Serology (trans 3) Flashcards
SEROLOGY
Uses antigens (Ag) and antibodies (Ab) to detect microorganisms
In vitro Ag-Ab reactions
Application to the diagnosis of infectious diseases
Culture is the gold standard in the detection of infectious diseases but often time serology is important. Serology is not really inferior to culture but it serves as an adjunct or as a tool to help in our diagnosis
Role of Serology in Laboratory Diagnosis:
If culture is not possible
Early or presumptive diagnosis
To know a particular serotype
To compare actual and convalescence sera (“Paired Sera”)
To compare baseline vs. post-treatment titers
Role of Serology in Laboratory Diagnosis: If culture is not possible
o Because some are rare to find or if present, is not easily isolated and identified by other available techniques.
o E.g. spiral bacteria, Syphilis, rickettsial diseases, leptospirosis, enteric fever, brucellos, viral hepatitis, rotavirus infection
Role of Serology in Laboratory Diagnosis: Early or presumptive diagnosis
o To give empiric treatment (geared towards the most probable causative/etiologic agent); ex: if you have throat swab, the result will be released after 3 days, but if you have serologic testing, the result will be handy
Role of Serology in Laboratory Diagnosis: To know a particular serotype
Ex: In culture, you will only determine Vibrio cholerae, but with serology you will know what serotype of V. cholerae is present
Role of Serology in Laboratory Diagnosis: To compare actual and convalescence sera (“Paired Sera”)
o To determine the rise in titer
o To identify and if indicated, serotype a pathogen that has been isolated by culture
Role of Serology in Laboratory Diagnosis: To compare baseline vs. post-treatment titers
To know if the treatment has been effective
Serological Tests
Antibody tests – use known antigen suspensions to detect and measure antibody present in the patient’s serum in response to infection.
Antigen tests – use specific antisera to identify microorganisms by detecting their antigens in specimens or isolates.
IMMUNO-SEROLOGIC REACTIONS Primary Reactions a. Fluorescence Immunoassay b. Radioimmunoassay c. Enzyme Immunoassay d. Sol Particle Immunoassay Secondary Reactions a. Precipitation b. Agglutination c. Complement Fixation d. Neutralization Tertiary Reactions
Primary Reactions
o Combination of antigen-antibody (Ag-Ab)
o Non-visible reaction in vitro
Ex: Fluorescence Immunoassay, Radioimmunoassay, Enzyme Immunoassay, and Sol Particle Immunoassay
Secondary Reactions
o Demonstrable Ag-Ab reaction in vitro
Ex. Precipitation, Agglutination, Complement Fixation, and Neutralization
Tertiary Reactions
o Immunologically present in vivo
o Biologic reaction is detectable
Ex. Phagocytosis, opsonization, chemotaxis
IMMUNO-SEROLOGIC REACTIONS
PRIMARY REACTIONS: Fluorescence Immunoassay
Widely used in the serological diagnosis of bacterial, viral, fungal, and parasitic diseases
Usually sensitive and gives reproductive results
Also referred to as Fluorescent Antibody Test (FAT)
PRINCIPLE: Fluorochrome (color) + UV light => fluorescence
**Fluorescent Dyes (fluorochrome) illuminated by UV light are used to show the specific combination of an Ag with its Ab. The Ag-Ab complexes are seen fluorescing against a dark background. The intensity of the fluorescence is directly proportional to the amount of substance in the sample
IMMUNO-SEROLOGIC REACTIONS
PRIMARY REACTIONS: Fluorescence Immunoassay
TECHNIQUES
- Direct/Single Layer Immunofluorescent Assay
2. Indirect/Double Layer Immunofluorescent Assay
IMMUNO-SEROLOGIC REACTIONS
PRIMARY REACTIONS: Fluorescence Immunoassay
TECHNIQUES - Direct/Single Layer Immunofluorescent Assay
Used to detect and identify unknown antigen in specimens ex: Group A Streptococcus, pathogens in CSF, viruses, Yersinia pestis
Specimen is placed on a microscope slide and fluorescent labelled specific antibody is added, allowing time for the antigen to react. Preparation is then washed, leaving only the labelled antibody combined with the antigen. Ag-Ab complexes are seen fluorescing upon microscopy.
Called direct because the fluorescent dye is directly labelled or attached to the Ab
Glycerol or mineral oil is used as immersion fluid because it does not fluoresce like normal immersion oil
Fluorochrome + UV light -> Ag-Ab reaction (basis for having fluorescence)
Fluorescein isothiocyanate -> yellow green fluorescence
Rhodamine -> red fluorescence
IMMUNO-SEROLOGIC REACTIONS
PRIMARY REACTIONS: Fluorescence Immunoassay
TECHNIQUES - Indirect/Double Layer Immunofluorescent Assay
Ab is labelled indirectly
Unlabeled Ab + Ag + fluorescent labelled against anti-species globulin + UV light. The unlabelled or unattached Ab combines with Ag and the Ag-Ab complex is detected by adding a fluorescent labelled anti-species globulin which binds to the unlabelled Ab and results to fluorescence.
