1. Labeled Immunoassay
Lab. 2

2. Immunoassay
An immunoassay is a test that uses antibody and antigen complexes [immuno-complex] as a mean of generating a measurable result
Immuno refers to an immune response that causes the body to generate antibodies,
and assay refers to a test
The assay takes advantage of the specific binding of an antibody to its antigen
The antibodies used must have a high affinity for the antigen
Immunoassays derive their unique specificity, sensitivity, and flexibility from three important properties of antibodies:
Their ability to bind to an extremely wide range of natural and man-made chemicals, biomolecules, cells, and viruses
Exceptional specificity for the substance to which each antibody binds.
The strength of the binding between an antibody and its target

3. Immunoassay Both the presence of antigen or antibody can be measured
For measuring hormones such as insulin, the insulin acts as the antigen
When detecting infection the presence of antibody against the pathogen is measured
For numerical results the response of the object being measured must be compared to standards of a known concentration
This is usually done through the plotting of a standard curve on a graph paper, and then the quantity of the unknown is found from the curve

4. Types of Tests Visible Change Observable Reaction Name of Test
Clumping
Particulate antigen reacts with corresponding antibody; antigen may be in form of RBCs (hemagglutination, latex, or charcoal coated with antigen).
Agglutination
Precipitates
Soluble antigen reacts with corresponding antibody
Precipitation
Complement activation, hemolysis
Competition between two antigen-antibody systems (test and indicator systems)
Complement fixation (CF)
Visible microscopic fluorescence
Fluorescent-tagged antibody reacts with antigen-antibody complex in the presence of ultraviolet light.
Immunofluorescence
Color change indicates enzyme substrate reaction.
Indirect EIA for quantification of an antigen or antibody enzyme and substrate
Enzyme-linked immunosorbent assay (ELISA)
Detection of antibodies of specific mobility
Electrophoresis separation of antigen subspecies
Immunoblot (eg, Western blot [WB])
Light scatter proportionately increases as numbered size of immune complexes increases.
Measures either antigen or antibody in solution through the scattering of a light beam; antibody reagent used to detect antigen IgA, IgG, IgM; concurrent controls are run to establish amount of background scatter in reagents and test samples.
Rate nephelometry

5. Antigen-antibody interaction
Antigens are substances that stimulate and subsequently react with the products of an immune response. They may be:
Enzymes,
Toxins,
Microorganisms (eg, bacterial, viral, parasitic, fungal),
Tumors,
Autoimmune factors
Antibodies are proteins produced by the body's immune system in response to an antigen or antigens.
The antigen-antibody response is the body's natural defense against invading organisms

6. Antibody Structure V Light Chain Light Chain Heavy Chains Constant SS
Antigen binding site V SS SS
Light
Chain
Light Chain
Constant
Heavy Chains

7. Antigen Binding
Antigen 2
Antigen 1
Antigen 3
Variable
Light
Heavy

8. covering the fields of:
There are now hundreds of immunoassays for dozens of analytes including:
covering the fields of:
Developments in antibodies, labels, and automation have resulted in highly specific and sensitive assays
Hormones
Tumor markers
Antibodies
Drugs
Cardiac markers
Endocrinology
Oncology
Hematology
Toxicology
Immunology
Infectious diseases

9. Constituents of a Labeled Immunoassay
For detection of an analyte, the following are usually a part of the assay:
Labeled analytes
Specific antibody
Standards or calibrators
A method to separate the bound from free components
A method for detection of the label

10. 1- Labeled Analytes
A labeled analyte is used to detect whether or not specific binding has taken place
The label used in immunoassay:
must not alter the reactivity of the molecule
and it should remain stable for the shelf life of the reagent
Labels attached to analytes and antibodies can be:
Radioactive
usually iodine-125 (radioimmunoassay),
Enzymes
such as alkaline phosphatase and horseradish peroxidase, (enzyme immunoassay or enzyme-linked immunosorbent assay [ELISA]),
Chemiluminescent
e.g. acridinium ester
Fluorescent
e.g. fluorscein

11. Methods of coupling indicator labels to antigen or antibody
The Methods include:
Binding to amino acids
The radioactive isotope iodine is covalently linked to tyrosine residues present on antibodies and most antigens

12. Methods of coupling indicator labels to antigen or antibody
Using glutaraldhyde
It is a bifunctional reagent that covalently cross links two aminoacids together, reacts with amine groups
Fluorochromes or enzymes may be coupled to antigens or antibodies using glutaraldhyde

13. Methods of coupling indicator labels to antigen or antibody
Biotin-Streptavidin system
Biotin is a vitamin that can bind tightly to either avidin or streptavidin (proteins)
The natural attraction of biotin to these two proteins is a property that has been exploited to facilitate coupling of indicator molecules to antigens or antibodies
At the end of the assay, a conjugate of streptavidin linked to a signal- generating substance is added
Examples of suitable conjugates are streptavidin-alkaline phosphatase, streptavidin-HRP, streptavidin-125I, streptavidin fluorescein

