Enzyme Linked Immunosorbent Assay (EIA) | Human Genetics

The below mentioned article provides notes on Enzyme Linked Immunosorbent Assay (EIA).

Enzyme immunoassay (EIA) was first described by Engvall and Perlmann (1971, 1972), and by von Weemen and Schuurs (1971, 1972). The technique was derived from the work of Nakane and Pierce (1967) which showed that antibodies could be labelled with enzymes for use in histochemical staining of tissues and that of Catt and Tregear (1967) using solid-phase for radioimmunoassay.

(In enzyme immunoassays, antibodies or antigens are conjugated to enzymes instead of radioac­tive labels in such a manner that the immuno­logical and enzymatic activities of each moiety are maintained.

The degradation of a substrate by the enzyme, measured spectrophotometrically, is proportional to the concentration of the unknown “antibody” or “antigen” in the test solution.

The two fundamentally different systems which have been developed for EIA are:

1. Homogeneous enzyme immunoassay or enzyme multiplied immunoassay technique (EMIT), and

2. Heterogeneous enzyme immunoassay or enzyme linked immunosorbent assay (ELISA).

The general principles of EIA and its spe­cific applications for detecting haptens, anti­gens and antibodies have been described in detail in a number of reviews.

Homogeneous Enzyme Immuno­assay:

The principle of homogeneous enzyme immunoassay is to link a hapten to an enzyme in such a way that the enzyme activity is altered when the hapten combines with the antibody, as illustrated in Fig. 15.1. The assay system consists of a fixed concentration of enzyme-labelled hapten, a fixed concentration of antibody, an enzyme substrate and the test sample.

If the hapten is present in the test sample, it would combine with the antibody leaving the enzyme-labelled hapten free to degrade the substrate. The amount of sub­strate being degraded can be read in a spec­trophotometer. The main applications of EMIT are for detecting drugs, hormones and other low molecular weight substances.

EMIT offers simplicity of protocol and does not require separation and washing steps. However, it has been found difficult to apply this method for the detection and determina­tion of high molecular weight antigens with the degree of sensitivity required.

Moreover, its sensitivity also depends on four other factors:

(a) The detectability of enzymatic activity;

(b) The change of the enzymatic activity upon binding of the antibody to enzyme- hapten conjugate;

(c) The binding affinity for the antigen and antibody and

(d) The susceptibility of the assay to inter fering substances in the test sample, such as endogenous enzymes, proteases, cross- reacting antigens or enzyme inhibitors.

Heterogeneous Enzyme Immuno­assay:

The heterogeneous enzyme immunoassay involves the initial adsorption of an antigen or antibody to an insoluble carrier surface. The relevant antigen or antibody is then incubated in this sensitized carrier. This is followed by the addition of enzyme-labelled antiglobulin. Excess immunoreactants in each step of the procedure described in the above are removed by washing before the addition of the sub­strate.

The concentration of the product from the enzyme reaction is directly proportional to the concentration of antigen or antibody in the test sample. This technique is commonly used for the detection and measurement of large molecular weight proteins, such as carcinofetal proteins, viral, bacterial and parasitic antigens and for the monitoring of immune status. The advantages of using ELISA for detecting antigen and antibody are mainly:

(a) Its convenience;

(b) The labelled immunoreagents are sta­ble for long periods;

(c) The precautions and disposal proce­dures required for radioisotopes are unneces­sary; and

(d) The use of chromogenic substrates for the enzyme labels permits visual interpretation of test results. On the other hand, there may be a loss in the reactivity of the antibody upon its conjugation to the enzyme which limits sub­strate detection.

The most commonly used ELISA methods can be divided into (a) non-competitive and (b) competitive assays.

A. Non-competitive ELISA Technique:

Non-competitive ELISA technique is potentially more sensitive and is more widely used. The technique has several variations:

(i) The direct ELISA method for the detection of antibody;

(ii) The double antibody sandwich method for the detection of antigen; and

(iii) The antibody class capture assay for the detection of class specific immunoglobulin.

B. Competitive ELISA Technique:

Competitive ELISA technique can be employed to detect either the antigen or the antibody. The detection of antigen using this method involves a reaction step in which unlabelled and enzyme-labelled antigen compete for a limited number of antibody binding sites.

