RIA AND ELISA

RIA AND ELISA

RADIOIMMUNO ASSAY(RIA)

1. INTRODUCTION:

• Radioimmunologicassay techniques are superior to most analytical procedures with regard to sensitivity, precision, general applicability, and experimental simplicity.
• Both for diagnosis and for monitoring of therapy this method has greatly advanced our understanding of endocrine physiology.
• Besides its use in clinical chemistry, radioimmunoassay is also employed in pharmacology, toxicology, and in pharmacokinetics of new drugs.
• RIA  is very sensitiveinvitro assay technique  used to measure the concentration of  antigen  by use of antibodies.
• It can be seen as the reverse of a radio binding assay,  which quantifies an antibody by the use of corresponding antigens.
• It requires special precautions and licensing, since radioactive substances are used.It has been  supplanted by ELISA, where the antigen antibody rections is measured by using colorimetric signals instead of radioactive signals.
• The RAST test (Radio allegosorbent test) is an example of RIA . It is to detect the causative allergen of an allergy.

2. History:

• It was developed in the 1950s by Rosalyn Yalow and Solomon Aaron Berson working at the Bronx Veterans Administration Hospital affiliated with the Mount Sinai School of Medicine.[1] 
• In 1977, Rosalyn Sussman Yalow received the Nobel Prize in Medicine for the development of the RIA for insulin: the precise measurement of minute amount of such a hormone revolutionized the field of endocrinology.
• ·         By allowing the precise measurement of blood levels of hormones the mechanism of hormone deficiency diseases could be identified, and better treated.[2]
• Yalow and Berson refused to patent the assay, because they felt that it should be freely available to the field of medicine.[2] 
• Berson would have shared the prize, but he had died before the award was given.
• With this technique, separating bound from unbound antigen is crucial.
• Initially, the method of separation employed was the use of a second "anti-antibody" directed against the first for precipitation and centrifugation.
• Use of charcoal suspension for precipitation was extended but replaced later by Drs. Werner and Acebedo at Columbia University for RIA of T3 and T4.[3] An ultramicro RIA for human TSH was published in BBRC (1975) by Drs. Acebedo, Hayek et al.[4]

3.  SALIENT FEATURES OF RADIOIMMUNO ASSAY:

• They belong to a class of extremely sensitive methods of analysis.
• Sample required for assay is usually very small eg: 1ml of serum.
• Concentration upto the nano gram rangei.e.,10-9g can  be measured accurately.
• A large number of harmones and drugs which find their abundant usage in a bad way,namely: cortisol, insulin, morphine, barbiturates.
• Incidence of interferences observed in the RIA are fairly insignificant by virtue of the highly specific hapten  - antibody complexation reaction .
• Exception do occur when two 5-substituted  barbiturates present  together  cannot be  assayed  by this method , obviously   due to  interference.

4. THEORY:  

4.1 PRINCIPLE:

• Basic  principle  of  RIA  utilizes the reaction  between  an antigen  {hapten}  and its  specific  antibody.
• Now , a micromolecule { hapten}  drug serves as hapten , can be normally  made  antigenic  by  coupling  them chemically  to macromolecular  substance.
• The hapten that used for coupling is obtained from the non antigenic  compound   (eg: morphine,cortelol)  which  conjugate covaently to a carrier molecule to render it antigenic

4.2 IMPORTANT  TERMS:

4.2.1 SPECIFICITY:

 It is lowest concentration  of a compound  that can be  detected  in undiluted                    body  fluids  .Generally  known as”cut off  level” / “detection  limit”. 

 4.2.2 SENSITIVITY:

          It is defined as –“ the degree  to which an assay  can  distinguish  1 compound  from  another  compound of same  nature”.

4.2.3 AFFINITY:

           Affinity is de defined  as –“ strong  bond  between  antigen  and  antibody”.

