Reactions of Antigen-Antibodies in Bacteria

In this article we will discuss about the Reactions of Antigen-Antibodies in Bacteria.

When an antigen combines with its specific antibody in presence of an electrolyte (NaCl) in an optimum concentration at an optimal pH and temperature (37°C), the antigen and antibody complex forms an insoluble precipitate.

Important tests for antigen-antibody reactions include precipitation, agglutination, complement fixa­tion, neutralisation, and labelled assays (immuno­fluorescence, enzyme immunoassay, radioimmuno­assay) (Table 9.1). These reactions are greatly impro­ved by the availability of highly specific monoclonal antibodies.

Antibody titre:

The term titre is used to denote the highest dilution of the serum at which antibody activity is demon­strable, which is usually expressed as the reciprocal of the dilution of the serum, e.g. 64, when antibody was detected at a final serum dilution of 1 in 64. The antigens and antibodies taking part in the reaction are also measured by their mass weight, e.g. mg nitrogen.

Tests for antigen-antibody reaction:

1. Precipitation reaction (Table 9.2):

When a soluble antigen reacts with its specific antibody at an optimum proportion, pH and temp, Ag-Ab complex forms an insoluble precipitate, e.g.

(a) Simple precipitation tests:

Ring test, slide test, slide flocculation test (VDRL), tube flocculation (Kahn) test.

(b) Immunodiffusion:

Single diffusion in one dimension, double diffusion in one dimension, double diffusion in two dimensions, counter-immunoelectrophoresis.

2. Agglutination:

It is an antigen-antibody reaction in which an antibody (usually patient’s serum and soluble) combines with a particulate antigen in presence of an electrolyte and at an optimal temperature and pH resulting in visible clumping (aggregation of the particles, such as agglutination of bacteria, erythro­cytes, latex particles etc.), e.g.

(a) Slide and tile agglutination test:

Detection of isolated bacteria by slide agglutination test.

(b) Tube agglutination test:

Widal test.

(c) Heterophile agglutination test:

Weil-Felix reaction, Paul-Bunnel test, Streptococcus MG agglu­tination test.

(d) Anti-globulin test:

Coombs’ test.

(e) Micro-titration agglutination:

Indirect passive haemagglutination test, haemagglutination inhibi­tion (HI) antibody test, reverse passive haemagglu­tination (RPHA) assay.

3. Complement fixation test:

Wassermann reaction (WR), immobilisation test, immune adherence test, cytolytic or cytocidal reaction.

4. Neutralisation test:

When an antibody (anti­toxin) combines with specific antigen (toxin), the biological effects of the toxin (antigen) are neutra­lised e.g. Neutralisation test (guinea pig inoculation) and neutralisation in vivo (agar gel precipitation test), both tests are done for detection of diphtheria toxin.

5. Labelled Immunoassay:

(a) Radioimmunoassay (RIA) antibodies labelled with radioactive substances like iodine — ¹²⁵I or ¹³¹I.

(b) Immunofluorescence (commonly used antibody marker is fluorescein isothiocyanate).

(c) Enzyme-linked immuno-sorbent assay (ELISA): Enzyme like peroxidase, alkaline phosphatase, etc. are coupled to antibody.

VDRL Slide Flocculation Test:

Uses:

(i) VDRL test provides a simple, rapid con­venient and economical, procedure for serologic testing for diagnosis of syphilis.

(ii) It has high sensitivity. Test is non-specific. It can be used for rapid and exact quantitative titration of reactive serum samples for screening and moni­toring treatment.

(iii) It is well-suited for mass surveys.

Reagents:

1. VDRL Antigen:

It is an alcoholic solution and contains cardiolipin 0.03 per cent, purified lecithin 0.21 per cent, and cholesterol 0.9 per cent. The antigen is serologically standardised by comparison with an antigen of known reactivity. The antigen is dispensed in 0.5 ml quantities (with a sufficient excess for convenient withdrawal of the required amount) in sealed glass ampoules which should be stored in a cool dark place.

2. Buffered saline solution:

The buffered saline has the following composi­tion:

Formaldehyde …. 0.5 ml

Na₂HPO₄ …. 3.037 gm.

KH₂PO₄ …. 0.170 gm.

NaCl …. 10.0 gm.

Distilled water …. 1000ml

and has a pH of 6.0 + 0.1. 3.

VDRL slide:

Glass slides (2 x 3″) with 12 paraffin rings or cavities of approximately 14 mm inside diameter are used. Similar slides with permanently fixed ceramic rings (5 rings) (Fig. 9.7) should be more convenient but are somewhat expensive.

Procedure:

1. Preparation of Serum:

Serum is inactivated for 30 minutes at 56°C. Sera to be tested more than 4 hours after being heated; should be reheated at 56°C for 10 minutes.

