ILP z3185356 CCL2 05Sep08

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--Shijun Lin 00:28, 18 November 2008 (EST)

ILP z3185356 - CCL2 Immunoassay on CSF Samples 05Sep08

Kit used: Human CCL2 Immunoassay R&D Systems, DuoSet Lot 1148862 Catalogue Number DY279 Expiry 28 Sep 2009


Materials Provided:

Capture Antibody - (Part 840204, 1 vial) 180ug/mL of mouse anti-human IL-6 when reconstituted with 1.0mL of PBS

Detection Antibody - (Part 840205, 1 vial) 18ug/mL of biotinylated goat anti-human MCP-1 when reconstituted with 1.0mL of Reagent Diluent

CCL2 Standard -(Part 840206, 2 vials) 130ng/mL of recombinant humam MCP-1 when reconstituted with 0.5mL of Reagent Diluent

Streptavidin-HRP -(Part 890803, 1 vial) 1.0mL of streptavidin conjugated to horseradish-peroxidase


Materials Required:

PBS - 137 mM NaCl, 2.7mM KCl, 8.1 mM Na2HPO4, pH 7.2-7.4, 0.2 um filtered

Wash Buffer - 0.05% Tween 20 in PBS, pH 7.2-7.4

Reagent Diluent - 1% BSA in PBS. pH 7.2-7.4, 0.2 um filtered

Substrate Solution - 1:1 mixture of Colour Reagent A (hydrogen peroxide) and Colour Reagent B (Tetramethylbenzidine

Stop Solution - 2 N Sulphuric Acid


Aim

To detect CCL2 in elevated levels in CSF of patients with symptoms of delirium using pre-prepared ELISA kit.

Hypothesis

CCL2 is released into CSF of patients with symptoms of delirium in amounts detectable by ELISA. Therefore, CCL2 levels detected in the experimental group will be elevated as compared to the control group.

Methods

Reagent Preparation:

A) Wash Buffer 1. Warmed to r.t.p. and dilute 20mL of wash buffer concentrate with 500mL RO water in a measuring cylinder before being stored in 500mL bottle

B) Capture Antibody 1. Add 55uL of Capture Antibody to 9945uL of PBS

C) Detection Antibody 1. Add 55.5uL of Detection Antibody to 9944.5uL of Reagent Diluent

D) Streptavidin 1. Add 50uL of Streptavidin to 9950uL of Reagent Diluent

E) CCL2 Standard 1. Reconstitute CCL2 standard with 0.5ml of Reagent Diluent to produce a stock solution of 130ng/mL and mix by gentle agitation for 15 minutes 2. 3.84uL of Reconstituted CCL2 was added to 496.2uL of Reagent Diluent 3. Serially dilute stock solution in Reagent Diluent to produce 7 standards; high standard of 1000pg/mL and a low standard of 15.6pg/mL 4. Use pure Reagent Diluent for the 0pg/mL standard


Standard 1 - 1000 pg/mL

Standard 2 - 500 pg/mL

Standard 3 - 250 pg/mL

Standard 4 - 125 pg/mL

Standard 5 - 62.5 pg/mL

Standard 6 -31.2 pg/mL

Standard 7 - 15.6 pg/mL

Standard 8 - 0 pg/mL


Sample Collection:

1. Patient samples were removed from -80oC freezer and allowed to thaw to r.t.p prior to start of the experiment
2. Excess patient material was stored in box labelled “Refrozen” and returned to -80oC


Plate Preparation:

1. Add 100uL of Capture Antibody to each well and cover plate with cling wrap. Incubate overnight at r.t.p
2. Wash each well with 300uL of Wash Buffer 3 times and blot against clean paper towels to remove excess solution
3. Block plates by adding 300uL of Reagent Diluent to each well and cover with cling wrap. Incubate for 1 hour at r.t.p. 4. Repeat wash as in step 2
5. The plates are now ready for sample addition


Steps Taken:

1. Add 100ul of standard or patient CSF to each well and cover plate with cling wrap. Incubate for 2 hours at r.t.p.
2. Wash each well with 400uL of Wash Buffer 3 times and blot against clean paper towels to remove excess solution
3. Add 100uL of Detection Antibody to each well and cover with an adhesive strip. Incubate for 2 hour at r.t.p.
4. Repeat wash as in step 2
5. Add 100uL of Streptavidin to each well and cover plate with cling wrap. Incubate for 20 minutes at r.t.p. and protect from light
6. Repeat wash as in step 2
7. Add 100uL of Streptavidin to each well and cover plate with cling wrap. Incubate for 20 minutes and protect from light
8. Repeat wash as in step 2
9. Add 100uL of Substrate Solution to each well and cover plate with cling wrap. Incubate for 20 minutes at r.t.p and protect from light.
10. Add 50uL of Stop Solution to each well. An observable colour change from blue to yellow occurred to indicate thorough mixing
11. Determine optical density of each well within 30 minutes using a microplate reader set to 450nm


Results

Refer to file Experiment Documentation CCL 2.doc

Discussion

No statistically significant difference in CCL2 levels were found in the CSF of patients with and without symptoms of delirium

References

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