5.  METHOD:

• Mix a fixed volume  (fixed concentration) of antiserum  containing  the specific antibody with  a constant  amount of  radiolabelled  antigen ,
• Incubate it  for   specified duration at  an appropriate  temperature  usually +4 C.
• A definite volume of the sample containing  the hapten to be measured  is added to the  reaction  test tube.
• The antibody reacts with both the radioactive and unlabeled hapten forming an  antibody  radiolabelled antigen and antibody unlabelled   antigen complexes.
• Since , both the radioactive  and  non radioactive  antigens (haptens)  are  more  or  less chemically and immunochemically same , they will eventually  compete  for the limited number of antibody sites available;  thus , the amount of  radioactivity that ultimately combines  with the antibody  will  be an inverse function of the amount of unlabelledhapten competing for these  sites
• The radioactivity falls because the unlabeled  antigen dilutes it  i.e., reducing the number of labeled  hapten combining with the antibody.
• The counts obtained from the radioactivity are used to determine the hapten concentration in the sample,the interpretation being done on the standard curve and
• RIA is anexquisitely  is  an sensitive assay method  that is capable of measuring with great accuracy  (hapten) concentrations  in nanograms  and pictograms  utilizing very small volumes of the sample.

5.1  ANALYSIS  BY COMPETITIVE   ANTIBODY  BINDING  OR ISOTOPICALLY   LABELLED  COMPOUNDS:

                 RIA is nothing but a competitive binding assay  with an principle of binding of labeled antigen to its specific antibody(Ab)  and ability of unlabeled antigen not only to compete in the reaction but also to displace the labelledantigen from antibody.

• Hence a labelled  antigen and antibody are present  in limiting factors & the concentrationsof the unlabeled antigens are increasing continuously.
• Thus  the % of the antibody-bound labeled antigen  declines progressively as the concentration of unlabelled antigens increasinesi.e.,as a consequence of saturation of the combining site on the antibody molecule.

DIAGRAMATIC REPRESENTATION:

  

• Ø Inorder to fulfill  the requirements of an ideal behaviour the following are essential:
  • Non –radioactive antigen (A) & radioactive antigen(A*).
  • Equilibrium constants of the binding of the labeled & unlabeled antign to antibody.
  • Antigen & antibody usually react in 1:1 ratio.
  • There are no cross reactions observed in the medium i.e., the antibody is specific only for the single antigen indicated in the reaction.
  • The main aimof RIA is to determine the concentration of unlabeled antigens
  • Before conducting RIA astandard curve should be plotted where the concentration of non labelled antigen in standard solution as a function of radioactivity
  • Saturation of antibody binding sites with radioactive (labelledantigens)adding knownconcentraton of hapten (unlabelled)in standard solution to the reaction mixture of unlabelled antigen from its bindig site on the antibody.
  • Hence a graph is plotted against (x-axis)concentration of unlabelled antigen and radioactivity(y-axis).Thus a close response curve is obtained.

GRAPH:

  

• If a labeled antigen form a substrate A is added to a plasma containing un labeled substrates(A)and a limited number of bindig sites(p).Then an dynamic equilibrium exist between(A)and(P).Thus the A*shall distribute itself evenly among A(unlabeled) 

• If the affinity between AandP are very high then A*will be added untilP is saturated at equilibrium.

(A-P)  +  (A*-P)

Total(A+A*)

Where,

(A-P) is antibody  labeled antigen- complex

(A*-P) is antibody unlabeled antigen –complex

• As the number of A are added ,it compete with the binding  sites  of (A*-P) & thus  reduce their concentration.
• On further  addition  it will cause the ( A*-P)concentration to reduce gradually.
• The reduction in (A*-P) complex can be predicted by  following  formula:

(A-P + A*-P)    ×100

Total(A+A*)      

GRAPH:

6. INSTRUMENTATION:

RIA

CENTRIFUGE

SCINTILLATION COUNTERS

GAMMA  COUNTers;

6.1 CENTRIFUGE:

• It is capable of  generating  1200-2500 rpm in swing  bucket  rotor  (or)  3500- 4000 rpm  using  a fixed  angled  head  rotor  are a employed for process. 
• But, swing bucket rotor  is for mostly  preferred  because the pellets are formed here, at the bottom of the test tube and supernatant layer is more easily removed .
• If the centrifuge have less gravitational force then the centrifugation time should be increased until suitable pellets are formed.