2. Preparation of Antigen Emulsion:

0.4 ml of buffered saline is pipetted into the bottom of a 1 oz. reagent bottle with flat or concave inner bottom surface. Add 0.5 ml of antigen, drawn from an ampoule into a 1.0 ml pipette graduated to tip, directly on to the saline while rotating the bottle on a flat surface.

Antigen is added drop by drop but rapidly so that it takes approximately 6 seconds to complete the delivery. Blow the last drop of the antigen and continue rotation of the bottle for 10 more seconds.

Then add 4.1 ml of buffered saline from a 5.0 ml pipette. Stopper the bottle and shake it vigorously for approximately 10 seconds. Temper­ature of buffered saline solution and antigen should be preferably between 23°C to 29°C during the preparation of antigen emulsion.

Maturation of the antigen increases the sensi­tiveness and this is almost complete in 15 to 30 minutes. The antigen emulsion may then be used during the whole day.

Under conditions of high temperature and low humidity, the antigen emul­sion is stored in a refrigerator but should be restored to room temperature before the test. Antigen emulsion should be shaken gently every time before use. This quantity (5.0 ml) is sufficient for 250 serum tests.

The preparation of antigen emulsion should first be examined by testing known reactive and non- reactive sera. This is done by adding one drop of antigen emulsion to 0.05 ml of each serum and completing the test as described under Quantitative Serum Test.

These tests should present typically reactive and non-reactive results, respectively and the number and distribution of antigen particles per microscopic field in the negative sera should be optimum. An unsatisfactory antigen emulsion should be discarded.

Qualitative Serum Test:

A volume of 0.05 ml heat inactivated (56°C for 1 hour) serum is pipetted into one ring of a VDRL glass slide. The serum should spread. Now add one drop (1/60 ml) antigen emulsion on to the serum by a syringe dropping 1/60 ml antigen per drop. Rotate the slide for 4 minutes. (If rotated by hand on a flat surface this movement should roughly circumscribe a 5 cm diameter circle 120 times per minute).

Reading and reporting the results:

The tests are read immediately after rotation under a microscope with low power objectives at 100 x magnification. The antigen particles are seen as small fusiform needles which remain more or less evenly dispersed in a non-reactive serum and aggregate into clumps in reactive sera.

No clumps or very slight — Non-reactive (N)

Small clumps — Weakly reactive (W)

Medium and large clumps — Reactive (R)

Zone reactions are recognised by the irregular clumping. Clumps are not compact and very small. Large clumps may be seen in the same field. In such cases, the results are reported on the basis of quan­titative reaction done on the same serum.

Quantitative Serum Test:

Quantitative test is performed on all reactive serum samples and on all samples showing weakly reactive or “rough” in the qualitative test. Successive two fold dilutions of the serum are made in 0.9 per cent saline and each dilution is treated as an individual serum and tested as described under Qualitative Serum Tests. The results are reported in terms of the highest dilution which gives a frank reactive reaction.

Reporting results of the Quantitative Tests:

The results are reported in terms of the highest dilution of the serum that produces a definite reaction (not weakly reactive).

Some examples are shown below (Fig. 9.8):

VDRL Antigen can also be used for the VDRL Tube Flocculation test and for testing spinal fluids.

Widal Test:

Principle:

It is used for detection of antibodies produced by the host against determinants on the surface of a bacte­rial agent in response to infection with Salmonella typhi and S. paratyphi. Patient’s serum show aggluti­nating antibodies that cause agglutination of the bacterial antigens. The patient’s serum is tested by agglutination for its titres of antibodies against H and O suspensions of S. typhi and S. paratyphi.

Materials required:

1. Small test tubes (Kalin tube). Dreyer tubes may be used for testing H-agglutination.

2. Antigen suspensions, prepared from suitable stock cultures in the laboratory, but generally commercially prepared suspensions are used.

3. 0.85% NaCl.

4. Water bath.

Procedure:

Patients serum is tested in a series of dilutions against each of the different antigen suspensions:

1. Eight tubes are taken for each series — seven for serum dilutions and the eighth for non-serum control (C).

2. Add 0.4 ml saline in tubes 1 to 7. Dilute patient’s serum 1 in 15 in saline and add 0.4 ml of this to tubes 1 and 2. Dilution of serum in tube 2 will be 1 in 30 (volume 0.8 ml). No serum or saline is placed in tube 8.

3. Mix the contents of tube 2 thoroughly and transfer 0.4 ml to tube 3. Repeat the process till tube 7 and discard 0.4 ml in the end. The serum dilutions in 7 tubes will be 1: 15, 1: 30, 1: 60, 1: 120, 1: 240, 1: 480, and 1: 960 and 8th tube serves as a control.

4. Add 0.4 ml of antigen suspension to each tube, start from tube 7 and working backwards to tube 1. So, the serum dilutions in tubes 1 to 6 will be now 1: 30-1:960. The four different antigens used are TO, TH, AO and AH.