The antibody is attached to a solid phase. This assay requires the labelling of a pure antigen preparation which, in some cases, may be difficult (if not impossible) to obtain. In addi­tion, the test sample in contact with the enzyme-labelled antigen may contain enzyme inhibitors, proteases or other interferants which may alter the activity of the enzyme used as label.

Competitive ELISA is most com­monly used for antibody detection. In this type of assay, the antigen is attached to a solid phase and the enzyme-labelled antibody is competitively inhibited by the added standard or test antibody for the immobilized antigen. The product concentration measured at the end of incubation is inversely proportional to the concentration of the standard or antibody present in the test sample.

A rapid, sensitive and quantitive kinetic- based enzyme-linked imunosorbent assay (k-ELISA) using cuvettes or using the Falcon assay screening test (FAST- ELISA) system has also been developed to quantitate antigen and antibody. In this system, the amount of indicator enzyme is measured by assaying the initial substrate-conversion velocities.

However, the principle of each assay system may be summarized as follows:

(a) An antigen or antibody is adsorbed to a solid phase.

(b) The addition of test sample to the “sensitized” solid phase.

(c) The addition of an enzyme-labelled antiglobulin.

(d) The incubation and washing steps.

(e) The addition of the enzyme substrate.

(f) The measurement of the amount of substrate being degraded by the enzyme.

The following is a summary of the essen­tial steps in non-competitive and competitive ELISA methods.

Non-Competitive ELISA:

Indirect system:

a. Adsorb the antibody to the solid phase.

b. Wash.

c. Add sample (containing anti­gen).

d. Wash.

e. Add enzyme-labelled specific antibody.

f. Wash.

g. Add enzyme substrate (o).

h. The product (•) α [Antigen] present in the sample.

Antibody Class Capture Assay:

a. Adsorb class specific antiglobu­lin to the solid phase.

b. Wash.

c. Add sample. (Class specific globulin in the sample attaches to the antiglobulin).

d. Wash.

e. Add antigen which attaches to any specific antibody.

f. Wash.

g. Add enzyme-labelled antibody.

h. Wash.

i. Add enzyme substrate (o).

j. The product (•) α [Antibody] present in the sample.

Double Antibody Sandwich:

a. Adsorb the antigen to the solid phase.

b. Wash.

c. Add sample. Antibody attaches to the antigen.

d. Wash.

e. Add enzyme-labelled antiglobu­lin.

f. Wash.

g. Add enzyme substrate (o).

h. The product (•) α [Antibody] present in the sample.

Competitive ELISA:

Detection of Antigen:

The product [Y] – [X] α [Antigen] present in the sample.

Detection of Antibody:

The product [Y] – [X] a [Antibody] present in the sample.

Practical Aspects of ELISA:

The Solid Phase:

Heterogeneous ELISA requires immobi­lization of either antigen or antibody to a solid phase. This permits the separation of immunologically reacted from unreacted mate­rial during the test. The use of preformed materials, such as tubes, beads, microtiter plates made of polystyrene, polypropylene, polyvinyl and other plastics as carriers have been found to give adequate, reproducible uptake of most antigens and antibodies, mere­ly by passive adsorption.

If a high uptake of the immunological reagents is needed, polyvinyl microtiter plates may be preferred. However, the polyvinyl microtiter plates tend to give higher back­ground, so are less suitable for visually read ELISA tests. It is also possible to covalently couple an antigen or antibody to cellulose, agarose, polyacrylamide, Sepharose beads and porous glass.

Adsorption of the antigen or antibody to nylon and to nitrocellulose membrane has also been adapted for the detection of viruses by the ELISA method.

It has also been reported that pretreatment of microtiter plates with poly-lysine increases adsorption of polysaccharide antigen and the binding of viral antigens.

Pretreatment of polystyrene microtiter plates with protamine sulphate increases the adsorption of DNA. However, it is impractical to analyse a large number of samples using the chemical cou­pling of the antigen or antibody to cellulose, agarose, Sepharose etc., for ELISA methods require washing steps involving centrifugation.

At present the use of specially designed microtiter plates made of polystyrene has a major advantage over other solid phase ELISA methods as the unreacted reagents can be sep­arated easily and several automated microtiter plate readers are available.