Called indirect because of the use of a fluorescent
labelled anti-species globulin which binds to the Ab.
An Anti-species globulin is an Ab that reacts specifically to any antibody of that particular species.
Ex: They injected to the body of the rabbit the human blood, then they extracted blood, then they developed anti-human.
*anti-human immune serum globulin + UV light -> Ag-Ab reaction (basis for having fluorescence)
IMMUNO-SEROLOGIC REACTIONS
PRIMARY REACTIONS: Fluorescence Immunoassay
TECHNIQUES - Indirect/Double Layer Immunofluorescent Assay
a. Indirect FAT to detect antigen – used in preference to direct FAT in detecting and identifying an antigen in a specimen because it leaves an antiserum free (unlabeled) for use in other serological tests and because the fluorescence is much brighter
b. Indirect FAT to detect antibody – detection of FTAAbs (Fluorescent Treponemal Anti-body Absorption), for Treponemapallidum, syphilis or autoimmune disease like SLE (systemic lupus erymatosus)
APPLICATION:
FA test for rabies
FTA-ABS
IMMUNO-SEROLOGIC REACTIONS
PRIMARY REACTIONS: Fluorescence Immunoassay
DIFF. PATTERNS FOR ANTI-NUCLEAR ANTIBODIES (ANA)
- An antinuclear antibody (ANA) test measures the amount and pattern of antibodies in your blood that work against your own body (autoimmune reaction).
- THE “RIM/PERIPHERAL” PATTERN
- THE “SPECKLED” PATTERN
- THE “NUCLEOLAR” PATTERN
IMMUNO-SEROLOGIC REACTIONS
PRIMARY REACTIONS: Fluorescence Immunoassay
DIFF. PATTERNS FOR ANTI-NUCLEAR ANTIBODIES (ANA) - THE “RIM/PERIPHERAL” PATTERN
This is more characteristic of systemic lupus erythematosus (SLE) than other autoimmune diseases. The rim or sides are glowing or highlighted. The nuclei have different patterns
Fluorescence Immunoassay
DIFF. PATTERNS FOR ANTI-NUCLEAR ANTIBODIES (ANA) - THE “SPECKLED” PATTERN
This is a pattern of antinuclear antibody test staining which is more characteristic of the presence of auto-antibodies to extractable nuclear antigens, particularly to ribonucleoprotein. It is speckled because the nucleus is filled with dots.
Fluorescence Immunoassay
DIFF. PATTERNS FOR ANTI-NUCLEAR ANTIBODIES (ANA) - THE “NUCLEOLAR” PATTERN
This is a pattern of staining in which the bright fluorescence is seen within the nucleoli of the Hep2 cells. This pattern is more suggestive of progressive systemic sclerosis (scleroderma). The nucleolus itself is the one glowing.
IMMUNO-SEROLOGIC REACTIONS
PRIMARY REACTIONS: Radioimmunoassay
Used when high specificity and sensitivity are required
PRINCIPLE: The radioactivity of a specific isotope labelled antibody or antigen is used to detect and quantify antigen or antibody in test specimens
IMMUNO-SEROLOGIC REACTIONS
PRIMARY REACTIONS: Radioimmunoassay
**Solid Phase RadioImmunoassay (RIA)
Specific antibody is coated on a solid phase such as the inner surface of a test tube. Test sample is added and time is allowed for the Ag to attach to the Ab. Tube is then washed and radioactive Ab is added which combines with the antigen. Radioactivity is measured to determine amount of Ag present.
Radioactive isotopes used in T3 and T4 (thyroid hormones)
o I131 – used in Radioactive Immuno Uptake (RAIU), most common
o I125 – also used in Radioactive Immuno Uptake (RAIU)
o H3
o C14
Used as reference technique for detecting HBsAg in serum and also for detecting anti-HA V IgM in the serum of patients with Hepa A
TECHNIQUES:
1. Competitive
2. Non-competitive
2 tests for IgE:
Radioallergosorbent test (RAST) – measures Ag-specific IgE where the ligand band is labelled anti-IgE
Radioimmunosorbent test (RIST) – a competitive RIA for total serum IgE; RIST activity is indirectly proportional to IgE concentration.
