Get Complete Information on Pregnancy Testing by Using the Immunological Methods

Pregnancy Testing by Using the Immunological Methods

1. Objective :

Detection of pregnancy testing by using the immunological methods.

2. Introduction :

Pregnancy is a stage of conception in women that can be detected in the early stage by using the immunological methods. However, this test is based on the presence of an hormone human chorionic gonadotropin (hCG). It is a glycoprotein consisting of a protein core with branched chains of carbohydrates which terminates often with sialic acid.

It is produced by trophoblastic cells of the placenta about 10 days after the conception, and found in the urine ol the pregnant women. The hCG is a dimmer consisting of two sub-units, alpha and beta.

The molecular structure of hCG is similar to the other glycoprotein hormones found in urine such as luteinising hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH).

After five weeks of last menstrual period (LMP), the concentration of hCG rises sharply in urine and attains its peak level at 10 weeks of gestation (LMP). During the first trimester it reaches to 1,00,000 mlU/ml (IU, international unit) which is more accurate and related to specific gonadotropic activity of 0.1 mg of dried standard kept at the National Institute for Medical Research (London).

There are several methods developed for pregnancy test but the most accurate and rapid ones are the immunological methods such as latex particle agglutination test, chromatographic immunoassay test, etc.

A. Latex-Agglutination Inhibition Test

1. Principle :

The basis of pregnancy testing is the competitive binding assay. Soon after conception level of hCG rises rapidly which is used as a marker for pregnancy testing. In this test, a small volume of urine is mixed with anti-hCG – antibody. Then hCG – coupled reagent is added and mixture is awaited for proper reaction. The non-pregnant woman does not contain hCG.

Hence, coaggulation of antibodies occurs because anti-hCG antibodies are free to react with latex coupled-hCG. On the other hand, no agglutination will take place after using urine from pregnant woman because the anti-hCG will be neutralised and will not react with latex-coupled hCG antigen.

2. Requirements :

i. First morning urine sample (of pregnant women in a sterile clean glass container), glass slides

ii. Dropper, a wooden mixing stick

iii. Anti- hCG antibody, antigen (suspension of polystyrene latex particle to which hCG has been coupled chemically)

3. Procedure :

(i) Collect urine of pregnant women in a sterile clean glass container which is supposed to be free from soap or detergent.

(ii) Put a drop of urine (specimen or control) on a glass slide using a clean pipette.

(iii) Take a drop of anti-hCG serum in a clean dropper and put on a clean glass slide.

(iv) Put one drop of urine to the anit-hCG serum, mix thoroughly for 30 seconds with mixing stick.

(v) Now add 1-2 drops of antigen (hCG-coated latex particle suspension) and mix with a clean stick thoroughly.

(vr) Rock the slide very slowly and gently for no longer than 90 seconds and observe agglutination.

4. Results :

If the urine does not contain hCG (non-pregnant woman), the added antiserum will not be neutralized. When the hCG-coated latex suspension is added, the still active antiserum reacts with hCG causing agglutination i.e. the clumping formation. Thus, the even suspension of the latex particles shows a positive result.

On the other hand, agglutination shows a negative result. In case of proteinuria a false positive result may occur.

5. Discussion :

Discuss the results following the text.

B. Haemagglutination Inhibition Test

1. Principle :

In this test anti-hCG serum and sensitized red cells are used. The anti-hCG serum is neutralised having no effect of the sensitized red cells. Therefore, the red cells are gathered at bottom of the tube giving the positive test. However, if the hCG is absent in urine, this reactions will not take place.

2. Requirements :

i. Sterile small test tubes

ii. Urine sample (from pregnant woman)

iii. Anti-hCG serum

iv. Sensitised red cells

3. Procedure :

(i) Collect urine sample in a clean glass vial as described earlier and dilute it with distilled water.

(if) Take 1-2 drops of anti-hCG serum in a glass tube.

(iii) Add 1-2 drops of urine (test sample) with anti-hCG serum and mix properly.

(iv) Add 1-2 drops of sensitized red cells in the suspension.

4. Results :

If the urine contains hCG, the anti-hCG serum will be neutralized and the sensitized red cells will not be affected. The red cells will gather on the bottom of the tube and form a ring giving the positive result. In contrast, if the urine lacks hCG, the active antiserum will agglutinate the red cells and form an evenly dispersed layers on the bottom of the test tube showing the negative result. This test is not affected by proteinuria.

5. Discussion :

Discuss the results following the text.

C. Chromatographic Immunoassay Method :

1. Principle :

The anti-(a) hCG is coated on a membrane which captures antibody on the test band region and goat anti-mouse on the control band region. The colloidal gold particles are coated with mouse anti-(P) hCG monoclonal antibody. During testing, the urine is allowed to react with the gold particles. The mixture moves along the membrane by capillary action chromatographically.

A purple-coloured band with a specific antibody (anti a-hCG antibody anti P)- colloidal gold particle complex will form on the membrane in the test band region. It gives a positive result. A negative result is obtained where a purple-coloured band in the test band region is absent.

A purple-coloured band in control region will always appear because the control region contains goat anti-mouse antibody which reacts with the mouse anti-(3 hCG monoclonal antibody coated gold particles. This does not occur with respect to the fact whether they have bound hCG or not.

2. Requirements :

i. Monoclonal anti-hCG colloidal gold

ii. Polyclonal anti-hCG coated on the membrane.

3. Procedure :

(i) Take out one testing strip from its pouch.

(ii) Carefully hold the strip vertically and dip into urine for 3 to 5 second (do not immerse the strip to pass the maximum line).

(iii) Removing the strip from urine sample, keep it on the non-absorbent sheet and wait for 5 minute for interpretation of the results.

4. Results :

The result is interpreted as below:

(i) Positive Test:

If there are two purple-colour bands on the strip, it shows that result of the specimen contains hCG and should be interpreted as positive result.

(ii) Negative Result:

Development of only one purple colour band in the strip shows that the urine specimen does not contain a detectable level of hCG and interpreted as a negative result.

(iii) Invalid Result:

If there are no purple-coloured bands present in the marked area of result window, the test result is invalid. If an insufficient volume of specimen is added into the test kit, the control band will not appear. This is because proper procedure has not been followed in the test or the test kit may have been deteriorated. In such a situation the test procedure should be repeated by using a new test kit.