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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


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