The mechanism of adsorption of proteins to plastic surfaces is not completely under­stood. It may be the result of hydrophobic interactions between non-polar protein side chains and the non-polar plastic matrix; van der Waals’ forces may also play a role in the binding. This may explain why different pro­teins do not bind with the same efficiency to the same plastic material.

However, the main disadvantages of using plastic as the solid phase are:

(a) The binding of antigen or antibody to plastic surface is only physically adsorbed, not covalently bound. Thus, desorption of protein may take place during incubation and wash­ing.

(b) Adsorbed proteins may undergo denaturation to some extent with loss of immunological activity. This will lower the accuracy of the assay and probably also affect its sensitivity.

(c) The plastic surface has only a limited capacity for adsorption but nevertheless can be coated easily with protein. The ease and rapidity of separation of unreacted reagents compensate for these drawbacks.

Immobilization of Antibody:

In assays where antibody is to be used for coating to the solid phase, IgG fraction, F(ab)₂ fraction, monoclonal antibodies and whole serum can be employed. The use of whole serum requires an appropriate dilution so as to eliminate non-specific interferants. IgG from a variety of species can be immobilized on sev­eral types of plastics.

The amount of antibody that can be adsorbed to plastic surfaces is not highly dependent on the antibody source, the conditions used for adsorption or the type of plastic used. Quantitative studies have shown that purified IgG binding to plastic surface is linear up to 1 µg/ml. Concentrations of IgG above this are adsorbed to a lesser degree.

IgG concentrations higher than 10 µg/ml do not increase the sensitivity of the immunoassay. This is apparently due to desorption of antibody from the plastic surface, or to steric hindrance caused by closely packed antibody on the plas­tic surface. However, for accurate results, it may be necessary to carry out a chequer board titration on each new antibody.

Immobilization of Antigen:

The types of antigen used for adsorption to the solid phase vary widely. It is not possible to predict which antigen preparation is most suit­able. If only crude mixture of antigens is avail­able, the only practicable coating is to carry out chequer-board titrations against positive and negative sera.

The antigen dilution that gives the best discrimination between the positive and negative sera is used in subsequent tests. If pure antigens are avail­able, the concentration required to produce optimum coating can be determined. It is “also possible to use insoluble antigens, including whole cells, for coating to the plastic surface, although insoluble antigens tend to adsorb less uniformly.

Choice of Coating Conditions:

Since adsorption of the antigen or anti­body to the solid phase is a passive process, the optimum concentrations of reagents for an ELISA should be determined for each coating substance. The optimum concentration of an antigen or antibody for coating is generally between 1 to 10 ug/ml.

Higher concentra­tions of protein used for coating tend to increase desorption during incubation and washing. This may give rise to poor assay reproducibility and reduce assay sensitivity. The coating buffers which have been most fre­quently used are 0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl (PBS) or 0.05 M carbonate buffer, pH 9.6.

The initial adsorption of most proteins to the solid phase at room temperature is rapid and usually complete within 2-3 hours. This is followed by a slower phase and maximum adsorption is attained within 4-6 hours. The conditions and adsorption time for coating antigens and antibodies to the solid phase vary according to their stability.

No general rules exist for highly efficient and reproducible coat­ing. Storage lifetime of coated plastic also varies with the coating proteins. The optimum incubation time and temperature for each new system are best determined by trial and error.

For conve­nience, microtiter plates may be sensitized overnight at 4°C and test sample and conju­gate incubated, for 2 hours each at room tem­perature. However, in many systems, ade­quate sensitization is achieved after 1-2 hours at 37°C and an incubation period of one hour is often satisfactory. Shorter incubation tends to yield less accurate results.

Test Sample:

A great many different samples from serum, faecal extracts, urine, saliva, tumor and plant extracts have been tested by ELISA tech­nique. Samples may be tested either at a single dilution or a series of dilu­tions. In some cases, dilution may be desired as it also serves as a simple method to mini­mize interfering factors presented in the test sample. The lower limit of sensitivity for detecting the sample by ELISA in most cases is approximately 100 pg to 1 ng.

Non-specific Reaction with the Sample:

Because of the complex nature of a vari­ety of samples, non-specific reactions are a frequent problem for enzyme immunoassay. A common cause of false positives is rheumatoid factor (Rh. F) in serum sample. Rh. F is an antiglobulin that recognises the F_c portion of altered immunoglobulins. It is not species specific.