14. 2- Production of Antibodies
The production of antibodies is an important process in the use of immunoassays because it is the antibody-antigen complexes that form the basic
Antibodies can be called depending upon the technique used to produce them either:
Monoclonal or
Polyclonal

15. a) Polyclonal Antibodies
May be produced in mammals such as rabbits or sheep
When a foreign substance enters the body, it stimulates the immune system to produce antibodies to the substance
Using this natural reaction, an analyte in as pure form as possible is injected into the animal stimulating the production of antibodies
Antiserum usually contains a mixture of antibodies that recognize and bind to the same antigen, but they may attach to different epitopes Ag
Multivalent Antigen complex
Polyclonal Antibodies
Antibody-Antigen complex

16. b) Monoclonal Antibodies
Monoclonal antibodies production result in very specific antibodies that bind only to one antigen epitope, which in turn reduces the occurrence of false positives in the immunoassay
Multivalent Antigen complex
Monoclonal Antibodies
Antibody-Antigen complex Ag

17. 3- Standards or calibrators
Calibrators are solutions with known values that establish the relationship between the amount of signal produced in the assay and analyte concentration
By running a set of calibrators, a calibration curve is set up in the instrument’s software and correlates certain values of signal to known analyte concentrations
By comparing levels of signal produced by patient samples to this calibration curve, a patient analyte concentration value, or result, can be determined

18. 4- Separation Methods
In most assays, once the reaction between antigen and antibody has taken place, there must be a way of separating reacted from unreacted analyte
Unreacted analyte can be removed by:
Adsorption on particles such as dextran-coated charcoal
These adsorb out the smaller unbound molecules, which are then separated from bound molecules by centrifugation or filtration
The amount of label remaining in the supernatant provides an indirect measure of analyte present in the patient's sample
Another means of separation involves precipitation of antigen- antibody complexes
Complexes can be precipitated by adding concentrated solutions of ammonium sulfate, or ethanol

19. Currently, most immunoassays use a solid-phase stage for separation
4- Separation Methods
Currently, most immunoassays use a solid-phase stage for separation
Numerous substances, such as polystyrene test tubes, microtiter plates are used for this purpose
Antigen or antibody is attached by physical adsorption, and when specific binding takes place, complexes remain attached to the solid phase

20. 5- Methods for detection of label
The last step common for all immunoassays is detection of the labeled analyte
The method depends on the label;
e.g. 125I is easily detected by a γ-counter
Enzymes are generally used to produce coloured products from colourless substrates that can be determined easily using a spectrophotometer
Automated plate readers are commercially available which make reading large numbers of samples relatively easy

21. Quality Control in Immunoassays
It is essential that quality control procedures be established
This is done to limit random errors, such as
temperature fluctuations
minor changes in the concentration of reagents
and changes in detector efficiency
A negative control and a positive control should be run
This serves as a check on the quality of the reagents to make sure that the label is readily detectable under current testing conditions

22. Methodological Principles
Competitive Immunoassays
Noncompetitive Immunoassays
Heterogeneous Immunoassays
Homogeneous Immunoassays

23. immobilisation surface
1- Competitive Immunoassays
Based on competition between the analyte in the sample and a labeled analyte for a limited mount of antibody L S P
Enzym.
reaction
Product
measurement
Incubation
Coating
Specific Ab Ag L
antigen- enzyme conjugate
immobilisation surface

24. 2- Noncompetitive Immunoassays
Noncompetitive immunoassays (Sandwich) generally provide the highest level of assay sensitivity and specificity
The reaction mixture typically includes an excess of labeled antibody, so that all analyte is bound
The amount of antibody-antigen complex is then measured to determine the amount of analyte present in the sample
The labeled antibody, is directly proportional to the amount of antigen present in the sample

25. Heterogeneous versus Homogeneous Immunoassays
3- Heterogeneous Immunoassay
Methods that require separation of bound Ab-Ag* complex
4- Homogeneous Immunoassay
Those that do not require separation.
Homogeneous methods have been generally applied to the measurement of small analytes such as abused and therapeutic drugs
Since homogeneous methods do not require the separation of the bound Ab-Ag* from the free Ag*, they are generally much easier and faster to perform

26. Types of Immunoassays (Heterogeneous)
Within the categories of competitive, noncompetitive, homogenous, and heterogeneous, there are specific types, which include:
Radioimmunoassays (RIAs)
utilize a radioactive label (usually 125I, 3H or 14C), which emits radiation that can be measured with a beta or gamma counter