IMMUNO-SEROLOGIC REACTIONS PRIMARY REACTIONS: Enzyme Immunoassay Involves a colorimetric reaction which indicates the end result Darker color = higher concentration Enzymes used: o Alkaline Phosphatase (ALP) – used in ELISA o Horseradish Peroxidase – used in ELISA o Glucose Oxidase o B-galactosidase
TECHNIQUES:
- Direct
- Indirect – to detect and assay antibody
- Sandwich/double Ab – to detect antigen (the bun is the antibody, the patty is the antigen)
- Competitive binding
- Enzyme inhibition
IMMUNO-SEROLOGIC REACTIONS
PRIMARY REACTIONS: Enzyme Immunoassay
**Enzyme-linked Immunosorbent Assay (ELISA)
- PRINCIPLE: Uses an enzyme system to show the specific combination of an antigen with its antibody
Used in diagnosis of microbial infections and is specific, sensitive, and requires only a small amount of specimen
Results are spectrophotometrical
A change in color indicates the presence of an antigen or antibody
Application: Screening test for HIV
IMMUNO-SEROLOGIC REACTIONS
PRIMARY REACTIONS: Enzyme Immunoassay
**Enzyme-linked Immunosorbent Assay (ELISA)
Antigen is attached to bottom of well => Patient’s serum is added. If antibodies to the antigen are present, they will bind to antigen. Plate is washed. => antihuman globulin (AHG) with enzyme attached is added. Plate is again washed. AHG will remain only if antibody is present. => Substrate of enzyme is added. If enzyme is present, a colored product will be formed. Color indicates the antibody is present. (The intensity of the color is directly proportional to the amount of substance)
- Double Antibody ELISA
Well of microtitration plate is coated with specific Ab, and specimen containing Ag is needed. After allowing the Ag to combine with the Ab, plate is washed and enzyme labelled Ab is added which attaches the antigen. After washing, a substrate is added which is hydrolyzed by the enzyme to give a color change.
Used in the diagnosis of rotavirus infection in young children - Indirect ELISA
Used in the diagnosis of several parasitic infections and is being increasingly used in the diagnosis of bacterial and fungal infections.
IMMUNO-SEROLOGIC REACTIONS
PRIMARY REACTIONS: Enzyme Immunoassay
**TYPHIDOT - PRINCIPLE: Indirect ELISA
For detection of IgM and IgG to Salmonella typhi
First known qualitative Ab detection test against S. typhi
Used in early diagnosis of Typhoid fever
Indicate stages of typhoid infection (acute, convalescence, previous exposure)
Can diagnose with a single serum specimen (but culture is still the gold standard in diagnosing typhoid fever)
Salmonella Ags on beads + Px serum + enzyme labelled AHG (IgM and IgG) + substrate (chromogenic substance) => (+) color formation
IMMUNO-SEROLOGIC REACTIONS
PRIMARY REACTIONS: Enzyme Immunoassay
**TYPHIDOT - Interpretation of Result in Typhoid Fever
- Acute or Primary Infection: IgM (+), IgG (-)
- Past or recurrent infection of receipt of typhoid vaccination: IgM (-), IgG (+)
* *IgM = Primary Antibody Response
* *IgG = Secondary Antibody Response
IMMUNO-SEROLOGIC REACTIONS
PRIMARY REACTIONS: Sol Particle Immunoassay
Usea colloidal particles in a liquid consisting of metal or insoluble metal compound as label or tag
Metal will go on top of the organism (metallic impregnation)
Colloidal Particles Used: Gold Silver Silver Iodide Barium Sulfate TECHNIQUES: 1. Homogenous 2. Heterogenous
- Remember that in serology, we are just dealing with a few microliters so you cannot afford to have an over or under amount. There will be presence of prozone and postzone.
- The manner of reporting in serologic testing is reactive or nonreactive because it means that there is Ag-Ab reaction. Not the usual positive or negative because we are not saying that it is confirmed.
“In the case of antibody excess, the prozone phenomenon occurs, in which antigen combines with only one or two antibody molecules, and so no cross-linkages are formed. At the other side of the zone, where there is antigen excess, the postzone phenomenon occurs, in which small aggregates are surrounded by excess antigen, and again no lattice network is formed”
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS:
Used to detect and identify antigens in specimens and to detect and quantify antibodies in serum
Antigens and antibodies involved are in soluble form and combine to form a visible precipitate
Precipitin: the insoluble compound thrown out in solution after two reactants are mixed
Precipitin can take the form of a line or an arc
Tube precipitin, gel diffusion, counterimmunoelectrophoresis
Types of Precipitation tests:
- Single Diffusion, Single Dimension (Ouidin Test)
- Single Diffusion, Double Dimension (Radial Immunodiffusion)
- Double Diffusion, Double Dimension (Ouchterlony Technique)
- CounterImmunoelectrophoresis (CIE)
- Immunoelectrophoresis (IEP)
- Rocket Electrophoresis (Laurell Techniques)
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Precipitation test
Single Diffusion, Single Dimension (Ouidin Test)
Test antigen is carefully layered on to a clear antiserum in a precipitin tube
After incubation, if corresponding antigen is present and the proportion of antigen to antibody is optimal, a line of visible precipitin will form between the two layers of liquid
(+): precipitation line
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Precipitation test
Single Diffusion, Single Dimension (Ouidin Test)
Specific antibody is incorporated into the agar gel and wells are cut to contain the antigen
A ring of precipitation (dotted line) forms around a well that contains the corresponding antigen
The higher the concentration of the antigen, the larger the ring (diameter) of precipitation
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Precipitation test
Single Diffusion, Single Dimension (Ouidin Test) - Two methods:
Fahey Method: kinetic diffusion; allows the reaction to be in progress and measures the amount of precipitate after the reaction is over (no time limitations)
Mancini Method: endpoint diffusion; measures the amount of reaction after a predetermined time (with time limitations)
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Precipitation test
Double Diffusion, Double Dimension (Ouchterlony Technique)
Has two diffusing areas (to the left and right)
Ag and Ab diffuse towards each other, a visible precipitate line forms where they meet in optimal proportion
Thickness of line precipitation is semi-quantitative measure of the amount of antigen-antibody combination
Three patterns:
a. Identity – forming smooth curve; antigen in the sample is same with known antigen
b. Partial Identity – precipitation line merge with spur formation (like the leg of rooster); antigen in the sample has some similarities with the known antigen
c. Non-identity – precipitation lines cross and intersect; antigen in the sample is different from known antigen
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Precipitation test
Double Diffusion, Double Dimension (Ouchterlony Technique)
**Ouchterlony Technique. The patient’s serum is in the center well. B and C are reactive with the patient’s serum. A, D, and E are non-reactive.