6.2 RADIOACTIVE COUNTERS:

These are of  2 types :

• Ø Gamma counters
• Ø Scintillation counters         Used based upon the type of radio isotope.

6.2. 1GAMMA COUNTERS:

• Used mostly for gamma emitting isotopes.

Eg: I125most commonly used.

6.2.2 SCINTILLATION COUNTERS:

• Used mostly for counting β energy  emitting isotopes.
• Eg:1)tritinium(3H)

               2)Carbon(14C )isotopes

• These are of 2 types 1) Fluorescent scintillation counters

  2) Liquid scintillation counters

6.2.2.1 FLUORESCENTSCINTILLATION COUNTERS:

• In this process, the absorbed energy produces flash of light. When a particle passes through the material it collides with atomic electrons, exciting them to higher energy levels.
• After a very short time the electrons fall back to their natural levels causing emission light.

Types:  a) sodium iodide crystal scintillation detectors(γ counters)

                       b) organic liquid scintillation detectors (β counters)

a)sodium iodide crystal scintillation detectors:

It commonly occurs as a well detector which has a hole drill in the end of a cylindrical crystal to accept a test tube.

• As it is hygroscopic, the crystal is sealed in an Al can with a transparent Quartz window at one end through which blue-violet (420nm) scintillators are detected.
• The photos of gamma emitters in the sample easily penetrate the specimen tube and are thin, low density can and enter the crystal where they are absorbed in the thick, high density sodium Iodide.
• A well counter is not suitable for measuring β radiation, as it can not penetrate the sample container or Al lining of the wall.
• The crystal is usually a circular cylinder machined from a single crystal of sodium iodide to which a small amount of Thallium is added to improve performance.
• The high atomic number of iodine and high density of sodium iodide(3.7gm/cm3) favours the absorption of gamma radiation. For this reason a well counter is always referred to as gamma counter.
• For a typical well detector, the counting efficiency for I125 is approximately 70%.
• organic liquid scintillation detectors: organic liquid scintillation detector measures radio activity by recording scintillations occurring with in a transparent vial that contains the unknown samples and liquid scintillators. Because the radio nuclide is mixedwith the liquid scintillator the technique is ideal for pure β emitters.
• Countingefficiency range from between 60% for H3 to 90% for  C14.
• The liquid scintillator is known as the scintillation cocktail and contains 2 components, the primary solvent and the primary scintillator.
• The primary solvent is usually  inexpensive and is chosn for its efficiency in absorbing and transferring  radiation energy.It is usually one of the aromatic hydrocarbon :Toulene or xylene.
• The primary scintillator absorbs the energy from the primary solvent and converts  into light.
• A common primary scintillator is 2,5-diphenyloxazole which emits UV light of 380 nm.
• The other components can be added to liquid scintillator as a secondary solvent to improve solubility or  surfactant  to stabilize or emulsify the sample.
• A secondary scintillator may be added to absorb the ultraviolet photons of the primary scintillator and reemit the energy at a longer wavelength.

b) LIQUID SCINTILLATION COUNTER:

• Most disadvantageous technique.

6.3  DISADVANTAGES:

1)    Non specificity of the technique

2)    Non sensitivity of the method

3)    Involvement of the process of extraction, purification and concentration of the specimen under investigation

4)    Heat treatment of the specimen resulted invariably in degradation and destruction of the substances

5)    Many processes involved ultimately make the analysis rigorous and un necessarily sluggish

6)    Usage of liquid scintillation technique, hence the gamma counters are comparatively simpler technique.