5. Incubate the tubes in a rack in water bath at 37°C for 2 hours. Then the rack is brought out from water bath and kept at room temperature for 30 minutes. H-agglutination is noted at this point. Then the rack is again kept in water bath at 37°C for another 2 hours, followed by preservation in refrigerator for overnight period before O-agglutination is noted.

Results:

1. H-agglutination, appears as large flakes is read by viewing the tubes under good light against a dark background with the aid of a magnifying lens (Figs. 9.9 to 9.11). O-agglutination form fine granular deposit, best viewed with a concave mirror (Figs. 9.12 and 9.13). If necessary, the tubes can be gently rotated, to swirl up granules from the deposit.

2. The titre of the serum is the highest dilution of serum giving visible agglutination.

Remarks:

1. A progressive rise in titre between first and second week after onset of fever is highly significant. A positive or a negative result in a single test is not significant.

2. Since the antibodies are detected only after seven to ten days of illness, test should be done later.

3. The serum of some uninfected subjects causes agglutination at dilutions of about 1 in 60. So titres are considered significant when agglutination occurs in serum dilution above 100.

4. H-agglutinins tend to persist longer than O-agglutinins. Persons immunized with TAB vaccine may show high titres of antibodies to all the antigens and so only a marked rise in titre is considered signi­ficant.

5. Early administration of antibiotic cause arrest of titre.

Rapid Slide Test:

Commercial kits are available for the purpose:

1. Place 0.01 ml serum on each of five squares on a glass plate.

2. Add 0.03 ml of antigen (1 drop) from a bottle of antigen suspension. Antigen is thoroughly mixed before use.

3. Mix well by a stick (toothpick). Then lift the plate and slowly tilt back and forth for 2-3 minutes.

Reading:

Result is read in a viewing box or against a flat black background. Test with diluted serum is done to find out titre.

Vi. Agglutination (Fig. 9.13):

Antibodies to Vi-antigen appear during typhoid fever but disappear after recovery from disease. Vi antibodies are detected in current or recent infection but soon disappear. Vi antibodies persist in carrier state.

Antibody to Vi antigen (1: 10 or above) is present in serum of most carriers (Fig. 9.13):

Convalescent carriers and some individuals with sub­clinical infection (healthy carriers) of typhoid fever continue to excrete organisms in faeces, less commonly in the urine for about 2 months to a year.

Incidence of chronic carriers is lower in paratyphoid than typhoid fever. In addition to detection of antibody to Vi antigen in patient’s serum repeated stool cultures may be required to detect the bacilli. Aspirated bile and duodenal drainage cultures are helpful.

Latex Agglutination Test (Fig. 9.14):

Latex or polystyrene particles coated with Ag or Ab react with specific corresponding Ab or Ag to produce clumping of the particles. Based on this phenomenon, many diagnostic serological tests have been introduced: e.g. detection and semi-quantitative assay of ASO, RF, C-Reactive protein, ANF, etc. The test for RF is described below.

Principle:

RF slide test for detection of rheumatoid factors is based on the principle of agglutination. The test specimen is mixed with RF latex reagent and allowed to react. If RF (anti-human IgM) is present within detectable levels then a visible agglutination is observed. If RF is absent below detectable levels, then no agglutination is observed.

Reagent required:

1. RF reagent:

A uniform suspension of polysty­rene latex particles coated with suitably modified Fc fraction of IgG. The reagent is standardised to detect 10 IU/ml of RF or more.

2. Positive control serum, reactive with the RF reagent.

3. Negative control serum, non-reactive with the RF reagent.

Each batch of reagents undergoes rigorous quality control at various stages of manufacture for its specificity, sensitivity and performance.

Reagent contains 0.1% Sodium azide as preser­vative. Avoid contact with skin and mucosa. On disposal, flush with large quantities of water.

The reagent can be damaged due to microbial contamination or on exposure to extreme temper­ature. It is recommended that the performance of the reagent be verified with the positive and negative controls provided with the kit. Shake the RF latex reagent well before use to disperse the latex particles uniformly and improve test readability.

Specimen:

Only serum should be used for testing. Should a delay in testing occur, store the sample at 2-8 °C. Samples can be stored for up to a week. Do not use haemolysed serum.

Material required:

1. RF latex reagent, Positive control, Negative control.

2. Glass slide with six reaction circles. Samples dispensing pipettes, Mixing sticks, Rubber teat.

3. Stopwatch, Test tubes, a high intensity direct light source, isotonic saline.

Procedure:

Bring reagent and samples to room temperature before use.

(a) Qualitative Method:

1. Pipette one drop of serum onto the glass slide using the disposable pipette.

2. Add one drop of RF latex reagent to the drop of serum on the slide. Do not let the dropper tip touch the liquid on the slide.

3. Using a mixing stick, mix the serum and the RF latex reagent uniformly over the entire circle.

4. Immediately start a stopwatch. Rock the slide gently, back and forth, observing for agglutination macroscopically at 2 minutes.

