl of Dilution A is diluted into 390 μl of Dilution Buffer to prepare final dilution (400x) and mixed well. The diluted set of standards are not stored.

Quality Controls HIGH, LOW

The Certificate of Analysis is referred to for current Quality Control concentration.

Each Quality Control (QC) 400x is diluted with the Dilution Buffer just prior to the assay in two steps as follows: Dilution A (10x): 10 μl of QC is added into 90 μl of Dilution Buffer and mixed well. Dilution B (40x): 10 μl of Dilution A is added into 390 μl of Dilution Buffer to prepare final dilution (400x) and mixed well. The diluted Quality Controls are not stored.

Conjugate Solution Conc. (50x) The working Conjugate Solution is prepared by adding 1 part Conjugate Solution Concentrate (50x) with 49 parts Conjugate Diluent. Example: 0.25 ml of Conjugate Solution Concentrate (50x) + 12.25 ml of Conjugate Diluent for use of all 96-wells. Only the volume needed for the test is prepared and mixed well.


Wash Solution Conc. (10x) Wash Solution Concentrate (10x) is diluted ten-fold in 900 ml of distilled water to prepare a 1x working solution, e.g. 100 ml of Wash Solution Concentrate (10x) + 900 ml of distilled water for use of all 96-wells.

Sample

The kit measures cystatin C in serum, plasma (EDTA, citrate, heparin), urine and cerebrospinal fluid. Samples are assayed immediately after collection and should be stored at -20°C. Thawed samples are mixed thoroughly just prior to the assay and repeated freeze/thaw cycles are avoided, which may cause erroneous results.

Samples (serum, plasma) are dliuted400x with the Dilution Buffer just prior to the assay in two steps as follows: Dilution A (10x): 10 μl of sample is added into 90 μl of Dilution Buffer. Mix well (not to foam). Vortex is recommended. Dilution B (40x): 10 μl of Dilution A is added into 390 μl of Dilution Buffer to prepare final dilution (400x) and mixed well.

Samples (CSF) are diluted 1600x with the Dilution Buffer just prior to the assay as follows:

Dilution A (40x): 10 μl of sampleis added into 390 μl of Dilution Buffer and mixed well. Dilution B (40x): 10 μl of Dilution A is added into 390 μl of Dilution Buffer to prepare final dilution (1600x) and mixed well.

Protocol

1.100 μl of diluted Standards, Quality Controls, Dilution Buffer (=Blank) and samples are pippeted in duplicates into appropriate wells.

2. The plate is incubated at room temperature (ca. 25°C) for 30 minutes, shaking at ca. 300 rpm on an orbital microplate shaker.

3 The wells are washed 3-times with Wash Solution (0.35 ml per well). The plate is inverted and tapped strongly against paper towel after the final wash.

4.100 μl of Conjugate Solution is added into each well.

5. The plate is incubated at room temperature (ca. 25°C) for 30 minutes, shaking at ca. 300 rpm on an orbital microplate shaker.

6. The wells are washed 3-times with Wash Solution (0.35 ml per well). The plate is inverted and tapped strongly against paper towel after the final wash.

7. 100 μl of Substrate Solution is added into each well.

8. The plate is incubated for 10 minutes at room temperature. The incubation time may be extended [up to 20 minutes] if the reaction temperature is below than 20°C. Do not shake with the plate during the incubation.

9. The colour development is stopped by adding 100 μl of Stop Solution.

10. The absorbance of each well is determined using a microplate reader set to 450 nm, preferably with the reference wavelength set to 630 nm (acceptable range: 550 - 650 nm). Subtract readings at 630 nm (550 - 650 nm) from the readings at 450 nm. The absorbance should be read within 5 minutes following step 9.

LDH Assay

Reagent Preparation

1. Assay buffer preparation: dissolve the Cell-Based Buffer Tablet in 100ml of distilled water.

2. LDH Diaphorase: 1 each vial LDH Diaphorase : Reconstitute with 150µl Assay buffer OR 1 each vial LDH Diaphorase: Reconstitute with 600µl Assay buffer

3.LDH Reaction solution: to make 10ml of Reaction Solution,sufficient for use on one 96-well plate,add 100µl of the following to 9.6ml of the assay buffer: -NAD+

-Lactic Acid

-INT

-Reconstituted diaphorase

LDH Standard - Reconstitute the contents of the LDH Standard in 1.8ml of Assay Buffer

- Obtain 6 clean test tubes

- Label them 1 through to 6

Previous samples

No samples for patients 5, 7, 11, 20

Pt 1: CSF 500µL X2; BLOOD 500µL; One unlabelled tube 500µL

Pt 2: CSF 100µL

Pt 3: CSF 500µL; BLOOD 500µL

Pt 4: CSF 500µL; BLOOD 500µL

Pt 6: CSF 500µL X2; BLOOD 500µL X2

Pt 8: CSF 500µL

Pt 9: CSF 500µL; BLOOD 500µL

Pt 10: CSF 500µL; BLOOD 500µL

Pt 12: CSF 500µL

Pt 13: CSF 500µL; BLOOD 500µL

Pt 14: CSF <500µL; BLOOD <500µL

Pt 15: CSF 500µL; BLOOD <500µL

Pt 16: CSF <500µL; BLOOD <500µL

Pt 17: CSF 50µL; BLOOD <500µL

Pt 18: CSF 500µL; CSF<500µL; BLOOD <500µL.