                        Eg:     131I, 124I, 125

6.4 Radioimmunoassay and  Related Procedures in Medicine:

• Radioassay procedures for the measurement of substances such as hormones, vitamins and drugs in the body fluids and tissues, above all in the blood, are now  the  front rank of medical applications of radioactive materials.
• These procedures, which are carried out on specimens in the medical laboratory and do not involve the administration of any radioactive material  to the patient, are now widely employed in the routine diagnosis and investigation of disease, whilst their use in research has led  to important advances in many branches of medicine.
• Typical of radioassay methods is radioimmunoassay, which depends on the antigen-antibodyreaction between the substance  to be measured and the antibodies in an antiserum against that substance produced in a guinea-pig, rabbit, sheep or other animal.
• The importance of radioimmunoassay was recently underlined by the award of the Nobel Prize in medicine for1977  to Dr. Rosalyn Yalow of the United States of America  for her pioneer work on the method over the past  two decades, particularly in relation  to the measurement of protein hormones.
• This symposium was the  t h i rd on the subject  to have been sponsored by the IAEA. The first  took place in Vienna in 1969 and the second in Istanbul in 1973. During the four years  from 1973  to 1977, the growing commercial availability of reagents and kits for radioassays brought them into routine use.
• This in turn led  to an increasing awareness of the need  for assay standardization and quality control and  to an increasing attention  to techniques of assay data analysis.
• The burgeoning demands made on assay services stimulated interest in the possibilities  for automation of assay procedures.
• Promising new methods were developed, notably solid-phase radioassay and radioreceptor assay.
• At the same time there was a resurgence of interest in alternative assay methods not based on the use of radioactive materials, which made a critical re-examination of the entire subject desirable.
• The symposium provided opportunities for an exchange of information of all of these topics.
• The introductory lecture by Dr. W.D. Odell of the USA, entitled "We Don't Look at Hormones the Way We Used  t o ", drew attention to the very great conceptual changes that have arisen in relation  to hormones in recent years, largely as a result of the applications of radioassay procedures in endocrinology.
• Dr. Odell emphasized that bioassay, radioimmunoassay, radioreceptor assay and the consideration of molecular structure provide four distinct approaches  to the quantification of hormones and that it is not  to be expected that these approaches will yield comparable results.
• However, the results taken together may permit an understanding of disease not previously possible.

6.5            APPLICATIONS:

1)    The technique of  RIA has revolutionized research and clinical practice in many areas: 

• Blood banking
• Diagnosis of allergies
• Endocrinology

2)RIA is also used in non clinical areas such as

                                       a)Food safety

                                        b)Environmental monitoring

3) RIA is used in the assays of

             Plasma levels of: 1)Harmones

                                         2)Digitoxin or digoxin I patients receiving these drugs

3) For the presence of  hepatitis B surface  antigen (HBsAg) in donated blood;

4) Anti-DNA antibodies in systemic  lupuserythromatus.(SLE).

6.6RIA OF MORPHINE:

6.6.1Synthesis of Immunogen:

• The morphine is first converted to  3-o-carboxymethyl-morphine by reacting the  free base with sodium –p-chloroacetate in absolute ethanol.
• The product II is coupled to bovin-serum albumin by dissolving the former in and
• distilled water containing the latter, maintaining the pH of resulting mixture to 5.5
• 1-ethyl-3-(3-dimethylamino propyl)carbidimide was added.
• Incubate over night at room temperature and dialyzed against distilled water for purification.
• The resulting product carboxy-methyl-bovine-serum conjugate is then labeled with tritium.

6.6.2ANTISERUM  PRODUCTION:

• The immunogen, carboxy-methyl-bovine-serum is emulsified with freunds adjuvant.
• Initial immunization doses are  injected  into albino rabbits.
• Later the booster doses are injected after the period of  6 weeks.
• The antiserum titre is determined with each booster dose injection and is duly harvested when the titre value is maximum.
• This is diluted suitably and employed in RIA.

6.7 RIA OF INSULIN:

• The RIA in insulin is based upon  thecompetiotion between unlabeled insulin in standard solution  and radio iodinated insulin for the limited binding sites for specific antibody .
• At the end of incubated period the soluble insulin –antibody complex is separated from free insulin using  dextran coated charcoal suspension.
• By measuring the proportion of radio iodinated insulin bound to the antibody in the presence of varying amounts o reference standard solution of insulin, the concentraton of insulin, unknown sample is interpolated.