However, this problem can be overcome by the use of F(ab)₂ fragments which do not bind Rh. F in the assay or removal of Rh. F by the addition of aggre­gated IgG to the sample diluent, or the addi­tion of N-acetyl cysteine to the test sample to break down Rh F.

The adsorption of proteins to the solid phase is non-specific. Thus, during the incu­bation of an enzyme conjugate with the immo­bilized antigen and antibody, the enzyme con­jugate may also be adsorbed directly onto the solid phase.

However, this non-specific adsorption of the enzyme-conjugate can be minimized by the addition of a nonionic deter­gent, such as Tween 20 or Triton X-100, or the addition of bovine serum albumin or gelatin to the diluent or washing solution. These treatments do not interfere with the antigen-antibody reaction but prevent or block the non-specific factors binding to the plastic surface.

Choice of Enzymes for Conjugates:

A variety of enzymes has been used as labels for enzyme immunoassay. The choice of enzymes depends on the requirements of a particular assay.

The important criteria for the choice of the enzyme are:

(a) High specific activity;

(b) Excellent stability of the enzyme;

(c) Simple and reproducible procedures for the covalent coupling to antigens or anti­bodies;

(d) High yield and stability of the conju­gates;

(e) Absence of endogenous enzymes and interfering factors in the test sample.

In tests employing labelled antibodies, it is often possible to label the whole immunoglo­bulin fraction with the enzyme. If specificity is to be ensured, it is preferable to use pure anti­body prepared by affinity chromatography. The two most commonly used enzyme labels in enzyme immunoassay techniques are alkaline phosphatase and horseradish peroxidase.

Glutaraldehyde and periodate have been widely used as cross- linkers of enzymes and antigens or antibodies. Glutaraldehyde cross-links two proteins via the epsilon amino groups of lysine by formation of a Schiff’s base. The mechanism for glutaralde­hyde cross-linking involving polymerization products of glutaraldehyde has been proposed by Richards and Knowles (1968).

The use of periodate for coupling two proteins requires the presence of a carbohydrate moiety in one of the protein molecules. The carbohydrate is oxidized by sodium periodate and the resulting aldehyde groups are allowed to link with amino groups of the other protein.

The high affinity constant of biotin binding to avidin also provides a good system for enzyme immunoassay. In this system, a specif­ic antibody is labelled with biotin and enzyme- labelled avidin used as an indicator. Another useful conjugate is pro­tein A, a cell wall component of Staphylo­coccus aureus, which selectively binds to the F_c region of most mammalian IgGs. Since the binding of protein A to antibody does not affect the antigen and antibody reaction, protein A labelled with enzymes is a very useful analytical reagent for quantitation of antigens and antibodies.

Washing of the Solid Phase:

After completion of the immunochemical reaction steps, the removal of unbound or loosely bound reagents from the solid phase is required. The washing procedures are critical and it is important to ensure that all wells of microtiter plates or tubes are treated exactly the same way.

Methods that have been used to remove excess reagents from the solid phase are washing with:

(a) 0.9% NaCl;

(b) 0.9% NaCl containing 1% human serum albumin;

(c) 0.9% NaCl containing 0.05% Tween 20;

(d) 0.015 M phosphate-buffered saline, pH 7.2 containing 0.15% Tween 20; or

(e) Phosphate-buffered saline containing 0.05% Tween. Washing can be carried out by simple decantation or aspiration. Air bubbles must not be trapped in the wells. The plate with all wells filled with -washing solution is left for 2 – 3 minutes, after which the procedure is repeated.

Choice of Substrate:

The choice of substrate is governed by the enzyme label used. Most of the assays found to be clinically useful in diagnosing infectious dis­eases use chromogenic substrates, such as p- nitrophenyl phosphate for alkaline phos­phatase and o-phenylene diamine or 3,3′, 5,5′-tetramethyl benzidine (TMB) for horseradish peroxidase.

TMB is considered to be a better substrate for peroxidase than o- phenylene diamine because its apparent molar extinction coefficient is higher than that of o- phenylene diamine. In addition, TMB has no mutagenic activity and does not appear to be carcino­genic. Fluorogenic and radioactive substrates and chemiluminescence have also been used in enzyme immunoassay to enhance sensitivity.

