The following points highlight the top two types of serology for recognition of antigen and antibody. The types are: 1. Agglutination 2. Precipitation.
Serology: Type # 1. Agglutination:
(a) Widal Test (Fig. 8.1):
In the routine laboratory diagnostic Widal reaction, the patient’s serum is tested simultaneously with the antigen of each organism likely to be responsible for enteric fever in the particular region e.g. Salmonella typhi or S. paratyphi A or B or C. Additional information can be obtained by testing separately for H and O agglutinins.
Thus, Widal test generally involves parallel tests with different Salmonella group or organisms and also different forms of the same organism.
In addition to the test with typhoid-paratyphoid organism, it is the practice of many laboratories to test also for Brucella melitensis agglutinins and, if considered necessary, with non-motile strains of proteus, OX19, OX2 and OXK for typhus fever, thus increasing the number of parallel tests. This is called “Triple Widal“.
Master (1: 15) dilution of the patient’s serum is first prepared, thereafter a series of doubling dilutions are made in narrow tubes. Then, 0.4 ml of the bacterial antigen is added in each tube.
To observe H type (loose cotton wool) agglutination, it is usually sufficient to incubate at 37°C for 2 hours and leave for half an hour at room temperature, “Large flake” clumping or agglutination can easily be detected with naked eye. The flocculi can also sediment rapidly and the deposit is quite perceptible in the narrow tube (Dreyer’s tube).
Compact agglutination of O type settles down in round bottom tubes (Felix tubes) kept in the first row of test tube rack and its readings should be made after 4 and 24 hours of incubation as these forms of reactions develop slowly. The control tube contains saline only. The clumps are small and granular, visible with the help of hand lens.
O agglutination is always positive as O antigen is common to all Salmonella. If O and H agglutinations are positive, then it is diagnosed as Typhoid—since the causative agent, S. typhi, contains both O and H antigens.
If AH agglutination is positive, then it is considered as paratyphoid—caused by S. paratyphi A.
(b) Weil-Felix Test:
It is an agglutination test which detects anti-rickettsial antibodies that cross-react with certain strains of proteus. The basis of this test is the sharing of an alkali-stable carbohydrate antigen of some rickettsial with O antigen of certain non-motile strains of proteus, P. vulgaris OX19 and OX2 and P. mirabilis OXK. False positive reaction may occur with typhoid fever, liver disease, proteus infection (UTI or other infection) etc.
(c) Brucellosis Test:
This is a tube agglutination test in which equal volumes of serial dilutions of the patient’s serum and the standardised antigen (a killed suspension of a standard strain of Br. abortus) are mixed and incubated at 370°C for 24 hours or 50°C for 18 hours. A titre of 160 or more is considered significant. Most patients with acute Brucellosis develop titres of ≥ 640 by 3-4 weeks of illness.
The same agglutination technique of Widal test is also applicable to diagnose Brucellosis, Typhus fever in man. A titre of 1:40 or 80 International units (IU) per ml. is considered positive for Brucellosis and a titre of 200 or a peak titre of 1000-5000 is positive for Typhus fever.
(d) Cold Agglutination Test:
In case of primary atypical pneumonia caused by Mycoplasma hominis, the serum may agglutinate at low temperature group O human red blood cells. To a series of doubling dilutions of patient’s serum (1: 8 to 1: 2048) an equal volume of 0.2 per cent of suspension of washed group O human erythrocytes is added.
The mixtures are kept in the refrigerator at 0°-4°C overnight. Agglutinations are observed with naked eye. A titre of 1: 32 to 1: 64 is considered significant.
(e) Paul-Bunnel Test:
An agglutinin for sheep red blood cells is present in the serum and is of diagnostic significance during and after the attack of infectious mononucleosis.
The patient’s serum is heated at 55°C for 20 minutes and a series of doubling dilutions of this serum is made from 1: 16 to 1: 1024. The control tube contains saline only 0.5 ml of a 1 per cent suspension of saline washed sheep red blood cells is added to each tube containing diluted serum.
The tube were thoroughly shaken and incubated at 37°C for four hours. Tube showing agglutination of red blood cells is the final dilution of the serum. A suggestive titre is 1: 128; a significant titre is 1: 256.