(b) Semi-quantitative Method:

1. Using isotonic saline prepare serial dilutions of the serum sample positive in the qualitative method 1: 2, 1: 4, 1: 8, 1: 16, 1: 32, 1: 64 and so on.

2. Pipette each dilution of the serum sample onto separate reaction circles.

3. Add one drop of RF latex reagent to each drop of the diluted serum sample on the slide. Do not let the dropper tip touch the liquid on the slide.

4. Using a mixing stick, mix the sample and the latex reagent uniformly over the entire circle.

5. Immediately start a stopwatch. Rock the slide gently, back and forth, observing for agglutination macroscopically at 2 minutes (Fig. 9.14).

Interpretation:

Qualitative method:

Agglutination is a positive test result and indicates the presence of rheumatoid factors in the test specimen. No agglutination is a negative test result and indicates the absence of rheumatoid factors in the test specimen.

Semi-quantitative method:

Agglutination in the highest serum dilution corresponds to the approxi­mate amount of rheumatoid factors in IU/ml present in the tests specimen.

To calculate the RF in IU/ml, use the following formula:

RF (IU/ml) = S x D

where, S = Sensitivity of the reagent, i.e. 10 IU ml

D = Highest dilution of serum showing agglutination.

Notes:

1. Markedly lip-emic, haemolysed and contamina­ted serum samples could produce non-specific results.

2. Use of plasma rather than serum can lead to false-positive results.

3. Do not read results beyond two minutes.

4. Rheumatoid factors are not exclusively found in rheumatoid arthritis but sometimes in syphilis, systemic lupus erythematosus, hepatitis, hypergammaglobulinaemia also.

5. It is recommended that results of the test should be correlated with clinical findings to arrive at the final diagnosis.

6. The RF reagent is free from pro zone effect at RF levels between 10 IU/ml to 2300 IU/ml of RF con­centration.

7. RF reagent is sensitive to the presence of IgM RF with heterogeneous specificity.

Summary:

Autoantibodies are sometimes produced by the human body against self-antigens. The precise role that this aberrant immunity plays in the patho­genesis of certain rheumatoid diseases is unknown. The presence of these autoantibodies serves as credible marker of the disease.

In rheumatoid arthritis, diagnostically useful autoantibodies termed as “Rheumatoid factors” (RF) can be detected which are immunoglobulin’s of the class IgM, IgG, IgA and IgE. Practically, IgM class RF with specificity to human IgG (Fc) is the most useful prognostic marker of RA.

The clinical significance of RF determinations consists in differentiation between rheumatoid arthritis, in which RF of modified IgM class have been demonstrated in the serum of approximately 80% of the cases examined and rheumatic fever, in which RF are almost always absent. The agglutination test is most frequently used because of its greater sensitivity and simplicity.

Indirect Haemagglutination Assay (IHA):

Principle:

When a particulate antigen is mixed with its antibody in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated. Agglutination occurs optimally when antigens and antibodies react in equivalent proportions.

This test measures as little as 1-10 μg/ml of anti­body. RBCs are used in the test as they are extremely convenient passive carriers of antigen and give a visible reaction and many antigens can be coated on complex surface of RBC. RBCs used can be of Human group “O” as it does not contain antigen and of Sheep (or RBCs of Horse, Monkey, Turkey Guinea-pig, etc. can be used).

Reagents:

1. Phosphate buffer solution (PBS) (pH 7.4) – 0.15 M

2. Alsever’s solution:

Sodium citrate: 0.08 g

Dextrose: 2.05 g

Sodium chloride: 0.42 g

Distilled water: 100 ml

Adjust pH to 6.1 with 10% citric acid (autoclave at 10 pounds for 15 minutes).

3. Tannic acid (TA):

Dissolve 1 mg in 20 ml of normal saline and store at 4°C. (1: 20,000 dilution).

4. Rabbit Serum Saline (RSS):

1% Rabbit serum in PBS.

Preparation of sensitized RBC’s:

1. To collect sheep blood in Alsever’s solution.

2. To wash the RBCs thrice in PBS by centrifugation at 1,000 rpm.

3. The RBCs are packed at 2500 rpm by washing with PBS (pH 7.2) for 5-10 minutes.

4. Make 2.5% suspension of RBCs in PBS (pH 7.2) (make up to 2 ml.).

5. Add equal volume of Tannic acid.

6. Incubate at 37°C for 15 min. in a water bath.

7. Wash the RBCs thrice with PBS.

8. For sensitisation of RBCs add 1.5 ml of tanned cells to 1.5 ml of antigen [diluted in PBS (pH 6.4)] (Antigen is titrated first against positive serum of known titre by chequer board and finally minimum antigen concentration which gives agglu­tination with known positive serum at same dilution is used).