Graph:

6.7.1 REAGENTS REQUIRED:

            1.Phosphate buffer pH-7.4

            2.Albumin –phosphate-EDTA-buffer

            3.Human insulin standard working solution

            4.Anti insulin serum

            5.I125-radio iodinated insulin.

            6 .Insulin free serum.

            7. Dextran coated charcoal.

6.7.2 PREPARATION OF STANDARD CURVE:

• A set of 18 tubes are selected  and divide them into 9groups containing two in each group.
• Different volumes of  albumin-phosphate-EDTA buffer is added to the tubes to get different concentrations of the standard.
• Working human  insulin standard solution is added to  the tubes in the required volumes.
• The contents are mixed thoroughly and  gently to avoid foaming.
  • ml of Anti insulin serum is added to the  tubes 3-18.and contents are mixed gently( tubes I & 2are blank).
  • Incubate in refrigerator at 2-4 0 for one hour.
  • 0.1 ml of  I125 radioiodinated insulin is added to tubes 1-18,mix gently incubate in refrigerator 2-4 for   3-5 hr.
  • 0.1ml of insulin free serum is added to 1-18 tubes.
  • 1 ml of dextran coated charcoal suspension is added to all the tubes and mixed for the uniform suspension of charcoal  and maintained at room temperature for 10 min.

• Tubes are centrifuged at 3000-3500 rpm for 30 min at room temperature.
• Clear supernatant liquid is separated and counted for radioactivity for 1-5 min.    

ENZYME - LINKED IMMUNOSORBENT ASSAY (ELISA)

1.INTRODUCTION TO ELISA:

• ELISA, or enzyme-linked immunosorbentassay,arequantitative immunological procedures in which the Ag-Abreaction is monitored by enzyme measurements.
• The term ELISA was first used by Engvalland Peterperlmann in 1971.
• The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity
• ELISA is the abbreviation of  ENZYME-LINKEDIMMUNOSORBENT ASSAY
• It is useful & powerfulmethod in estimating ng/mLtopg/mLordered materialsin the solution .

2.HISTORY:

• Before the development of ELISA the only option for conducting an immunoassay was radioimmuno assay (RIA); a technique using radioactively labeled antigens / antibodies.
• Because, radioactivity posses a potential health threat safer alternative  wassoughted.
• In 1971, Peterperlmannand Engvallat Stockholm university in Sweden and AntonschuursandBauke van weemen in the Netherlands; independently published papers about the methods to perform ELISA in which non –radioactive signals are used.

3.DEFINITION:

          ELISA is a biochemical technique used mainly in immunology to detect the presence of an Ab/ antigen in a sample.

4.BASIC PRINCIPLE OF ELISA:

• Performing an ELISA involves atleast one Ab with specificity for a  particular Ag .
• The sample with an unknown amount of Ag is immobilized on a solid support .(usually on an polyterenmicrotitter plate) either nonspecifically or specifically.
• After Ag is immobilized the detector Ab is added forming a complex with the Ag.
• The detected Ab can be covalently linked to an enzyme or can itself be detected by a 2 0 Ab which is linked to an enzyme through biconjugation between each step the plate is typically with a mild detergent solution to remove any proteins are Ab that are not specifically bound.
• After final wash step the plate is developed by adding  an enzymatic substance to produce a visual signal which indicates the quantity of Ag in the sample.

antibody

   

antigen

   

5.ANTIGEN:   

Any molecule that induces production of antibodies when introduced in the body of an animal is calledantigen         (OR)      

any thing ´, foreign to the immune system.

e.g. bacteria, viruses, (or their parts), pollen, etc. 

carbohydrates

proteins

6.ANTIBODY ( Ab):

 Proteins produced by the immunesystem which help defend against antigens

  

7.MATERIALS  NEEDED:

1)Testing sample,                      2)Antibody (1st, 2nd) / Antigen

3)Polystyrenemicrotiterplate     4)Blocking buffer

5)Washing buffer                        6)Substrate                    7)Enzyme

8.SPECIMEN SAMPLES FOR ELISA:

1. Urine                                  3)semen
2. supernatant culture             4)stool

 DIAGRAMATIC REPRESENTATION OF METHOD OF ELISA :

  

9.TYPES OF ELISA:

                    ELISA

INDIRECT ELISA METHOD

SANDWICH METHOD

COMPETITIVE METHOD

REVERSE METHOD

DIRECT ASSAY

INDIRECT ASSAY

9.1 INDIRECT ELISA METHOD:

The steps of the general, "indirect," ELISA for determining serum antibody

concentrations are:

• Apply a sample of known antigen of known concentration to a surface, often the well of a microtiter plate.
• The antigen is fixed to the surface to render it immobile.
• Simple adsorption of the protein to the plastic surface is usually sufficient.
• These samples of known antigen concentrations will constitute a standard curve used to calculate antigen concentrations of unknown samples.
• Note that the antigen itself may be an antibody.

•  A concentrated solution of non –interacting protein, such as bovine serum albumin(BSA) or casein, is added to all plate wells. This step is known as blocking, because the serum proteins block nonspecific adsorption of other proteins to the plate.

• The plate wells or other surface are then coated with serum samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards .Since antigen immobilization in this step is due to nonspecific adsorption, it is important for the total protein concentration to be similar to that of the antigen standards.

• The plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins.

• Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. These secondary antibodies are conjugated to the substrate specific.

• Wash the plate, so that excess unbound enzyme antibody conjugates are removed.

• Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal.

• View/quantify the result using a spectrophotometer, spectrofluorometer, or other optical/electrochemical device.

9.1.1.ADVANTAGES:

1. Versatile ,since many 20 Ab are available commercially.

1. Immunoreactivity of 10Ab is not affected by labeling.

1. Sensitivity is increased because, each 10 Ab contain several epitopes. That can be bound by the labeled 20Ab allowing signal amplification.

9.1.2.DISADVANTAGES:

1. The method of antigen immobilization is non-specific.

1. Cross reactivity may occur.

9.2 SANDWICH ELISA:

• A less common variant of this technique, called "sandwich" ELISA, is used to detect  sample antigen.

• The steps are as follows

• Prepare a surface to which a known quantity of capture antibody is bound.
• Block any non- specific binding sites on the surface.
• Apply the antigen containing sample to the plate.
• Wash the plate, so that unbound antigen is removed.
• Apply primary antibodies that bind specifically to the antigen.
• Apply enzyme linked secondary antibodies which are specific to the        primary antibodies.
• Wash the plate, so that the unbound antibody enzyme conjugates are removed.
• Apply a chemical which is converted by the enzyme into a color or fluorescent or
• electrochemical signal.
• Measure the absorbance or fluorescence or electrochemical signal (e.g., current)
• of the plate wells to determine the presence and quantity of antigen

9.2.1. ADVANTAGES:

1. The method of Ag immobilization is specific
2. Quantity of Ag immobilized is improved.

9.2.2. DISADVANTAGES:

• This method is expensive.

9.3 COMPETITIVEELISA METHOD:

• A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:

• Unlabeled antibody is incubated in the presence of its antigen.
• These bound antibody/antigen complexes are then added to an antigen coated well.
• The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence competition.")
• The secondary antibody, specific to the primary antibody is added.
• This second antibody is coupled to the enzyme.
• A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
• For competitive ELISA, the higher the original antigen concentration, the weaker the Eventual signal.

(Note: that some competitive ELISA kits include enzymelinked antigen rather than enzyme linked antibody. The labeled antigen competes for primary antibody binding

sites with your sample antigen (unlabeled). The more antigen in the sample, the less

labeled antigen is retained in the well and the weaker the signal)

9.3.1. ADVANTAGES:

• Ability to use crude or impure samples and it can still selectively bind any Ag that may be present.

9.3.2. DISADVANTAGES:

• Higher the original Ag concentration, weaker will be the eventual signal.
•  

9.4 REVERSE ELISA:

• This technique utilizes solid phases made up of an immunosorbent polysterene with     4-12 protruding ogives.
• The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogenous) are carried out by dipping in the ogives in microwells of standard micro plates pre filled with reagents.