Pt 19: CSF 500µL; Blood 500µL

Pt 21: CSF 500µL; BLOOD 500µL

Pt 22: CSF 500µL X4; BLOOD 500µL.

Pt 23: CSF 500Œúl, BLOOD 500µL X3.

Pt 24: CSF 500µL X5; BLOOD 500µL X3.

Pt 25: CSF 500µL X4; BLOOD 100µL; 50-100µL, 50µL

Pt 26: CSF 500µL X6; BLOOD <500µL X6.

Pt 27: CSF <500µL X7; BLOOD <500 µL X2.

Pt 28: CSF <500µL X7; Blood <500µL X3.

Pt 29: CSF <500µL X19, Blood <500µL X2.

Pt 30: BLOOD <500µL X3

Pt 31: CSF <500µL X7; BLOOD <500µL X9.

Pt 32: CSF <500µL X7, BLOOD <500µL X9.

Pt 33: CSF <500µL X3

Pt 34: CSF 50µL X30; 50-100µL X30; 100µL X15. BLOOD 50µL X12.

Pt 35: BLOOD 50µL X10; <500µL X 6; 500µL X6

Pt 36: CSF 50µL X8; 100µL X 40; BLOOD 50µL X8, 500µL X4.

Pt 37: CSF 50µL X10; 100µLX39; BLOOD 100µL X26, 500µL X3, 100µL

Pt 38: CSF 50µL X10, 100µL X37, BLOOD 50µL X7, 500µL X3, 100µL X2.

Pt 39: CSF 50µL X10, 100µL X10.

Pt 39: CSF 50-100µL X28; BLOOD 50-100µL X10; 500µL X3.

Pt 40: CSF 50µLX8, 50-100 µL; BLOOD 100 µL X6, 50-100 µL X4, 500 µLX4.


Pt 41: CSF 50µL X10, 100µL X 41; BLOOD 100µL X11, 500µL, 50µL X2


Pt 42: CSF 50µL X 20, 100µL X33, BLOOD 50µL X10, <500 X4, 500µL X3.


Pt 43: CSF 50µL X10, 100µL X43 BLOOD 100µL X8, 500µL X2


Pt 44: CSF 50 µL X10, 100 µL X45. BLOOD: 100µL X8, 500µL, <500 µL


Pt 45: CSF 50 µL X10, 100 µL X54 BLOOD: 100µL X12, 500 µL X2.


Pt 46: 50µL X10, 100 µL X72. BLOOD: 50-100 µL X10, 500 µL X5.

Pt 47: CSF 50µLX16;100µLX26 BLOOD 100µLX27

Pt 48: CSF 100µLX7 BLOOD 100µLX4;200µLX3

Pt 49: CSF 100µLX21; BLOOD 200µLX15;500µLX3

Pt 50: CSF 100µLX4;200µLX6;500µLX17 BLOOD 200µLX9

Pt 51: CSF 50µLX10; 100µLX6 BLOOD 200µLX16

Pt 52: CSF 100µLX18;200µLX19;500µLX11 BLOOD 100µLX17

Pt 53: CSF 50µLX6; 100µLX1

Pt 54: BLOOD 100µLX10; 200µLX3

Pt 55: CSF 200µLX25;500µLX9 BLOOD 100µLX33;200µLX3

Pt 56: BLOOD 200µLX13

Pt 57: BLOOD 200µLX19

Pt 58: BLOOD 200µLX13

Pt 59: BLOOD 200µLX19

Pt 60: BLOOD 200µLX14

Pt 61: BLOOD 200µLX19

Pt 62: BLOOD 200µLX21

Pt 63: BLOOD 200µLX16

Pt 64: BLOOD 200µLX12

Pt 65: BLOOD 200µLX25

Pt 66: BLOOD 200µLX7

Pt 67 CSF 200µLX8 BLOOD 200µLX18

Summary of samples collected (ROSH)

Patient68 CSF 100μLX5;200μLX38 BLOOD 100μLX13


Patient 69 CSF 200μLX22;500μLX8 BLOOD 100μLX12;200μLX3


Patient70 CSF 50μLX5;100μLX10;200μLX15;500μLX4 BLOOD 100μLX9;200μLX2


Patient 71 CSF 50μLX10;100μLX20;200μLX10;500μLX8 BLOOD 50μLX5;100μLX5;200μLX3


Patient 72 CSF 50μLX10;100μLX20;200μLX20;500μLX5 BLOOD 50μLX10;100μLX5;200μLX5