9. Incubate at 37°C for 30 minutes in water bath.

10. Centrifuge at 1,000 rpm for 10 minutes.

11. Wash with 1% Rabbit Serum Saline (RSS).

Procedure (Figs. 9.15, 9.16):

1. The test sera is inactivated at 56°C for 30 minutes.

2. 1 per cent RSS is used for serial dilutions of serum samples.

3. Make 4-fold dilutions of all serum samples in Perspex plates with the help of micro diluters starting from 1: 8 to 1: 2,048 (take 50 of diluted serum).

4. Two controls, i.e. tanned RBCs control and antigen control should always be put with every test.

5. Add 0.025 ml (one drop of a micro diluter) of sensitized RBC’s in each well (except in antigen control well). Mix by shaking.

6. Incubate for 1 hour at 37°C and then overnight at 4°C.

7. Agglutination, characterised by uniform mat of cells covering the entire bottom of the well, suggests positive reaction and button (of RBCs) suggest negative reaction (Fig. 9.16). So the titre of antibody will be the highest dilution of antibody which will give agglutination reaction.

Remarks:

1. Tanning of RBCs is important, as it exposes the surface receptors and also reduces the electrostatic charges. RBCs are negatively charged and so are always repelling each other. To cause enhancement of agglutination, if the concentration of tannic acid (TA) is too high, it will reduce the specificity of the test as auto-agglutination occurs.

2. Rabbit Serum Saline (RSS) should be used because it prevents auto-agglutination of tanned cells and acts as a stabilizer by balancing out the agglu­tinating effect of TA resulting in greater sensitivity of the test. Rabbit serum should also be inactivated at 56°C for 30 minutes.

3. Plates are thoroughly washed and cleaned and serum should be free of contamination.

4. Sometimes the test shows false-positive results, because of the presence of arti-facts in plates or insufficient stability of RBCs or because of auto­-agglutination of cells by excessive tanning or by using excess of antigen. So all these should be standardised properly and time given for each step should be adhered to — to avoid false-positivity.

5. False-negative results do occur when the concentration of antigen is low, or by under-tanning RBCs. So proper precautions should be taken.

6. Cold centrifuge should preferably be used for all steps as the test is very sensitive to high temper­atures.

7. Instead of normal saline (as it can resist the changes in pH) PBS is used as the test is very sensi­tive to changes in pH.

8. Preferably fresh RBCs should be used, as preserved cells are less sensitive than (preservative formalin) fresh ones.

9. Alternatives for tannic acid are Bisdiazotised benzidine and chromium chloride.

Anti-Streptolysin O (ASO) Test (Tube Dilution Method):

Principle:

It is a neutralisation test used to measure the ability of a patient’s serum to neutralize the erythrocyte lysing capability of streptolysin O, an extracellular enzyme produced by Streptococcus pyogenes during infection.

Materials:

1. Antigen:

Streptolysin O — available commer­cially, is reconstituted before use.

2. ASO buffer:

Phosphate buffered saline (pH 6.4).

3. RBC suspension:

5% suspension of RBCs in ASO buffer: Human blood group ‘O’ or rabbit erythrocytes.

4. Water bath.

Procedure:

1. Serum dilution (Table 9.1)

Serum is at first diluted in 3 master dilution tubes as follows:

Master dilution tube I (1:10):

A volume of 0.2 ml. serum is added to 1.8 ml ASO buffer.

Master dilution tube II (1: 100):

A volume of 0.05 ml of serum is diluted in 4.95 ml ASO buffer.

Master dilution tube III (1: 500):

A volume of 0.5 ml of 1: 100 dilution serum from master dilution tube II is transferred master dilution tube III containing 4.5 ml of ASO buffer. Now place the diluted sera in tubes as shown in Fig. 9.15. Then add buffer to the tubes as shown in Table 9.3. The dilution in different serum tubes will be 12, 50, 100,125,166, 250, 333, 500, 625, 833, 1250, 2,500.

2. Addition of antigen:

Add 0.5 ml of the antigen to all the tubes.

Incubate at 37°C for 15 minutes.

3. RBC suspension:

After incubation add 0.5 ml of washed 5% RBCs, shake and incubate at 37°C for 1 hour.

4. Control:

Controls used with this test are cell control, antigen control and a standard serum control.

Results:

1. Reading can be taken as such but centrifugation of tubes makes reading easier.

2. The titre of ASO is the highest serum dilution causing no haemolysis. Significant titre is considered to be 250 or more.

Remarks:

1. Serum is to be inactivated at 56^o C for 30 minutes before use.

2. A rise in ASO is detectable after 1 week of infection and very high titres are observed between third and fifth week.

3. False-positive results are observed due to incre­ased levels of B lipoproteins in serum or contamina­tion of serum by S. aureus or Pseudom species or by oxidation of ASO.

