Manual of basic techniques for a health laboratory - libdoc.who.int

11. Immunological and serological techniques 33911.5 Determination of b-human chorionic gonadotropin(b-hCG) in urine by the agglutination inhibitiontechnique11.5.1 Materials and reagents●●●●●●●Test platesStirring rods, wooden sticks or rotatorTest-tubes, 75mm ¥ 12mmTest-tube rackAnti-b-human chorionic gonadotropin (anti-b-hCG) antibodyLatex hCG reagent (suspension of latex particles coated with hCG)Negative control● Positive controls (strongly and weakly positive).The above-mentioned reagents are usually supplied as part of a commercial testkit.11.5.2 Method1. Bring the urine samples and reagents to room temperature.2. Add one drop of each of the urine samples and each of the controls to the testplates.3. Add one drop of anti-b-hCG antibody to each of the samples and each of thecontrols. Mix carefully.4. Mix the latex hCG reagent suspension well; apply one drop to the testsamples and rotate the plates or mix with stirring rods or wooden sticks (one persample).5. After 3 minutes examine the plates and compare the reactions of the test sampleswith those of the controls.A positive reaction (pregnant or b-hCG present) is indicated by the absence ofagglutination. A negative reaction (non-pregnant or b-hCG absent) is indicatedby the presence of agglutination.Semi-quantitative analysisSemi-quantitative analysis may be desirable in some cases where the production ofb-hCG may have a pathological cause. This may occur in both pregnant and nonpregnantpatients.Make a twofold dilution of the positive urine samples and examine as described insteps 2–5 above. The highest dilution that does not cause agglutination is the titre.The results for this semi-quantitative analysis are reported in IU/ml which can beobtained by multiplying the dilution factor of the highest dilution that does notcause agglutination by the sensitivity or limit of detection of the method, as statedby the manufacturer.11.6 Quantitative determination of IgA, IgG and IgM byradial immunodiffusion11.6.1 Materials and reagents●●●Siliconized glass plates 8cm ¥ 12cm, or Petri dishes (glass or plastic)Boxes with tightly fitting lidsHole-punch with inner diameter of 2mm