Ouchternoly Test. Wells are cut into an agar surface and a drop of the patient’s serum is placed into the center well. Solutions of known antigens are placed into the peripheral wells. Antibodies from the patient’s serum diffuse outward from the center cell as do the antigens from their individual wells. the serum has antibodies to antigens C and H since there are precipitate lines formed.
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Precipitation test
CounterImmunoelectrophoresis (CIE)
Uses electrophoresis to increase the speed with which the antigen and antibody travel in the agar gel
A line of precipitation forms where the two meet in optimal proportion
At pH 8.6, the antigen migrates to the anode (+) electrode, and the antibody to the cathode (-) electrode
The pH (8.6), purity, and ionic strength of the agar are some factors that influence the movement of the antibody and antigen
Specific antibody is placed in the positive electrode (anode) end of the plate, and the unknown antigen in a well at the negative (cathode) end
An electric current is applied and the antigen and the antibody move toward each other
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Precipitation test
Immunoelectrophoresis (IEP)
Useful for identification of monoclonal proteins (Bence Jones protein seen in patients with multiple myeloma)
Monoclonal proteins are more associated with malignancy than polyclonal
Utilizes both double diffusion and electrophoresis
The longer the magnitude, the higher the level of antigen concentration
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Precipitation test
Rocket Electrophoresis (Laurell Techniques)
Antiserum is incorporated into the agar and the unknown antigen is placed in the well and electrophoresed
The total distance of antigen migration and precipitation is directly proportional to antigen concentration
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Agglutination Reactions
Antigens involved are particulate antigens. (In precipitation reactions, soluble antigens are used)
Compared with other serological tests, it is easier to perform, require no special equipment, and is usually less expensive.
PRINCIPLE:
Agglutination is the visible clumping together of bacteria, cells or particles, by an antigen combining with its specific antibody. The resulting clumps are referred to as agglutinates
In tests used to detect antibody in a patient’s serum, a known antigen suspension is used. The antigen particles are agglutinated if the serum contains the corresponding antibody.
AGGLUTINATES = POSITIVE RESULT
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Agglutination Reactions
Types of Agglutination Reactions
a. Direct Agglutination
b. Indirect Agglutination/Antiglobulin Technique (Coomb’s test)
c. Passive Agglutination
d. Reverse Passive Agglutination
e. Agglutination Inhibition
f. Hemagglutination
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Agglutination Reactions
Direct Agglutination
The antigens present in the patient’s serum directly reacts with the antibodies, and agglutinates are seen
Positive result: Visible agglutination
Blood Typing
o The antigens present on the surface of the RBCs will react with the antibodies in the reagents (anti-A and anti-B)
o Antigen A + Anti-A = agglutination
o Antigen B + Anti-B= agglutination
Widal Test
o Performed for the diagnosis of typhoid fever
o Principle: Direct Agglutination
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Agglutination Reactions
Indirect Agglutination/Antiglobulin Technique (Coomb’s test)
Anti-human IgG is added to bridge the gap between cells
Used to demonstrate incomplete antibodies
Usually used in detecting Hemolytic Disease of the New Born or Hemolytic Transfusion Reaction
Indirect agglutination test. The procedure involves the use of anti-human IgG to detect the presence of IgGs that may bind to the body’s RBCs and cause hemolysis. Anti-human IgG is produced by immunizing non-human species with human serum. Anti-human IgG binds to human antibodies, commonly IgM and IgG. When they bind to human antibodies fixed into RBC antigens, a visible agglutination results.
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Agglutination Reactions
Indirect Agglutination/Antiglobulin Technique (Coomb’s test)
Used to check for the presence of antibodies which were not detected in the first and second phases of crossmatching
This is the last step involved in cross-matching. Thus it is called indirect because multiple phases have to be gone through before reaching this last step.