Reading and Interpretation of Results:

Results can be read either spectrophoto- metrically for quantitative measurements or by eye to determine the presence or absence of colour products as positive or negative results, respectively. If the results are read spec- trophotometrically, then it may be expressed as absorbance values, where values greater than a certain reference value are positive and below which are negative.

There is a direct relationship within a certain range of antigen or antibody concentrations in the serum against the absorbance measured. At high concentrations of antigen or antibody, the dose response curve levels off because the amount of protein coating the solid phase or the amount of enzyme conjugates becomes a limiting factor.

In enzyme immunoassay, a certain back­ground is always obtained with normal sera. This may be attributed to specific or non-specific binding of IgG in normal serum to the coated plastics and probably to other unknown protein-protein interactions as well. In such cases, a range of background has to be estab­lished for each type of assay.

Adequte stan­dardization is the key to high accuracy in enzyme immunoassay. It can only be achieved by employing reference preparations. In tests for antibody, a reference sample consisting of pooled sera from a group of individuals with high antibody titre should be used. Dilutions of this sample and dilutions of a reference nega­tive serum must be included in each assay.

Antigens are often more difficult to standard­ize unless they are available in a purified form. For crude antigens, activity in the test is the most important property and can be expressed in terms of a reference preparation even though this preparation may not itself be pure.

An Example of the Indirect Microtiter Plate ELISA for the Detection of Antibody:

Equipment:

1. Polystyrene microtiter plates

2. Multi-channel micropipette

3. Micropipette

4. Test tubes

5. Humid box

6. Spectrophotometer or microplate reader.

Reagents:

1. Antigen

2. Enzyme-antibody conjugates

3. Test samples

4. Reference positive and negative sam­ples

5. Coating buffer, pH 9.6, consists of 1.59 g Na₂CO₃, 2.93 g NaHCO₃ and 0.2 g NaN₃ in 1 litre of distilled water. The pH is adjusted with 1 M NaOH to 9.6.

6. Washing solution is phosphate buffered saline/Tween (PBS/T), pH 7.4, which contains 8.5 g NaCl, 1.096 g Na₂HPO₄.2H₂O, 0.315 g NaH₂PO₄.H₂O, 0.5 ml Tween 20 and 0.2 g NaN₃ in 1 litre of distilled water. The pH is adjusted to 7.4 with 1 M HCl.

Conjugate Preparation and Purification:

Coupling of alkaline phospha­tase using one-step glutaraldehyde method:

(a) Add 2 mg antiserum (IgG fraction) in 1.0 ml 0.01 M phosphate buffer containing 0.15 M NaCl, pH 7.2, (PBS) and 5 mg alkaline phosphatase (at least 1,000 units/mg protein) and mix at room temperature.

(b) The mixture is dialyzed against 2 litres PBS at 4°C overnight with one change of buffer.

(c) Add 20% glutaraldehyde to yield a final concentration of 0.2% (v/v). Mix well; allow to incubate for 2 hours at room temperature. Dialyze against 2 litres PBS containing 1 mM MgCl₂ at 4°C overnight with 2 changes’ of the same buffer.

(d) Transfer the dialysis sac to 2 litres 0.05 M Tris-HCl, pH 8.0, containing 1 mM MgCl₂. Dialyze extensively with several changes of Tris-HCl, pH 8.0, containing 0.02% sodium azide and 1 mM’ MgCl₂.

(e) Dilute conjugate to 4.0 ml with Tris buffer containing 1.0% bovine serum albumin and 0.02% sodium azide. Store in the dark at 4°C until used. Usually this conjugate can be dilut­ed 1: 500 or more for use.

Coupling horseradish peroxi­dase using periodate method (Wilson and Nakane, 1978):

(a) Dissolve 4 mg horseradish peroxidase in 1 ml water. Add 0.2 ml of freshly prepared 0.1 M sodium periodate. Stir for 20 minutes at room temperature.

(b) Dialyze against 2 litres 0.001 M sodium acetate buffer, pH 4.4, overnight at 4°C.

(c) Add 20 ul 0.2 M carbonate buffer, pH 9.5. Mix well and immediately add 8 mg antiserum (IgG fraction) in 1.0 ml 0.01 M carbo­nate buffer, pH 9.5. Stir for 2 hours at room temperature.