Rose-Waller Test:

(Differential Sheep Cell Agglutination Test)

Principle:

It is an agglutination (anti-globulin) test using sheep RBCs sensitized with a sub-agglutinating dose of rabbit anti-sheep erythrocyte serum IgG. Rheuma­toid factor, 19 S IgM, will combine with the fixed 7S IgG and produce agglutination.

Materials required:

1. Sheep RBCs.

2. Anti-sheep RBC serum (A-SRBC):

The titre of antiserum which fails to agglutinate the SRBCs should be determined.

3. Sensitized RBCs:

2% RBC suspension is added to an equal volume of diluted antiserum with con­stant stirring. This can be used within few minutes

Procedure:

1. A serial two-fold dilutions of patients serum (0.2 ml) is made in saline in ten test tubes, such as 1: 4, 1: 8, 1:16, 1: 32 and 1: 64. The last tube is kept as control for SRBC suspension.

2. A volume of 0.2 ml of sensitized RBCs is added to all tubes and incubate at 37°C for one hour and then at 4°C for 2 hours.

3. Known positive and negative serum is included as control.

4. One row of ten tubes is taken as control in which un-sensitised SRBCs are added to diluted patient’s serum for detection of heterophile anti­bodies.

Findings:

1. If the cells are agglutinated then that serum dilution is taken as the positive. The highest dilution of serum showing definite haemagglutination is taken as the end point.

2. The serum titre is determined by taking the ratio between the dilution agglutinating un-sensitised and the sensitized RBCs. It is expressed as the reciprocal of the highest dilution of patient’s serum showing agglutination.

3. A significant titre is 1: 16 or above.

Remarks:

1. The serum is to be inactivated.

2. Controls with un-sensitized SRBC should always be included and there should be no agglu­tination in these tubes.

3. Ratio’s of 1: 8 considered as doubtful and less than that is not significant.

4. Latex particles coated with human gamma-­globulin can be used instead o£ sheep RBCs.

5. For determining the dilution of the antiserum to be used for sensitisation of RBCs different dilutions tested should be from 1/100 to 1/6,400 and from 1:140 to 1/8,960 by making two fold dilutions.

Elisa Test:

Principle and uses:

See Textbook at Microbiology by Dr. P. Chakraborty.

The ELISA (enzyme-linked immunosorbent assay) is so reamed as the test technique involved use of an enzyme system and an immunosorbent. The absorbing —material may be particulate e.g. cellulose or agarose or a solid matrix, e.g. micro-wells, membranes etc.

(A) Plate format test:

Materials required:

1. Micro-tie plate or tray.

2. Wash buffer (PBS with triton).

3. Sample diluent (PBS with bovine albumin).

4. Conjugate, substrate and stop solution (Table 9.4).

5. Strongly reactive control.

6. Non-reactive control.

Coat the wells of a PVC micro-titre plate with Ag:

(i) Fill the wells with Ag at a conc. of 1-10 μg/ml in carbonate/bicarbonate buffer (pH 7.4).

(ii) Cover the plate with an adhesive plastic and incubate at 4°C.

(iii) Remove the coating solution containing unbound Ag and wash the wall twice with 200 μl PBS Triton.

Blocking and adding samples:

(i) Remaining protein-binding sites in the coated wells is blocked by adding 200 μl blocking buffer, 5% non-fat dry milk/PBS, per well.

(ii) The plate is covered with adhesive plastic and incubated for 2 hours at room temper­ature or, overnight at 4°C.

(iii) Wash wells with 200 μl PBS-Triton twice as above.

(iv) 100 μl of appropriately diluted samples are added to the previously demarked wells. Standards in duplicates or triplicates and blanks are concurrently tested for calcula­ting accurate quantitative result. After addition of samples, blank solution and controls, the plates are incubated at 37°C for 60-90 minutes.

(v) The wells are washed twice with 200 μl PBS with triton.

Addition of 2nd step antibody (conjugate):

(i) 2nd step antibody (i.e. antibody against the antibody to be detected in the sample) conjugated with Alkaline phosphatase or Horseradish peroxidase is to be appropria­tely diluted according to its titration result done beforehand.

(ii) 100 μl of the 2nd step antibody conjugate is added to the reaction wells, where 1st Ab present in serum is already fixed. Incubation is done for 1 hour.

(iii) Repeat washing as above.

Addition of substrate:

(i) Prepare substrate solution. Substrate depends on the enzyme conjugated with the 2nd antibody. Add 50 μl of the substrate solution in each well. Keep the plate at room temperature in dark for 60 minutes.

(ii) Add appropriate stop solution in 50 μl quantity.

Reading:

The colour developed is measured (optical den­sity) using appropriate filter.

Result:

Qualitative:

Cut off value is calculated from the OD of blank and controls. A test result above the cut-­off value is taken as reactive.

Quantitative:

Graph is prepared using OD values corresponding to controls. By plotting the OD value of test on the graph, quantitative estimation could be done.

Modifications:

Above basic protocol may be modified according to the need:

(i) To detect Ag, coating of wells is done with corresponding monoclonal antibody.