The point of this test is to find out if your patient has antibodies against red cells – either antibodies against any red cell antigen at all (which is what the antibody screen looks for), or antibodies against the particular unit of red cells you have chosen to give the patient (which is what the cross-match is for). The DAT is looking to see if your patient’s red cells are coated with antibody; the IAT is looking to see if your patient has antibodies in his or her serum.
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Agglutination Reactions
Passive Agglutination
A soluble Ag is artificially attached to a particulate carrier (e.g. cells, latex, bentonite, celloidin, or charcoal)
Converting a precipitating test to an agglutinating test
Chemically link soluble antigen to inert particles such as latex or RBC
These are tests in which the specific antibody or known antigen is attached to inert particles or cells. When the known antigen or antibody combines with its corresponding antibody or antigen in the specimen, the articles or cells are agglutinated.
The carrier particles or cells are used only to show that an antigen antibody reaction has occurred. The role in the reaction is therefore passive.
Particle carriers include: RBCs, polystyrene latex, bentonite, charcoal
Used in the detection of: Rheumatoid factor, antinuclear antibody in LE, Ab to group A Streptococcus antigens, Ab to Trichinella spiralis
The substances and cells used as carriers in passive slide and tile agglutinations tests include:
- Latex Particle – These are polystyrene particles that can be coated with either known antigen or specific antibody (e.g. Antistreptolysin O (ASO) slide test – This detects significant rises of ASO antibody in the serum of patients with post-streptococcal complications. Antibody coated of latex particles are used in several tests including detection of extracellular bacterial antigens in cerebrospinal fluid.)
- Carbon Particle – These are coated with cardiolipin antigen and used in the rapid plasma regain (RPR) card test to screen for cardiolipin antibodies in the sera of patients with syphilis.
- Stabilized Staphylococcal strains – Most strains of Staphylococcus aureus produce on their outside surfaces a substance called protein A on to which specific antibody can be bound.
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Agglutination Reactions
Reverse Passive Agglutination
Antigen binds to soluble antibody coated on carrier particles and results in agglutination (e.g. detecting cholera toxin)
When the Abs (not the Ag, as in Passive agglutination) are attached to particulate carriers
Purpose is to detect the antigens
Application: C-Reactive Protein/CRP (most elevated during inflammation), RF (Rheumatoid factor) determination
For the detection of microbial antigens such as: Group A and B Streptococcus, Streptococcus aureus, Neisseria meningitides, Haemophilus influenza, Cryptococcus neoformans, Mycoplasma pneumonia, Candida albicans, rotavirus
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Agglutination Reactions
Reverse Passive Agglutination
Rheumatoid Factor Latex Test
**IgM + IgG rheumatoid arthritis => Agglutination for (+) result; In reverse passive agglutination, the antibodies are attached to the carrier
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Agglutination Reactions
Agglutination Inhibition
Homologous soluble Ag inhibits agglutination of another Antigen-coated particle
Used in most pregnancy test
1st step: soluble Ag in patient sample + known Ab reagent
2nd step: particulate Ag is added
Positive reaction: No Agglutination (because the test is agglutination INHIBITION)
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Agglutination Reactions
Hemagglutination
Agglutination of RBC due to antibody, viruses, bacteria, or other biologic substance.
It is not the antigen of RBC but the artificially attached Ag (after undergoing treatment) that are made to react with the Ab.
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Agglutination Reactions
Hemagglutination
**Viral Haemagglutination
Some viruses and microbes contain proteins which bind to erythrocytes causing them to clump together
NDV, Adenovirus III, AIV, IBV, Mycoplasma
Reading the results
Titer – The maximum dilution that gives visible agglutination
The end point is the well with the lowest concentration of the virus where there is haemagglutination
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Complement Fixation
Used to detect and quantify antibodies that do not agglutinate or precipitate when reacted with its antigen but can be demonstrated by its use, or fixation, of complements.
Principle: Using the actions of the complement proteins in vivo to detect antibodies or antigens not typically detected by other tests. Complements bind to an Ab-Ag complex and when this Ab-Ag complex with complements bound to it are attached to cells, they cause cell lysis.
Steps:
The patient’s serum (remember that serum is devoid of cellular contents) is inactivated and serially diluted.
The serum is reacted with a known antigen in the presence of complements. If the corresponding Ab is contained in the serum, it will combine with the Ag. The Ab-Ag complex will then use up the complements in the serum. RBCs are added to the serum.
If the Ab-Ag complex has already used up the complements then no hemolysis results. Thus this indicates the presence of the Ab in question.
The highest dilution of serum that prevents hemolysis is the antibody titer.
Positive result: No Hemolysis
IMMUNO-SEROLOGIC REACTIONS SECONDARY REACTIONS: Complement Fixation Two systems involved in Complement Fixation: Test system/Bacteriolytic system Indicator system/Hemolytic system
Components:
Known target Ag reagent (ex. beef heart extract, bacterial Ag) *In RPR, they use cardiolipin because it is a beef heart extract
Complement - Best source: Guinea pig serum
Hemolysin or Amboreceptor - the Ab used to sensitized indicator cells (Best source: Rabbit antisera)
Indicator cells - Sensitized Sheep RBCs
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Complement Fixation
Use of complement fixation tests
Most complement fixation tests are specific, not always very sensitive. They give inconclusive results when the test serum contains anti-complementary substances (common in sera from patients in tropical countries especially when testing is delayed).