(d) Add 0.1 ml fresh sodium borohydride solution (4 mg/ml water). Allow to stand for 2 hours at 4°C.

(e) Add an equal amount of saturated ammo­nium sulphate solution. Centrifuge. Wash pre­cipitate twice with 50% saturated ammonium sulphate. Dissolve the precipitate with a mini­mum amount of PBS, pH 7.2, and dialyze extensively against the same buffer.

(f) Bovine serum albumin is added to yield a final concentration of 1% (w/v). Add an equal volume of glycerol. Store at 4°C or -20°C. The working strengths of conjugates are deter­mined by testing dilutions of the conjugates in wells of a microtiter plate coated with 100 ng/L of IgG.

In an indirect microtiter plate ELISA, the conjugate dilution to be used is determined by the appropriate concentration of antigen, with reference positive and nega­tive sera. The purpose of conjugate dilution is that a clear distinction between positive and background (negative) a value is obtained after 30 minute incubation with the substrate at room temperature.

Substrates:

For Alkaline Phosphatase Conju­gates:

Diethanolamine buffer, pH 9.8, consists of 97 ml of diethanolamine, 800 ml of water, 0.2 g of NaN₃ and 0.1 g of MgCl₂.6H₂O. Adjust the pH to 9.8 with 1 M HCl and bring the volume to 1 litre with distilled water.

Immediately before use, dissolve 1 mg/ml of p-nitrophenyl phosphate in the above buffer at room temperature. It must be used on the same day. Keep the solution away from light.

Reaction stopping solution: 3 M NaOH.

Read absorbance of the test samples and standards at 405 nm.

For Peroxidase Conjugates:

Prepare a phosphate-citrate buffer, pH 5.0, by mixing 24.3 ml 0.1 M citric acid, 25.7 ml 0.2 M Na₂HPO₄ and 50 ml water. Adjust pH to 5.0.

Immediately before use, dissolve 40 mg o-phenylenediamine in 100 ml of the above buffer and add 40 ul 30% hydrogen peroxide. Alternatively, 3.3’m 5.5′-tetramethyl benzi­dine (TMB) may be used as a substrate for per­oxidase.

The working solution is prepared by the addition of 1 ml of TMB (5 mg previously dissolved in 1 ml dimethyl sulfoxide) and 8 ul 30% hydrogen peroxide to 49 ml 0.1 M sodi­um acetate/acetic acid, pH 5.5. Both o- phenylenediamine and TMB are light sensitive and must be used at once. Sodium azide should not be used in solutions when peroxi­dase is used as an enzyme, since it inhibits the peroxidase reaction.

Reaction stopping solution: 2.5 M H₂SO₄ or 2.5 M HCl.

Read absorbance of test samples and stan­dards at 492 nm for o-phenylenediamine or 630 nm for TMB.

Procedure:

1. Coating: Apply 100 ul antigens (1-10 ug/ml) in coating buffer to each well of a polystyrene microtiter plate except wells A-l and B-l which are used as blanks. Incubate at room temperature for 3 hours. The plate may then be stored at 4°C for several days before use. Plate should be covered during incubation and storage.

2. Prior to innoculation of test samples, wash the plate 3 times with 200 ul PBS/T. The washing solution should sit in the wells for 2-3 minutes during each wash.

3. Add 100 ul of test samples diluted with PBS/T. Incubate for 2 hours at room temperature.

4. Wash the plate 3 times with PBS/T after incubation.

5. Add 100 ul of enzyme-labelled antiglobu­lin diluted in PBS/T. Incubate for 2 hours at room temperature.

6. Discard the solution when the incubation is over and wash 3 times with PBS/T.

7. Add 100 ul of enzyme substrate solution into each well of the plate and incubate at room temperature until the color is visible. Avoid strong light during the incubation period.

8. Stop the reaction by adding 50 ul of stop­ping solution to each well.

9. Read the absorbance of the contents of each well.

Conventional Chromosome Banding Technique:

The history of human cytogenetics over the past 35 years has been punctuated by the introduction of new technology (described pre­viously) followed by the identifications of increasing number of aberrations, smaller in extent and often associated with less striking phenotypic changes.