(f) Rose Waaler Test or Test for Rheumatoid Arthritis or Serum Factor:
Sheep red blood cells sensitized with rabbit anti-sheep erythrocytes serum are agglutinated by the sera of 70-80 per cent patients with rheumatoid arthritis and positive reactions occur in some 2-5 per cent of normal subjects. Serum factor is macroglobulin which is an index of inherited metabolic disturbances which predisposes an individual to rheumatoid arthritis and is an antibody to denatured gamma globulin.
Previously inactivated patient serum is diluted in two-fold steps from 1: 2 to 1 :1024.
Two sets of dilutions are required:
To one set is added an equal volume of sensitized red blood cells and the second set receives the same volume of unsensitised red blood cells of sheep and acts as control.
The tubes are incubated at 37°C and then placed in the refrigerator until the cells have completely settled. The last tube or last well of Perspex plastic plate, if used, shows a definite haemagglutination as the end titre. Serum titre of 16 or more is taken as positive in rheumatoid arthritis.
(g) Latex Agglutination Test for Rheumatoid Arthritis:
To one drop of diluted patient’s serum on the latex agglutination plate, one drop of latex antigen is added and mixed thoroughly with the plastic stick. Latex agglutination indicates that the test is positive.
(h) Coagglutination Test:
Staphylococcus aureus (Cowan 1 strain) cell wall protein gets attached with Fc fragment of specific IgG molecule directed against a specific antigen and agglutinates bacteria that have that antigen. This test is of diagnostic value. This co-agglutination reaction is highly specific but may not be as sensitive for detecting small quantities of antigen as latex agglutination test.
(i) Coombs’ Test (Anti-Globulin Test):
This test was devised by Coombs, Mourant and Race for detection of anti-Rh antibody. Coombs’ test is of two types – direct and indirect. In direct Coombs’ test, the sensitisation of RBC with incomplete antibodies takes place in vivo, as in haemolytic disease of the newborn due to Rh incompatibility, when washed RBC of infants is mixed with a drop of Coombs’ serum, agglutination results.
In the indirect Coombs’ test, sensitisation of red cells with the antibody globulin is performed in vitro. Rh +ve RBCs are mixed with the serum to be tested for Rh-antibodies and then after washing, anti-globulin is added. If the test serum contains anti Rh-antibodies, agglutination will take place.
(j) Blood Group (Fig. 8.2):
Serology: Type # 2. Precipitation:
(a) Diagnose Syphilis:
i. Venereal Disease Research Laboratory (VDRL):
Venereal Disease Research Laboratory (VDRL) flocculation test is simple, rapid, sensitive screening test used to diagnose syphilis.
One drop (0.05 ml) of inactivated patient’s serum is placed on a VDRL glass plate and one drop of diluted VDRL antigen is added on the serum and the whole plate is rotated on VDRL rotator for 3 to 4 minutes, visible aggregation or precipitation indicates a positive reaction.
ii. Rapid Plasma Reagin (RPR):
Rapid Plasma Reagin (RPR) card test or RPR 18 mm circle card test. Stabilized suspension of VDRL antigen and sized charcoal particles is used as RPR card antigen which reacts with patient’s serum, not with CSF, to form flocculation—if positive—for syphilis. A finger prick blood is sufficient.
(b) Kahn Flocculation Test:
This test is done to diagnose syphilis.
The test for each serum is set up as follows:
(c) Complement Fixation Test (CFT):
It is frequently employed with soluble antigen for the detection of antibody, even 0.08 pg of antibody nitrogen can be detected. It is very sensitive and specific. Both bacterial and viral infections can be diagnosed with this test.
When antigen and antibody reaction is specific, the complement is fixed. This reaction cannot be seen with the naked eye; but it can be observed with the help of haemolytic system (Sheep R.B.Cs + Haemolysin); if the complement is fixed, there is no haemolysis (Positive); if the complement is free, there is haemolysis (test is Negative).
(d) Agar Gel Precipitation Technique:
In this test, the antigen and antibody diffuse in the gel and form a line of precipitation when they meet. When this basic principle is adopted in electrophoresis apparatus, it is known as Counterimmuno electrophoresis (CIEP).