These difficulties, combined with the considerable time it takes to perform CFTs, have lead to the development of simpler techniques to replace these tests. For example, the VDRL and RPR tests have replaced the Wasserman CFT in the investigation of syphilis
Complement Fixation Test. Complement causes lysis of the RBCs. (+) Reaction = no hemolysis because complement has been bound (fixed) by the reaction between antigen and antibody. (-) Reaction = hemolysis because no antibody was present to bind with the antigen. Complement was therefore not fixed and was available to lyse the RBCs.
IMMUNO-SEROLOGIC REACTIONS
SECONDARY REACTIONS: Neutralization Tests
This is the reaction in which the antigenic activity is stopped (neutralized) by its specific antibody
Target: to detect toxins, viral agents, or antibodies to the toxin or viral agents
Types of Neutralization tests Toxin Neutralization o Schick Test – for Diphtheria o Dick Test – for Scarlet fever o Antistreptolysin O Titration Test (ASO Titration) – blood test to measure antibodies against streptolysin O, a substance produced by group A Streptococcus bacteria Virus Neutralization
OTHER SEROLOGIC TESTS
2 main groups: antibody tests and antigen tests
- Serologic Tests for Syphilis: Flocculation techniques
- Febrile/ Bacterial Agglutination Tests
- CRP (C-Reactive Protein)
- ASO (Anti-Strepsolysin-O)
- RF (Rheumatoid factor)
- Antinuclear Antibody (ANA) Fluorescence Patterns
- TORCH
- ELISA (Enzyme-linked Immunosorbent Assay)
OTHER SEROLOGIC TESTS - Serologic Tests for Syphilis: Flocculation techniques
VDRL (Venereal Disease Research Laboratory) Slide Test
o Standard screening test
o Non-treponemal test but detects reagin (substance found in patients with treponema)
RPR (Rapid Plasma Reagin)
o Detects CARDIOLIPIN antibodies in syphilis
o Does not use heat inactivation
OTHER SEROLOGIC TESTS - Febrile/ Bacterial Agglutination Tests
Widal Test
o Typhoid; principle: direct agglutination in test tubes
Weil Felix
o For heterophile antibodies to Rickettsial infections
o Proteus strains are used because they cross-react to Rickettsia antigens
OTHER SEROLOGIC TESTS - CRP (C-Reactive Protein)
What does C stands for? In 1930, it was discovered that there is an acute phase protein which reacts when you have inflammation. C is the polysaccharide capsule of pneumococcus. The level of CRP is found to be the highest among the other acute phase proteins.
OTHER SEROLOGIC TESTS - ASO (Anti-Strepsolysin-O)
This is a marker for streptococcal infection
Done by doing serial dilutions.
OTHER SEROLOGIC TESTS - TORCH
Toxoplasma, Rubella, Cytomegalovirus, Herpes Simplex Virus (usually in newborns)
Used as a panel for those with failure to thrive
OTHER SEROLOGIC TESTS - ELISA (Enzyme-linked Immunosorbent Assay)
Used for HIV and Hepatitis
You have to know the kinetics of the antigens and antibodies involved – when it rises and when it goes down. This is so you will know the important serologic markers to note in acute inflammations.
For example, with a patient who has an HIV antigen (p24 antigen) which rises early and also dies down early, you won’t be able to get accurate numbers if you do not get the specimen, use the incorrect method, have the wrong time frame - it may result in false negative results.
Note that the presence of increased antibodies will mean that the patient has already had a previous encounter of the antigen
In confirming for HIV, perform 2 ELISA and 1 western blot.
SOME NOTES ABOUT THE PROCEDURES IN SEROLOGIC TESTS:
o Note that the procedure are in very small amount (mL) so we have to very accurate in the delivery of the reagents and the volume of serum sample used because it may lead to prozone or postzone.
o Be sure to read on a specified time. Time lapsed could be a false reaction.
Check the volume of the specimen and reagents
PRO ZONE: excess antibody
POST ZONE: excess antigen
ZONE OF EQUIVALENCE: you should be here
o Make sure to also check the mixing procedure.
o Avoid contamination from other sites.
o Check the type of test indicated in the box because for example a positive reaction in direct agglutination is different with that of agglutination inhibition
o Know the shelf life of the reagent.
o Know also the significant titers for each tests and diseases
o Latex and Reagents are mixed to determine positive or negative reaction.