For identification of nor­mal chromosomes and detection of aberra­tions, the air dried chromosome preparations must be stained appropriately. The rapid development of in situ hybridization for the identification of specific chromosomes and chromosome region should not be allowed to overshadow the usefulness of conventional staining techniques, which continue to form the basis of modern cytogenetic analysis.

Conventional banding is comparatively simple to achieve and is inexpensive in relation to the in situ hybridization methods. It is capable of unequivocally identifying every normal human chromosome and the origin of most of the larger chromosome rearrangement. Its perfor­mance in specificity for the identification of small chromosome segments is, however, poor; therefore it should be used in conjunc­tion with in situ methods.

When we use Cytogenetic analysis?

Following are the general indications:

i) Confirmation or exclusion of the diag­nosis for known chromosomal syn­drome.

ii) Unexplained physchomotor retarda­tion with or without dysmorphic fea­tures.

iii) Abnormalities of sexual differentiation and development.

iv) Infertility.

v) Monogenic disorders associated with mental retardation and or dysmorphic features.

vi) Recurrent miscarriage or stillbirth.

vii) Pregnancies shown to be at risk of aneuploidy from the results of mater­nal serum screening or fetal ultra­sound scanning.

viii) Neoplastic conditions, particularly hematologic malignancies, for which the identification of specific chromo­somal aberrations may be valuable in diagnosis and management.

Methodology for Staining:

1. Solid Staining by Giemsa:

When the chromosomes are exposed to a Giemsa or similar stain that has an affinity for DNA, yielding a uniformly dark appearance to the chromatids. This technique has applications in the investigations of fragile sites, in the study of chromosome breakage syndromes and for scoring radiation damage, in the determina­tion of the extent of satellite polymorphism and in the measurement of individual chromo­somes.

Protocol:

Reagents:

(i) Giemsa stain

(ii) pH 6.8 buffer.

Methods:

(i) Add 5 ml of Giemsa stain to 45 ml of buffer in a staining jar

(ii) Place slides in stain for 5 minutes

(iii) Rinse slides in distilled water

(iv) Dry, mount and examine.

2. G-banding:

It is benchmark for the routine examinations of human chromo­somes, producing a characteristics light and dark banding pattern along the chromosomes. Each chromosome has a unique sequence of bar code-like stripes, allowing identification of individual homologous and the analysis of abnormalities of their structures by disruption of the normal banding pattern.

Protocol:

(i) Proteolytic digestion of the chromosome by trypsin enzyme

(ii) Stain the chromosomes with either Giemsa or Leishman stain.

[The dark band contains A-T rich DNA and light band contain G-C rich DNA.]

Reagents:

(i) Trypsin

(ii) Leishman stain

(iii) Phosphate buffered saline

(iv) Hank’s balanced salt solu­tions (2x)

(v) Buffer pH 6.8

(vi) Saline solution, 0.85% NaCl

Methods:

(i) Place Leishman stain in a staining jar (10 ml + 40 ml pH 6.8 buffer)

(ii) Place freshly prepared 0.05% trypsin solution in phosphate buffered saline in a staining jar

(iii) Place Hank’s balanced salt solutions (2x) in a staining jar

(iv) Age slides at 60°C overnight

(v) Place slides in Hank’s solu­tion for 5 minutes

(vi) Place slides in distilled water

(vii) Place slides in trypsin solu­tion for 5-20 seconds

(viii) Rinse slides in saline solu­tion

(ix) Stain slides in Leishman stain for 3-5 minutes

(x) Rinse slides in distilled water

(xi) Dry, mount and examine.

3. Reverse Banding:

A disadvantage of G-banding is in the definition of the light staining telomeric regions. Therefore R-banding resulting in the reverse of the banding pat­tern to that seen in the G-banding.

Protocol:

Reagents:

(i) Acridine orange

(ii) Phosphate buffer, pH 6.5

Methods:

(i) Place 50 ml of pH 6.5 phosphate buffer in a staining jar

(ii) Place 0.01% acridine orange solution in pH 6.5 phosphate buffer in a staining jar

(iii) Incubate slides in phos­phate buffer at 85°C for 5-10 minutes

(iv) Place slides in acridine orange solution at room temperature for 5 minutes

(v) Rinse slides in pH 6.5 phosphate buffer at room temperature

(vi) Mount in buffer and exam­ine under fluorescence microscopy and appropri­ate excitation and barrier filters.