27. Types of Immunoassays (Heterogeneous)
Enzyme linked immunosorbant assay (ELISA):
Direct, sandwich and competitive
Reaction components are absorbed or bound to the surface of a solid phase, commonly a well of a microtiter plate
Absorbance is measured using a micro-plate reader

28. Types of Immunoassays (Homogeneous)
Enzyme Multiplied Immunoassay (EMIT)
The drug in the sample and the drug labeled with G6PD compete for antibody binding sites
Binding inhibits enzyme activity, while free enzyme remains active to interact with the substrate
Enzyme activity/absorbance is directly proportional to drug concentration.
Substrate
Substrate reacting with enzyme
Drug blocking Drug attached to enzyme
Antibody
Drug
Enzyme with Drug attached

29. Immunoassay Results Qualitative Quantitative
Single point calibration at a specific cutoff
Results are either ‘positive’ or ‘negative’ (i.e. above or below the cutoff)
Quantitative
Provides numeric results that are an estimate the analyte concentration based on the measurement standards

30. Rubella IgM Test Kit

31. Intended Use
Rubella IgM ELISA test is an enzyme linked immunosorbent assay (ELISA) for the detection of IgM class antibodies to Measles (Rubella) in human serum or plasma.

32. Summary and explanation
Measles is an acute, highly contagious viral disease.
Although measles is usually considered a childhood disease, it can be contracted at any age.
Measles is spread by direct contact with nasal or throat secretions of infected people or, less frequently, by airborne transmission.

33. Summary and explanation
The presence of IgG antibody to Rubella virus is indicative of previous exposure or vaccination.
In individuals with acute measles, a significant increase in measles IgG antibody level is indicative of recent infection.
IgM antibodies to Rubella virus are often detectable with onset of the rash and typically persist for 4 weeks.
At least 80% of patients will be positive for Rubella IgM at 6 days and 100% at 16 days after onset of symptoms.

34. Principle
Diluted patient serum is added to wells coated with purified Rubella antigen.
Rubella IgM specific antibody, if present, binds to the antigen.
All unbound materials are washed away and the enzyme conjugate is added to bind to the antibody-antigen complex, if present.
Excess enzyme conjugate is washed off and substrate is added.
The plate is incubated to allow the hydrolysis of the substrate by the enzyme.
The intensity of the color generated is proportional to the amount of IgM specific antibody in the sample.

35. Procedure
Bring all specimens and kit reagents to room temperature (18-26 °C) and gently mix.
Place the desired number of coated strips into the holder.
Negative control, positive control, and calibrator are ready to use. Prepare 1:21 dilution of test samples, by adding 10 μl of the sample to 200 μl of sample diluent. Mix well.
Dispense 100 μl of diluted sera, calibrator and controls into the appropriate wells. For the reagent blank, dispense 100μl sample diluent in 1A well position. Tap the holder to remove air bubbles from the liquid and mix well. Incubate for 20 minutes at room temperature.
Remove liquid from all wells. Wash wells three times with 300 μl of 1X wash buffer. Blot on absorbance paper or paper towel.

36. Procedure
Dispense 100 μl of enzyme conjugate to each well and incubate for 20 minutes at room temperature.
Remove enzyme conjugate from all wells. Wash wells three times with 300 μl of 1X wash buffer. Blot on absorbance paper or paper towel.
Dispense 100 μl of TMB substrate and incubate for 10 minutes at room temperature. Add 100 μl of stop solution.
Read O.D. at 450 nm using ELISA reader within 15 min.
A dual wavelength is recommended with reference filter of nm.

37. Calculation of results
Check Calibrator Factor (CF) value on the calibrator bottle. This value might vary from lot to lot. Make sure you check the value on every kit.
Calculate the cut-off value:
Calibrator OD x Calibrator Factor (CF).
Calculate the Ab (Antibody) Index of each determination by dividing the O.D. value of each sample by cut-off value.
Example of typical results
Calibrator mean OD = 0.8
Calibrator Factor (CF) = 0.5
Cut-off Value = 0.8 x 0.5= 0.400
Positive control O.D. = 1.2
Ab Index = 1.2 / 0.4 = 3
Patient sample O.D. = 1.6
Ab Index = 1.6 / 0.4 = 4.0

38. Validation requirement and quality control
The O.D. of the Calibrator should be greater than
The Ab index for Negative control should be less than 0.9.
The Ab Index for Positive control should be greater than 1.2.

39. Interpretation of results
The following is intended as a guide to interpretation of Measles IgM test results; each laboratory is encouraged to establish its own criteria for test interpretation based on sample populations encountered.
Antibody Index Interpretation
< 0.9 No detectable Ab to Rubella IgM by ELISA.
0.9 – 1.1Borderline positive, follow up testing is recommended if clinically indicated.
> 1.1 Detectable antibody to measles IgM by ELISA.