Question

Which, if any, of the immunoassays we have discussed so far, have you used? What did you like about them, or dislike? If you haven’t used any so far, can you describe the methodology of some over the counter tests such as a home pregnancy test?

Answer 1

In clinical laboratories, Immuno assays play a vital role to identify cardiac markers, Thyroid testing, nutritional assessment, tumour markers and bonemarkers as well as analytical issues such as calibration.

Cardiacmarkers : 1. Creatinine kinase MB ( CK-MB) by using mass assay,

2. Myoglobin : rises after cardiac cell damage, the myoglobin appears in serum within 2 to 3 hrs after acute Myocardial Infraction ( AMI),

3.The Troponins : Troponins I and Troponin T of the tests immunoassays are specificity of assys of Troponins,

4. C-reactive protein (CRP) - more sensitive sandwich assay, suggested risk factors for first Myocardial Infraction,

5. Homocysteine : associated with risk factor for Atherosclerotic risk.

Thyroid testing : In 1995, the American thyroid association suggested that a measure of TSH an estimate for free thyroxine index (FT4) be used as screening tests for thyroid disease.

Most immuno assays systems are available for screening TSH , only two types of TSH assays are like first generation and second generation only difference is sensitivity, they are less and 95% sensitivity respectively.

Bone markers : Immuno assays are developed for a number of markers for bone resorption and dissolution but are not routinely used,

The markers are mainly C- Telopeptides, pyridinoline, and deoxy pyridinoline and bone alkaline phosphatase.

Anemia : Folate, vitamin B12, and Ferritin assays are useful ruling in or ruling out causes of lethargy and lack of energy,

Lipids: HDL and LDL are responsible for Atherosclerotic heart disease, App lipoproteins are responsible for transport cholesterol and other lipids in blood.

Tumor markers : PSA ( Prostate Specific Antigen) diagnostic test for prostate cancer, two analytical issues with this immunoassays that no universal standards, second is sandwich assays are done by using antibodies,

CEA ( carcino embryonic antigen) marker for colorectal cancer, false positive in cirrhosis, hepatitis and Ulcers.

CA ( cancer antigen) for breast cancer,

Nutritional assessment : The prealbumin value is useful for in detecting undernourished or malnourished patients.

In the overall above discussion, the immuno assays are useful for qualitative and Quantitative with simple, rapid, specific and sensitive methods. Newer offered for as diagnostic and monitoring patients.

Methodology for Home Pregnancy Test : Pregnancy is a stage of conception in a women that can be detected in the early stage by using Immunological methods, this test is based on the presence ofan hormone " Human chorionic gonadotropin " (hCG).

hCG is a glycoprotein produced by trophoblastic cells of the placenta that begin about 10 days after conception and found in the urine of only pregnant women.

The hCG consists of two subunits alpha and beta, molecular structure similar to other glycoprotein hormones found in urine such as LH,FSH and TSH.

There are several methods are developed for pregnancy test but most accurate and rapid ones are the Immunological methods, such as 1. Latex particle agglutination inhibition test,

2. Radio immuno assay for hCG,

3. Commercially available Pregnancy testing through kits.

Immunologic Pregnancy Test: ( In Direct Test)

Clinical significance : hCG appears in the Urine of a pregnant women within 2 weeks, after the first missed a menstrual cycle. This can be detected by pregnancy test.

Principle : Inhibition of agglutination

The urine is first treated with anti agglutination hCG reagent and then reacted with the latex particles coated with hCG, if the urine contains hCG, the anti- hCG will be neutralised.

Procedure : Bring all the reagents to room temperature,

- place one drop of urine on the ring of test slide,

- add one drop of anti - hCG reagent, mix well by using applicator stick,

- after about 39 seconds, add one drop of hCG latex antigen ,

- mix again with the applicator stick and spread out the pool of liquid uniformily within the entire area of the ring on the test slide, observe for two minutes,

- simultaneously perform the test on a negative control urine.

Observations :

PT's urine	Latex particles agglutinates within 2 minutes	Negative
Pt Urine	Homogeneous suspension, no agglutination	positive
Neg Control	Latex particles agglutinates within 2 minutes	Negative

Immunologic kit pregnancy test ( Direct Test)

The card has efficacy of detecting 25mIU/ml or greater, stored at 4- 5 degree centigrade, an anti hCG conjugate immobilised over a pad, when urine sample is applied, the hCG sample migrates and reconstitute and forms acomplex.

procedure : collect urine in a clean glass vial,

- morning sample have high conc of hCG,

- bring the kit and urine to room temperature,

- put three drops of urine into the center of sample well with the help of droplet,

- wait for 5 to 10 minutes

Results:

Possitive result : Two purple colored lines,

Negative : control line is positive,

No color : indicates wrong procedure, improper storage and exposure of the test device after opening the pouch and or expire of the card.