( COATED PLATE ASSAYS ...)Coated Plate Immunoassay on the MicroBeta - eg. TSH - IRMA

COATED PLATE ASSAYS

The MicroBeta is and ideal instrument for the counting of radioimmunoassays based on coated microtitration plates. The following method describes a typical assay protocol for a TSH assay. The data presented compare the standard immunoassay microtitration plates with those manufactured by PerkinElmer Life Sciences.

The basic assay procedure is straightforward:

(1) Coat the wells according to the standard method described* or use commercially coated plates.

(2) Carry out the immunoassay procedure as described** - eg react the antigen to the coated antibody.

(3) Add Optiphase HiSafe II into each well and place on a plateshaker for 5 minutes (or if the plate is dried, add Meltilex using the Meltipipette 1495-034/035, and heat in oven until liquified. Allow to reset and count).

(4) Place into the MicroBeta cassette (1450- 105) and count in the MicroBeta (remember to correct for crosstalk prior to counting the sample plates).

(5) Collect and analyze the data with MultiCalc.

As a comparison two other commercially available plates (Labsystems-Polysorp and Nunc-Maxisorp) were also assayed in parallel to the Wallac (1450-401) plate to show any observable differences in plate performance.

RESULTS

The graphs (Fig.1) clearly illustrate that the MicroBeta can be used in clinical chemistry laboratories, the sensitivity required for this assay can be obtained by the MicroBeta. Additionally there appears to be little difference between using the Wallac plate to using specially surface treated plates from other companies.

*COATING PROCEDURE

1. Coating Buffer formulation:
50 mM carbonate buffer, pH 9.6
22.5 mM Na₂CO₃
27.5 mM NaHCO₃
0.9% NaCl add water 90 ml

2. Add anti-TSH into coating buffer (antibody concentration 1.0 μg/200μL/well)

3. Dispense 200 μL/well

4. Incubate overnight in a moist chamber

5. Wash 3 x with the wash solution

6. Saturate 1 hour at room temperature with 200 μL of 50 mM TSA-buffer containing 0.5% BSA
Saturation Buffer formulation:
50 mM Tris = 0.606 g Tris (Sigma)
0.9% NaCl = 0.9 g
conc HCl 0.30 mL
7.5% BSA 6.66 mL
add 100 mL, pH 7.77

7. Aspirate to dryness

8. Cover with re-sealable seal and store at 4C

**ASSAY PROCEDURE OF TSH

Previously coated plates. Farmos 125I approx. 450 kBq/11 ml anti-TSH.

ASSAY PROCEDURE

1. Add 100 μL standard and samples in duplicates to the plates

2. Add 100 μL tracer

3. Incubate at room temperature for 2 hours (or overnight)

4. Wash 5 x with wash solution with a suitable plate washer

Wash solution formulation:
Tris-HCl 0.005 M (5 mM), pH 7.5
0.9% NaCl
0.005% Tween-20

5. Decant excess wash solution (& dry if using Meltilex)

6. Add scintillant to each well, shake for 5 minutes on a plate shaker

7. Count in MicroBeta

'"/>

Source:


Page: All 1 2
Related biology technology :

1. Counting Colonies or Plaques Directly from Images of Agar Plates
2. Isolation of Low Molecular Weight Digestion Products of the Human Platelet Thromboxane A2 Receptor, Rev A
3. Counting FlashPlates on the Wallac MicroBeta
4. Use of FlashPlate Technology for In Vitro Measurement of [35S]-GTPγS Binding in CHO Cells Expressing the Human 5-HT1B Receptor
5. Use of Novel FlashPlate Technology to Measure cAMP Accumulation in Chinese Hamster Ovary Cells Expressing Human -2 Adrenoreceptors
6. Use of FlashPlate Technology for In Vitro Measurement of 125I-Labeled TGF-1 Binding on Chimeric Extracellular Domain of Type II Transforming Growth Factor Receptor
7. In Vitro Measurement of the Second Messenger cAMP: RIA vs. FlashPlate
8. Direct Counting of Millipore MultiScreen Filtration Plates.
9. Evaluation of FlashPlate in a Helicase Assay
10. 96 Well Pipetting Precision and Accuracy Using the RapidPlate 96/384 Workstation
11. Sciclone ALH 3000 Liquid Handler: Functional Characterization of the High Volume Head - Part 1: Plate-to-Plate Precision Testing