(e) Fluorescent Antibody Technique:
There are two main methods used in immuno-fluorescent work: the direct and indirect methods. The direct method consists of bringing fluorescein tagged antibodies in contact with antigens fixed on a slide, allowing them to react, washing off the excess of antibody and examining under the fluorescence microscopy.
The site of union of the labelled antibody with the antigen can be determined by the apple green or orange yellow fluorescent area on the slide.
The indirect method is used to detect both specific antibody in sera and also, as a direct method, for identifying antigens. For the detection of antibody, a slide on which is a preparation of the antigen, is flooded with the specific serum, and after an interval, the excess serum is washed off.
The localisation of the antibody on the antigen preparation is then visualised under fluorescent microscope by levering the slide with the fluorescein labelled antibody specific for the serum proteins of the serum under test. The most suitable type of labelled antibody is that directed primarily against the globulin fractions of the test serum i.e. fluorescein labelled anti-globulin serum.
(f) Antinuclear Factor Test:
Sera that are able to induce the formation of Lupus Erythematosus (LE) cells contain the Antinuclear Antibody (ANF) which react with the nuclei of tissue cells. This factor present in the connective tissue diseases (Serum Lupus Erythematosus SLE) can be detected by the application of indirect fluorescent antibody technique.
(g) Antitoxin-Toxin Neutralisation Test:
Is very useful to detect the toxigencity of certain strains of bacteria e.g. Clostridium tetani in mice. The control mouse, which has already been protected passively by Anti-tetanus Serum (ATS) against tetanus, will survive, as the toxin is neutralized by anti-tetanus serum; the mouse which receives the toxin of toxigenic strain of CI. tetani, will show the symptoms and die of tetanus.
The same principle is applicable in skin tests. Schick skin test is to diagnose diphtheria and Dick test to diagnose scarlet lever.
(h) Anti-streptolysin-O (ASO) Titre:
Is useful for the detection of clinically significant levels of Anti-streptolysin-O antibodies found in the serum of persons following infection with a streptolysin-O producing strain of streptococci. The ASO titre, expressed in Todd units, is the reciprocal of the serum dilution which completely neutralized the streptolysin-O. A serum with a titre of 166 Todd units should show no haemolysis.
(i) Reverse Passive Hemagglutination (RPHA) Test:
This test is an useful sensitive test to detect Hepatitis B surface antigen (HBs Ag) in serum hepatitis patient. Besides, it is simple and specific.
A simple and convenient testing procedure to detect HBg Ag:
25 µI diluent should be added into U or V cavity plates; then 7 µl test specimen (patient’s serum) should be added into the same cavity and mixed; 25µl of sensitized sheep erythrocytes should be added and mixed again. The test should be kept at room temperature for 2 hours. (Fig. 8.3)
Reactive screening test result. The cell setting pattern will appear as a large diffused pattern; non-reactive test result—a distinct compact bottom of cells (Fig. 8.3).
(j) Enzyme Linked Immunosorbent Assay (ELISA) Technique:
In the micro-ELISA for detection of antibody, the antigen is coated on to the surface of wells in micro-titration plate. The patient serum is then added and time allowed for an antigen-antibody reaction to occur. An enzyme labelled specific anti-globulin is then added, which attaches to the antigen-antibody complexes.
Any untreated anti-globulin is washed away. An enzyme substrate is then added which is acted upon by the attached enzyme producing colour which can be measured colorimetrically or interpreted visually. Incubation with enzyme substrate produces a yellow orange colour in the test well, if anti-HIV is present in the sample.
Commonly used enzymes are Horse Radish Peri-oxidase (HRP) or phosphatase. Penicillinase prepared in India is also useful and cheaper than other enzymes.
ELISA test is widely used to diagnose viral (AIDS, Hepatitis), bacterial and parasitic diseases (Fig. 8.4).
(k) Western Blot Test:
This test is used to confirm HIV in patient sera screened positive for AIDS. This test is carried out only in AIDS Reference Laboratory (National Institute of Virology, Pune; All India Institute of Medical Sciences, New Delhi, India).
NEVA HIV1 and HIV2 is a whole blood based naked eye visible Agglutination Assay (NEVA) for the detection of antibodies against HIV1 and HIV2 in a drop of blood.