2.5(C), 5.0(D), 10(E), 20(F) and 40(G) µIU/mL. Store

at 2-8°C. A preservative has been added.

 Note: The calibrators, human serum based, were

calibrated using a reference preparation, which was

assayed against the WHO 2nd IRP 80/558.

B. TSH Tracer Reagent—13 mL/vial: One (1) vial

containing enzyme labeled affinity purified polyclonal

goat antibody, biotinylated monoclonal mouse IgG in

buffer, dye, and preservative. Store at 2–8°C.

C. Streptavidin Reaction Wells—96 wells: One 96-

well white microplate coated with streptavidin and

packaged in an aluminum bag with a drying agent.

Store at 2–8°C.

D. Wash Solution Concentrate—20 mL: One (1) vial

containing a surfactant in buffered saline. A preservative

has been added. Store at 2–30°C.

E. Signal Reagent A—7.0 mL/vial: One (1) bottle

containing luminol in buffer. Store at 2–8°C.

F. Signal Reagent B—7.0 mL/vial: One (1) bottle

containing hydrogen peroxide (H2O2) in buffer. Store

at 2–8°C.

Note 1: Do not use reagents beyond the kit expiration date.

Note 2: Opened reagents are stable for sixty (60) days when

stored at 2–8°C.

Materials [Required But Not Provided]

1. Pipette(s) capable of delivering 50 µL and 100 µL

volumes with a precision of better than 1.5%.

2. Dispenser(s) for repetitive deliveries of 0.100 mL and

0.300 mL volumes with a precision of better than

1.5% (optional).

3. Microplate washer or a squeeze bottle (optional).

4. Microplate luminometer.

5. Adjustable volume (200–1000 µL) dispenser.

6. Container(s) for mixing of reagents (see below).

7. Absorbent paper for blotting the microplate wells.

8. Plastic wrap or microplate cover for incubation

steps.

9. Vacuum aspirator (optional) for wash steps.

10. Timer.

11. Storage container for storage of wash buffer.

12. Distilled or deionized water.

13. Quality control materials.

Precautions

For in vitro diagnostic use

Not for internal or external use in humans or animals

All products that contain human serum have been

found to be nonreactive for Hepatitis B surface antigen,

HIV 1 and 2 and HCV antibodies by FDA required tests.

Since no known test can offer complete assurance that

infectious agents are absent, all human serum products

should be handled as potentially hazardous and capable

of transmitting disease. Good laboratory procedures for

handling blood products can be found in the Center for

Disease Control/National Institute of Health, “Biosafety

in Microbiological and Biomedical Laboratories,” 2nd

edition, 1988, HHS.

Specimen Collection and Preparation

The specimens shall be blood serum in type and the

usual precautions in the collection of venipuncture

samples should be observed. For accurate comparison

to established normal values, a fasting morning serum

sample should be obtained. The blood should be collected

in a plain red-top venipuncture tube with or without gel

606 Concise Book of Medical Laboratory Technology: Methods and Interpretations barrier. Allow the blood to clot. Centrifuge the specimen

to separate the serum from the cells.

Samples may be refrigerated at 2–8°C for a maximum

period of five (5) days. If the specimen(s) cannot be

assayed within this time, the sample(s) may be stored at

temperatures of –20°C for up to 30 days. Avoid repetitive

freezing and thawing. When assayed in duplicate,

0.100 mL of the specimen is required.

Reagent Preparation

1. Wash buffer

 Dilute contents of Wash Concentrate to 1000 mL

with distilled or deionized water in a suitable storage

container. Store at room temperature (20–27°C) for up

to 60 days.

2. Working signal reagent solution

 Mix equal volumes of Solution ‘A’ and Solution ‘B’ in a

clean container. Use within 60 minutes. For example,

add 1 mL of A and 1 mL of B for two (2) eight well

strips (A slight excess of solution is made. Discard the

unused portion).

Test Procedure

Before proceeding with the assay, bring all reagents, serum

references and controls to room temperature (20–27°C).

1. Format the microplates’ wells for each serum

reference, control and patient specimen to be assayed

in duplicate. Replace any unused microwell strips

back into the aluminum bag, seal and store at 2–8°C.

2. Pipette 0.050 mL (50 µL) of the appropriate serum

reference, control or specimen into the assigned well.

3. Add 0.100 mL (100 µL) of the TSH Tracer Reagent to

each well. It is very important to dispense all reagents

close to the bottom of the coated well.

4. Swirl the microplate gently for 20–30 seconds to mix

and cover.

5. Incubate 45 minutes at room temperature.

6. Discard the contents of the microplate by decantation

or aspiration. If decanting, tap and blot the plate dry

with absorbent paper.

7. Add 350 µL of wash buffer (see Reagent Preparation

Section), decant (tap and blot) or aspirate. Repeat

four (4) additional times for a total of five (5) washes.

An automatic or manual plate washer can be used.

Follow the manufacturer’s instruction for proper

usage. If a squeeze bottle is employed, fill each well

by depressing the container (avoiding air bubbles) to

dispense the wash. Decant the wash and repeat four

(4) additional times.

8. Add 0.100 mL (100 µL) of working signal reagent

solution to all wells (see Reagent Preparation

Section). Always add reagents in the same order to

minimize reaction time differences between wells.

9. Incubate for five minutes at room temperature in the

dark.

10. Read the RLU’s (Relative Light Units) in each well in

a microplate luminometer for at least 0.2 seconds/

well. The results can be read within 30 minutes of

adding the substrate solution.

Quality Control

Each laboratory should assay controls at levels in the

hypothyroid, euthyroid and hyperthyroid range for

monitoring assay performance. These controls should

be treated as unknowns and values determined in every

test procedure performed. Quality control charts should

be maintained to follow the performance of the supplied

reagents. Pertinent statistical methods should be employed

to ascertain trends. The individual laboratory should set

acceptable assay performance limits. Other parameters

that should be monitored include the 80, 50 and 20%

intercepts of the dose response curve for run-to-run

reproducibility. In addition, maximum absorbance should

be consistent with past experience. Significant deviation

from established performance can indicate unnoticed

change in experimental conditions or degradation of kit

reagents. Fresh reagents should be used to determine the

reason for the variations.

Results

A dose response curve is used to ascertain the concentration of TSH in unknown specimens.

1. Record the RLU’s obtained from the printout of the

microplate reader as outlined in following Example.

2. Plot the RLU’s for each duplicate serum reference

versus the corresponding TSH concentration in

µIU/mL on linear graph paper.

3. Draw the best-fit curve through the plotted points.

4. To determine the concentration of TSH for an

unknown, locate the average RLU’s for each unknown

on the vertical axis of the graph, find the intersecting

point on the curve, and read the concentration (in

µIU/mL) from the horizontal axis of the graph (the

duplicates of the unknown may be averaged as

indicated). In the following example, the average

RLU’s (15032) of the unknown intersects the

calibration curve at (6.04 µIU/mL) TSH concentration

(Fig. 22.24).