MAIN ANTIBODIES
Confirmation for HIV (ELISA)
Anti-GBM (Anti-Glomerular Basement Membrane)
o Kidneys
ANA or Anti-Nuclear Antibody
o Systemic lupus Erythematous (SLE)
Anti platelet Abs
o Idiopathic Thrombocytopenic Purpura (ITP)
Anti-Sm (Sm = Smith)
o Seen in 1/3 of SLE patients
Anti-sperm
o For those who have Antiphospholipid Antibody Syndrome (APAS)
WESTERN BLOT
Confirmation for HIV
o Patient’s serum is taken => substrate is added
Antigens can be “blotted” by Transverse Electrophoresis on to nitrocellulose sheets => bind non-specifically
Identified by staining with appropriately labeled antibodies
(+) bands = gp41 and/or p24 (HIV antigen)
o p24 is the antigen for HIV
o At 6 months, all would have sero-converted so it’s safer to check for antibody
There are criteria to compare with the standard
o Positive Control/Negative Control
o The antibody is more permanent than the antigen
Note: The specimen is usually BLOOD => separated into PROTEINS => further separated into RNA, then placed on an agar gel => blotted by TRANSVERSE ELECTROPHORESIS on a nitro cellulose sheet (bind non-specifically) => labeled with a specific ANTIBODY
DENGUE INFECTION
In dengue, the second attack is worse because of antibody enhancement.
IgG and IgM levels in dengue infection. If you had mosquito bite 4 days from now, you will have fever until 5 days and there is presence of NS1 Ag until about 6 days. IgM antibody will start to rise at day 4, while the IgG will only start to rise at day 14-21
DENGUE INFECTION: Immunological Response to Primary Dengue Infection
NS1 antigens (Non-structural 1)
o A glycoprotein essential for viral replication and viability
o Appears as early as day 1 after onset of fever and up to Day 6
o Circulate at high levels in serum during the entire clinical illness and in the first fever days of convalescence
o It is not detectable once anti-NS1 IgGAbare produced (corresponds to defervescence)
IgM antibodies
o First antibody to be produced
o Produced as early as day 4 & may persist up to 14 days
o Rise from 1-3 weeks, may persist up to 60 days
o May be detectable up to 6 months then gradually
decrease
IgG antibodies
o Second antibody to be produced after IgM
o Appears approximately 14 days after onset of symptoms
o Persist for life
o “Sometimes the basis for admission is CBC. Usually, the hemoglobin is constant while the hematocrit is rising, so there is hemoconcentration. The platelets then start to decline so after the patient is seen, it is usually when he has the lowest platelet count.”
MISCELLANEOUS SEROLOGY
Mycoplasma
Rabies
Chlamydia
Malaria
o Rapid Diagnostic Test – employed in the field or in massive blood donations; principle is finding the plasmodium lactate dehydrogenase (LDH)
Leprosy
o Lepromin test – similar to tuberculin test; positive for those with good immune system
o will also have a skin reaction – early (Fernandez reaction) and late (Mitzuda reaction)
Pregnancy Test – tests for hCG
Purified Protein Derivative (PPD) – Type IV: Delayed Hypersensitivity
TB Pathozyme
Moan Hemagglutination Test – for E. histolytica
Cryptococcal Ag
CSF Serology – Phadebact (brand name of kits for bacteriological diagnostics based on agglutination techniques) for knowing causative agent of bacterial meningitis; will result to a low CSF sugar (60-75% of the random blood sugar) compared to random blood sugar
Chronic Fatigue Syndrome- Epstein barr related
Pertussis
ANTIBODIES TO ANTIGENS OF FUNGAL DISEASES
Aspergillosis o Detect serum precipitin o CIE/Ouchterlony>/= 1:512 Candida o Detect serum precipitin o Ouchterlony Cryptococcus o latex agglutination (serum, urine, CSF): RF interferes Coccidioidomycosis o IgM early; IgG late
APPLICATION OF MOLECULAR DIAGNOSTICS
Nucleic Acid Hybridization Techniques
Nucleic Acid Amplification – PCR (Polymerase Chain Reaction)
Strain Typing and Identification
APPLICATION OF MOLECULAR DIAGNOSTICS: Nucleic Acid Hybridization
Involve using a labeled nucleic acid probe to identify related DNA or RNA molecules
Probe – a set of known genetic sequences
Northern Blot – Uses RNA probe (RNA is easily degraded)
Southern Blot – Uses DNA probe; named after Edward Southern
**General procedure for Northern Blot hybridization. The electrophoresis will allow the migration of the different specimens, and will also hybridize the probes. Note that the northern blot uses RNA.
APPLICATION OF MOLECULAR DIAGNOSTICS: Nucleic Acid Amplification: Polymerase Chain Reaction
Why do we want to amplify genetic sequences? Because it is easier to detect. An example is the TB PCR. Sometimes the patient has signs and symptoms of TB, history of exposure, chest x-ray, skin test is positive, BUT the sputum smear is negative for acid-fast bacilli. It’s because you need around ten thousand bacilli for it to become positive in the smear. For PCR, 1-10 bacilli can give a positive result.
PCR is now the trend. The culture will only based on phenotypic characteristic, PCR will base on genotypic characteristic (molecular diagnostic)
In nucleic acid, we use DNA because RNAs are easily degraded and contamination is usually expected in RNA.