4. Replication Banding:

This is sim­ply the modification of R-banding and it gives differential replication staining by exposing growing cells to Bromodeoxyuridine at differ­ent stages of the cell cycle. This method is generally used for identification of late repli­cating X-chromosome and structurally abnor­mal X-chromosomes.

Protocol:

Reagents:

(i) Bromodeoxyuridine

(ii) Hoechst 33258 stain

(iii) Giemsa stain

(iv) Phosphate buffer, 6.8 pH.

Methods:

(i) Prepare standard lympho­cyte culture (by using stan­dard tissue culture tech­nique)

(ii) Six hours before harvest, add Bromodeoxyuridine to a concentration of 1 × 10^-4 M

(iii) Harvest according to the standard protocol

(iv) Place slides in Hoechst 33258 stain (1 µg/ml in pH 6.8 buffer) under a mercury vapour lamp for 30 minutes

(v) Rinse slides well in distilled water

(vi) Place slides in 5% Giemsa stain in pH 6.8 buffer for 3-5 minutes

(vii) Rinse slides well in distilled water

(viii) Dry, mount and examine.

5. Q-banding:

This is actually quinacrine mustard banding gives a rather unclear indistinct bright and dull fluorescence along the chromosomes when exposed to uv light.

Protocol:

Reagents:

(i) Quinacrine dihydrochloride

(ii) Methanol.

Methods:

(i) Prepare fresh stian (2.5 gm quinacrine dihydro- chloride in 50 ml metha­nol) in a staining jar

(ii) Place slides in stain for 3-5 minutes

(iii) Rinse slides well in distilled water

(iv) Mount in distilled water, and examine using fluores­cence microscopy and appropriate excitation and barrier filters.

6. C-banding:

Or constitutive heterochromatin banding. Constitutive heterochromatin is found at the centromeric region of all the human chromosomes. The chromatin in this region has an increased proportion of highly repetitive satellite DNA compared with euchromatin regions and is more resistant to denaturation processes. This method can use­fully be employed as heritable chromosomes markers.

Protocol:

Reagents:

(i) Leishman stain

(ii) Hydrochloric acid (HC1)

(iii) Barium hydroxide

(iv) Saline sodium citrate

(v) Buffer, pH 6.8

Methods:

(i) Place Leishman stain (10 ml + 40 ml pH 6.8 buffer) in a staining jar

(ii) Place distilled water in a staining jar (4x)

(iii) Pretreat slides in 0.2 M hydrochloric acid for 2-5 minutes at room tempera­ture

(iv) Rinse slides well in distilled water

(v) Incubate slides in 5% bari­um hydroxide at 60°C for 2-5 minutes

(vi) Rinse slides well in distilled water

(vii) Incubate slides in 2x in saline sodium citrate at 60°C for 2-5 minutes

(viii) Rinse slides well in distilled water

(ix) Stain slides in Leishman stain for 3-5 minutes

(x) Rinse slides in distilled water

(xi) Dry, mount and examine.

7. Nucleolar Organizer Regions (NOR) Staining or Ag-NOR Staining:

The nucleolar organising regions of acrocen­tric chromosomes can be preferentially stained by the application of silver nitrate solution. This staining pattern allows identification of acrocentric derived chromosomes and also for the identification of bisatellited marker chro­mosomes. It has also been use in determining the parental origin of the extra chromosome in non-disjunctional human trisomies of the acrocentric chromosomes.

Protocol:

Reagents:

(i) Leishman stain

(ii) Silver nitrate solution (50%)

(iii) Gelatin solution.

Methods:

(i) Place Leishman stain (10 ml + 40 ml pH 6.8 buffer) in a staining jar

(ii) Place gelatine solution (2 gm gelatin in 99 ml of water), heat until dissolved, and then add 1 ml of formic acid

(iii) Place 50% AgNO₃ in dis­tilled water

(iv) Place 4 drops of AgN0₃ solution onto the slide, add 1 drop of gelatin solu­tion, and cover with a cover slip

(v) Incubate the slide at 60°C for 3 minutes

(vi) Remove the cover slip, and rinse it well in distilled water

(vii) Place the slide in Leishman stain for 2 min­utes

(viii) Rinse the slide well in dis­tilled water

(ix) Dry, mount and examine.