• The ogives each can be sensitized to a different reagent ; allowing the simultaneous detection of antibodies and different Ag for multi target assay analysis.

• One ogive is left unsensitized to measure the non specific reaction of the sample.

9.5. ELISA Reverse method & device (ELISA Rm&d):

• A newer technique uses a solid phase made up of an immunosorbent polystyrene rod with412protruding ogives (curved shape/feature).

• The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogenous) are carried out by dipping the ogives in microwells of standard microplates prefilled with reagents.

9.5.1. ADVANTAGES:

• The ogives can each be sensitized to a different reagent, allowing the simultaneous detection of different antibodies and different antigens for multi target assays;

• The sample volume can be increased to improve the test sensitivity in clinical (saliva,urine), food (bulk milk, pooled eggs) and environmental (water) samples;

• One ogive is left unsensitized to measure the non specific reactions of the sample;

• The use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not required, facilitating ready touse lab kits and onsite kits.

10. ENZYMES USED IN ELISA :

a)    Horseradish peroxidase(most commonly used)

b)   Alkaline Phosphatase

c)    γ-galactosidase

d)   Lactoperoxidase

e)    Tetra Methyl benzidine

• In case of peroxidase, the substrate hydrogen peroxide is converted into water and o2 in the presence of electron donors . (like diaminobenzidine or 4-chloronaphthol which themselves oxidized inthe reaction).

• Oxidation of diamino benzidine produces dark brown color while that of 4-chlorornaphthol yields purple color which is the basis of ELISA .

11. ENZYME SUBSTRATE:

• Initially the substrate should be colorless.
• After degradation by the enzyme it should be strongly colored or fluorescent.

12. COMPOSITION:

12.1   ENZYME:SUBSTRATE:CHROMOGEN:STOPPIN:-

Alkaline Phosphatasep-NPPp-NPP + diethandamine+ MgCl21 M NaOH Horse radishPeroxidaseH2O2Tetramethylbenzidine +Phosphate  ±Citrate buffer1 M H2SO4Horse radishPeroxidaseH2O2±  Phenylenediamine + HCl 1 M HCl

13.  ELISA KIT: 

13.1. Components of Kit: 

1. Pre- Coated, 
2. Stabilized 96-wellMicrotiter Plate

.                         3.  Sample Diluent

4. Standards and controls

5. Conjugated Detection

                                    6.Antibody

                                    7.10X WashSolution

                                    8. Substrate

13.2. ELISA PLATE:

1.                                                              i.      Microtitre wells
2.                                                            ii.      Generally 96 wells
3.                                                         iii.      Marked on oneside alphabetically 
4.                                                         iv.      Numerically onthe other side

                             All these are present with the kit.

13.2.1. PROCESS OF USING ELISA  KIT:

13.2.2. Collection and processing of serum: 

• Collect blood in a tube that does not contain any chemicals or anticoagulants.

• Collect 5mL of whole blood (for very small children collect 1mL).

• Place tube upright for 30-60 minutes then when firm clot has formed, centrifuge tube for 20 minutes at2500rpm.

• Remove serum with a pipette and place in a plastic storage tube (2-3mL microtube or cryovial).

• 5.If 5mL of blood was collected it will result in about2mL of serum.

13.2.3. TEST PERFORMANCE: 

• Using a clean Pipette , add 100 L of diluted serum sample (Dilute the sera to be tested1:100 in the sample diluents) to each well.

.

DIAGRAMATIC REPRESENTATION

• Incubate 1 hour at37°C .

• After incubation empty out contents of  wells into waste container.

• Using pipette, fill wells with washing buffer then empty out.

• Tap wells upside down on paper towel

• Distribute 100L of anti-human immunoglobulin-POD conjugate in each well.Incubate 30 minutes at37°C.

13.2.4. ELISA PLATE READY FOR READING:

• Measures the absorbance at 450nm
• With the help of ELISA READER.
• Calculate the absorbance for each sample and reference.
• We used Ascent Software for Calculation of the result.