(ii) To increase the sensitivity of Ab detection, sandwich method (Ag is captured between two layers of antibodies) is adopted where 2nd Ab used is a non-conjugated one, and a 3rd Ab used is within conjugated with enzyme and raised against the 2nd Ab in a different animal.

(iii) In competitive ELISA, used to detect an Ag, the coating is done with monoclonal Ab. The test sample and measured amount of Ab mixture is added in 1st reaction. Ab in sample competes with the bound Ab for binding the epitopic sites of the Ag.

After first wash unbound Ag and free Ab is washed out. The rest of the steps are similar to sandwich ELISA protocol. Here decrease in OD compared to control one (without free Ab in the 1st reaction) is propor­tional to the concentration of Ag.

(B) Elisa protocol for membrane format:

Liquid with Ab and Ag may flow through or move laterally in a membrane like nylon, nitrocellu­lose or other membranes. ELISA test may be perfor­med using these membranes as solid phase on which Ag or Ab is immobilised in a spot or line according to the need.

An absorbent material is placed below these membrane. The two membranes together forms the card on which the test is performed. Known Ag or Ab, according to the need, is impreg­nated on non-absorbent nylon or nitrocellulose membrane.

The absorbent membrane below serves to pull the reactants through the membrane and help to separate non-reactant components from Ag-Ab complex bound on the membrane.

Different tests:

1. Immuno-comb test.

2. Flow through test — e.g. spot test.

3. Lateral flow test — e.g. immuno-chromatographic strip test.

1. Immuno-comb test:

This is basically an indirect solid-phase enzyme immunoassay (EIA). The solid phase is a card with a number of projections (teeth), mimicing a comb (Fig. 9.17A).

Each tooth is impregnated on two spots:

Upper spot – by human IgG or IgM, as required, for internal control.

Lower spot – by purified Ag.

Procedure:

Initially, patient’s serum (treated and suitably diluted) is allowed to react with the Ag spot by immersing the tooth in the well of row ‘A’ of a developing plate (Fig. 9.17B) containing the test serum. If specific Ab is present, it binds with the Ag. Unbound components are then washed in row ‘B’.

In row ‘C’, captured Ab of test serum at lower spot and human IgG or IgM on the upper spot (internal control), will react with alkaline phosphatase (AP) labelled anti-human IgM antibody. In the next two rows, unbound components are removed by washing. In ‘E’ row, the bound alkaline phosphatase will react with chromogenic components. The results are visible as grey spots on the surface of the teeth of the card.

Interpretation (Fig. 9.18):

1. Positive — two spots develop on the card tooth.

2. Negative— the upper spot is developed only.

All specimens must produce an upper spot as internal control for validation of test.

2. Spot test (Membrane flow through test):

The flow through characteristic of nylon, nitrocellulose and other membranes, on which Ag or Ab is impregnated, is employed in Spot-ELISA test. This enhances the speed, sensitivity and simplicity of ELISA reaction process. Absorbent material below helps to pull the liquid reactants through the membrane and thus separates non-reactant compo­nents from Ag-Ab complexes (Fig. 9.19). This simplifies the washing steps.

Procedure:

According to the need Ag (e.g. synthetic peptide Ag of HIV-1 or HIV-2) is impregnated on a nylon or nitrocellulose membrane strip placed on an absor­bent membrane.

Undesirable epitopic sites of Ag are blocked by blocking solution (Hammersten Casein in 0.05%. Tween 20 in P.B.S.). Such test strips are readily available for many common sero-diagnostic tests, e.g. HIV. Tri-Dot detecting HIV-1 and HIV-2 Ab in patient’s serum (Fig. 9.19).

Test serum suitably diluted is dropped in ade­quate quantity on the reaction strip and allowed to incubate at room temperature for 10 minutes. This will allow formation of Ag-Ab complex on Ag/Ab spots, if the patient’s serum contains desired Ab/Ag, respectively. Any unbound material flows through towards underlying absorbent material.

As in conventional ELISA, a wash step is followed using dropping a wash buffer over the spots. Next, a conjugate (Antihuman IgG conjugated with collo­idal gold is added. This will cause development of colour spots over the Ag spots if the serum contains corresponding Ab. Control spot contain­ing human IgG will react with the conjugate to form colour spot declaring validity of the test (Figs. 9.20 and 9.21).

The procedure may detect Ag (e.g. P-24 Ag of HIV) if the corresponding monoclonal Ab is spotted on membrane and a second Ab is used to sandwich the Ag between two antibodies.

3. Lateral flow membrane test (Fig. 9.22):

This is an immuno-chromatographic test (rapid) for qualitative detection of Ag (e.g. Ag of P. falciparam) or Ab (eg. HIV-1 or HIV-2-Ab) in serum, plasma or whole blood. The strip consists of a nylon or nitrocellulose membrane, supported on an absor­bent membrane. The first one is impregnated by human IgG (control line) and Ag or Ab in test line.