PCR is quite expensive.
Uses a thermal cycler (taking temperatures per cycle)
Gene amplification technique intended for too small or too few organisms
The technique aims to amplify fragments in clinical specimens using enzymes and extracted DNA fragments through repeated thermal cycles
Ability to detect and identify organisms that cannot be grown in culture or are extremely difficult to grow or grow slowly
Steps are repeated 30-40 times and the result is a large molecule (amplification)
Can amplify a single copy of DNA by million fold in
APPLICATION OF MOLECULAR DIAGNOSTICS: Nucleic Acid Amplification: Polymerase Chain Reaction
Involves three processes with varying temperatures:
o Denature – 94oC
o Annealing – 50oC
o Extension – 60oC
- In the denaturation stage, the 5’ and 3’ ends are destroyed. The first step requires a high temperature to denature the dsDNA. This is typically done by briefly heating the sample to 92-94oC.
- Annealing stage. The second step requires lowering the temperature to allow annealing of the primers to the ssDNA. The optimal annealing temperature depends upon the melting temperature of the primer-template hybrid. If the temperature is too high the primers will not anneal efficiently, and if the annealing temperature is too low the primers may anneal nonspecifically. Determining the optimal temperature at this stage is critical.
- Extension stage. The third step requires DNA synthesis by DNA polymerase. To withstand the repeated exposure to high temperatures, a thermostable DNA polymerase is used for PCR. Taq polymerase from Thermus aquaticusis used because it can withstand high temperatures
APPLICATION OF MOLECULAR DIAGNOSTICS: Nucleic Acid Amplification: Polymerase Chain Reaction
The procedure requires that the DNA sequences that flank the desired DNA sequence be known, so that short oligonucleotide primers can be synthesized.
The DNA mixture is denatured into single strands by a brief heat treatment.
The DNA is then cooled in the presence of an excess of the oligonucleotide primers, which hybridize with the complementary ssDNA.
A temperature-resistant DNA polymerase, Taq polymerase, is then added, together with the four deoxyribonucleotide triphosphates, and each strand is copied.
The newly synthesized DNA duplex is separated by heating and the cycle is repeated. In each cycle there is a doubling of the desired DNA sequence in only 25 cycles the desired DNA sequence can be amplified about a million-fold.
DNA Gel electrophoresis is usually performed often after amplification of DNA via PCR, but may be used as a preparative technique prior to use of other methods such as PCR, cloning, DNA sequencing, or Southern blotting for further characterization.
The final product is an amplicon.
DNA Gel Electrophoresis is usually performed often after amplification of DNA via PCR, but may be used as a preparative technique prior to PCR, cloning, DNA sequencing, or Southern blotting for further characterization.
Electrophoresis
The next step after nucleic acid hybridization
Moves the molecules into the gel matrix and enables the sorting of molecules based on size and charge. A driving force is used to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.
Components:
o Electromotive force/Driving force – move molecules through the matrix
o Support medium – cellulose acetate, agrose gel, polyacrylamide gel, startch gel
o Buffer
o Sample
o Detecting system/stains
SUPPORT MEDIUM
A. Cellulose Acetate
B. Agarose Gel
C. Polyacrylamide Gel
D. Starch Gel
SUPPORT MEDIUM - Cellulose Acetate
Dry, brittle film with 80% air space (When the film is soaked on the buffer air spaces are filled with electrolytes, and the film becomes pliable.)
Can be stored for a long period of time
SUPPORT MEDIUM - Agarose Gel
could be used to separate DNA molecules ranging from several hundred nucleotides in length up to 10,000
Highly purified seaweeds
From Rhodophyta (red) algae (Genera Gelidium and Gracilaria)
Used for large nucleic acids
Stains and film are stored indefinitely
SUPPORT MEDIUM - Polyacrylamide Gel
Separates proteins
Toxic
Strong and elastic
SUPPORT MEDIUM - Starch Gel
Separates proteins
Not widely used due to technical difficulty in preparing the gel
*MRSA: Multiple resistant Staphylococcus aureus
*Through PCR, you will know if the MRSA is hospital-acquired or community-acquired. Community-acquired would have PVL (Panton-Valentine leukocidin) and it is more aggressive thus it is more severe and needs immediate treatment. Usually seen in prisoners (hygienic situation in the prison). Hospital-acquired is common in doctors (stethoscope). Transmission of disease is very common.
Applications of PCR
Epidemiologic – to determine the source of infection
To differentiate (E. histolytica and E. dispar look exactly the same, but we have to use PCR to differentiate them)
To measure the viral load – can be used in HIV, Hepatitis. Using ELISA, we can only determine if it is reactive or not. In PCR, we can have quantitative values.
Herpes simplex, Treponema, Trichomonas
TB detection – 16S rRNA
For reclassification – P. jiroveci, P. shigelloides
To differentiate wild types (usual type) vs. mutants