13.2.5. DETERMINATION:- 

A)   IgA and Ig Tests:-

• Plot the O.D. result of each reference , except for the negative reference on the vertical axis (Y-axis) in relation to the number of corresponding units on the horizontal axis (X-axis).
  • Using the absorbance value for each sample , determine the corresponding concentration of antibodies expressed in units/ml from the reference curve positive for IgM antibodies.

B)    IgM Tests :-

• We can calculate the cut off value A450nm sample / A450nm Positive limit reference the normalized value of the positive reference is 1.

• All samples whose value is comprised between 0.8 and 1.0 are consider eddubious and all samples whose normalized value is above 1.0 are considered.

13.2.6.Cut-off  VALUE:

• Provided in the kits by the manufacturer.

• The cut off value defines  arrange in which 90% of the normal population is negative  below the cut off value and 10% of the normal population is positive above the cut-off value.

• ELISA is semi- quantitative method.

Sample value=sample OD/cut-off OD

13.2.7. ADVANTAGES:

• Reagents are relatively cheap & have a long shelf life
• ELISA is highly specific and sensitive
• No radiation hazards occur during labeling or disposal of waste.
• Easy to perform and quick procedures
• Equipment can be inexpensive and widely available.
• ELISA can be used to a variety of infections.

13.2.8. DISADVANTAGES:

• Measurement of enzyme activity can be more complex than measurement of activity of some type of radioisotopes.
• Enzyme activity may be affected by plasma constituents.
• Kits are commercially available, but not cheap
• Very specific to a particular antigen.
• Wont recognize any other antigen
• False positives/negatives possible, especially with mutated/altered antigen.

13.2.9. LIMITATIONS:

• Results may not be absolute.
• Antibody must be available.
• Concentration may be unclear.
• False positive possible.
• False negative possible.

.13.2.10.  APPLICATIONS OF ELISA:

1. Screening donated blood for evidence of viral contamination by HIV

1. 2.        Measuring hormone levels HCG, LH, TSH, T3 and T4

1. It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds and eggs.

1. Used to determine the levels of Ab in faecal content.

1.  Higher sensitivity
2. Higher specificity
   1. The ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool for determining serum antibody concentrations (such as with the HIV test[8] or West Nile Virus).
   2. It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs.[9]
   3.  ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs.

ELISA (Enzyme-Linked Immunosorbent  Assay) plate.

10. DETECTION OF HIV  BY ELISA :

• The ELISA was the first screening test widely used for HIV because of its high sensitivity.
• In an ELISA, a person's serum is diluted 400-fold and applied to a plate to which HIV antigens are attached.
• If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum.
• A specially prepared "secondary antibody" — an antibody that binds to other antibodies — is then applied to the plate, followed by another wash. This secondary antibody is chemically linked in advance to an enzyme.
• Thus, the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate.
• A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence.
• ELISA results are reported as a number; the most controversial aspect of this test is determining the "cut-off" point between a positive and a negative result.
• A cut-off point may be determined by comparing it with a known standard.
• If an ELISA test is used for drug screening at workplace, a cut-off concentration, 50 ng/mL, for example, is established, and a sample that contains the standard concentration of analyte will be prepared.
• Unknowns that generate a signal that is stronger than the known sample are "positive." Those that generate weaker signal are "negative."
• Doctor Dennis E Bidwell and AlisterVoller created the ELISA test to detect various kind of diseases, such as Malaria, Chagas' disease, and Johne disease.[10] ELISA tests also are used as in in vitro diagnostics in medical laboratories.

.  12. The other uses of ELISA include:

• detection of mycobacterial antibodies in tuberculosis.
• detection of rotavirus in feces.
• detection of hepatitis B markers in the serum.
• detection of enterotoxin of E. coli in feces.

13)ELISA Qualitative/Quantitative:

a)Qualitative ELISA: 

• Determines antigen or antibody is present or absent.

b)Quantitative ELISA:

• Determines the quantity of the antibody .