Principle and Procedure:

(a) Ab Detection (Fig. 9.23):

For Ab detection, corresponding purified Ag is impregnated in test line. In the control line ‘human anti IgG-Ab’ is fixed for establishment of validity of the test. In the absorbent pad, colloidal gold conjugated to Ag and anti-human-IgG is loosely absorbed.

When sample is added to the absorbent pad (which is often arranged to dip in small well con­taining diluted sample), it moves up by capillary action towards the test and control lines. During this flow it dislodges the conjugates and drives towards the reaction lines.

If the sample contains desired antibody, it cross­links the fixed Ag in the test line with the Ag- colloidal gold conjugate producing a coloured line. Similarly, IgG of the serum cross-links at the control line, between fixed anti-human IgG and colloidal gold labelled antihuman-IgG, producing the colo­ured line, proclaiming validity of the test.

A reactive sample produces coloured lines both at test and control lines. A non-reactive result produce coloured control line only. If a test produces colours line at test site only, the result is to be considered as invalid.

(b) Ag detection:

Lateral flow membrane ELISA may be employed for detection of Ag also (eg. Ag of P. falciparum). The test is similar.

The differ­ences are:

(i) at test line, monoclonal antibody against the Ag to be detected is fixed.

(ii) in the absorbent pad, colloidal gold conjuga­ted with monoclonal Ab to the Ag and antihuman globulin is added. Reaction is depicted in Fig. 9.24.

Western Blot Test for HIV-1 Ab (Open Tray Procedure):

Principle:

The western blot test is much more specific than an ELISA, as it detects antibodies to specific proteins of HIV-1. The western blot assay begins with the same virus preparation used in the ELISA, but the individual viral proteins are then fractionated according to molecular size by SDS-PAGE (Sodium Dodecyl Sulphate-Polyacrylamide gel electro­phoresis).

These clearly separated bands are then blotted onto a nitrocellulose sheet which is then cut into strips. These strips are made to react with the ELISA positive serum samples, and if specific antibodies are present to the individual viral proteins, they will bind to them.

This binding is visualised by subsequent reaction to a conjugate and corresponding substrate. The viral proteins produce a banding pattern corresponding to core (p17, p24, p55), polymerase (p31, p51, p66) and envelope (gp41, gp120, gp160) proteins of HIV-1.

The viral glycoproteins produce broader bands at 41,120 and 160 KD.

A serum or a plasma sample that is positive in both the ELISA and the western blot is presumed to have come from a person exposed to HIV-1.

Materials required:

1. HIV-1 western blot strips: Pre-blotted strips with resolved HIV-1 specific viral proteins.

2. Wash buffer (PBS with Triton).

3. Sample diluent (PBS with bovine albumin).

4. Conjugate — Alkaline phosphatase labelled, affinity purified goat anti-human IgG (H+L).

5. Chromogen — Substrate (pNPP) for alkaline phosphatase.

6. Strongly reactive control-1 vial of heated inactivated human serum with a high titre of antibodies to HIV-1 antigen.

7. Weakly reactive control-1 vial of heat inacti­vated human serum with a low titre of anti­bodies to HIV-1 antigen.

8. Non-reactive control-1 vial containing normal human serum non-reactive for HIV-1 antigen.

9. Incubation trays-8 well trays for incubation of pre-blotted nitrocellulose strips.

Procedure (Fig. 9.25):

1. Place one HIV-1 western blot strip in each well of incubation tray.

2. Add 1 ml of sample diluent to each well.

3. Add 10 μl of specimen or control sample to each well.

4. Incubate the tray on a rocker or rotatory platform with gentle shaking for 30 minutes.

5. Aspirate the mixture from the wells into a tray containing disinfectant.

6. Wash for three minutes with gentle shaking with 1 ml/well of wash buffer. Repeat the washing three times.

7. Add 1 ml of conjugate solution and incubate with gentle shaking for 30 minutes at room temper­ature.

8. Aspirate the conjugate and do washing three times with wash buffer.

9. Add 1 ml of chromogen solution and incubate with gentle shaking for 10 minutes at room temper­ature.

10. Aspirate the chromogen and wash the strips two times with water.

11. Air dry the strips (for 30 minutes) completely before reading.

Interpretation of results:

Non-reactive control:

No HIV-1 specific bands should be observed.

Weakly reactive control:

Bands must be present at p24 and gpl20/gpl60 — other bands may or may not be present.

Strongly reactive control:

Bands must be present at p17, p24, p31, gp41, gp51, p66 and gp120/gp160. Other bands may or may not be present.

Remarks:

1. Non-reactive and weakly reactive controls must be run in each assay regardless of the number of samples to be tested. The non-reactive and weakly reactive controls are used to establish criteria for reactivity of bands.

2. In the first run of a kit, the strongly reactive control must be assayed.