IMMUNOLOGY SECTION
Test that been run in this bench
including
- Dengue serology
- Rapid Plasma Reagin (RPR) Test
- Treponema Pallidum particle agglutination
- Rheumatoid Factor Test
- Anti-Streptolysin O
- Widal/Weil Felix
- Cerebral fluid VDRL
Rapid Plasma Reagin (RFR) Test
Purpose: This test was done for the serodiagnosis of syphilis
Method:
Qualitative
- Using a disposable plastic pipette, dispense a drop of serum or plasma on to circle on a test card. Spread the sample over the entire area of the circle using the flat end of the pipette.
- Gently, shake antigen dispensing bottle before used.
- Using the dispensable bottle or needle assembly allow one free falling drop of antigen to drop on the test specimen.
- Rotate for 8 minute under humidifying cover on mechanical rotator T100
- Immediately after 8 minute, inspect the result visually in good light.
Semi Quantitative
- For each specimen to be tested, place 0.005 ml of 0.9%saline into the circles, numbered 2 to 5. A capillary (red line) or serological pipette 1ml or less maybe used.
- Using capillary with rubber bulb attached, place 0.05 ml of specimen onto circle 1
- Refill capillary to red line with test specimen and holding in a vertical position. Prepare serial two fold dilution by drawing saline and test specimen mixture up and down capillary.
- Transfer 0.05 from circle 2 to 3 to 4 to, mixing after each transfer. Discard 0.05 ml after mixing contents in circle 5
- Using a new stirrer for each specimen, start at highest dilution of serum and spread serum filling the entire surface of the circle. Proceed to circle 4,3,2 and 1 and accomplish similar spreading
- Gently shake antigen distending bottle before used. Holding in vertical position \, clear. Place one free falling drop onto each test area. Pick up the pre dropped antigen from bottle cap.
- Rotate for 8 minute under humidifying cover or mechanical rotator at 100 (+2rpm)
Qualitative test
- Medium and large aggregates -Reactive
- Finely dispersed aggregates -weakly reactive
- No aggregates visible, smooth gray -nonreactive
- Semi quantitative test
- Undiluted - reactive
- 1:2 -reactive
- 1:4 -reactive
- 1:8 -non reactive
- 1:16 -non reactive
Treponema Pallidum Particle Agglutination (TPPA) Test
Purpose: To detect antibodies of Treponema pallidum in serum or
plasma specimen
Principle:
SERODIA TPPA kit is manufactured
using gelatin particle carriers sensitized with purified pathogenic TP (Nichols
Strain). The test is based on the principle that sensitized particle are
agglutinated by the presence of antibodies to TP in human serum or plasma
Method
Qualitative Test
Well number
|
1
|
2
|
3
|
4
|
Sample diluents( µl)
|
100
|
25
|
25
|
25
|
Test specimen( µl)
|
25
|
25
|
25
|
25
|
Test specimen diluents
|
1:5
|
1:10
|
1:20
|
1:40
|
Unsensitized particles ( µl)
|
25
|
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Sensitized particles ( µl)
|
25
|
|||
Final dilution
|
1:40
|
1:80
|
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Mix content using plate mixer
|
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Interprete result
|
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Quantitative Test
Interpretation Of Result
Setting pattern of particle
|
Interpretation
|
|
Particle concentrated in the
shape of a button in the center of the well with a smooth round outer margin
|
(-)
|
Non reactive
|
Particle concentrated in the
shape of a compact ring with smooth round outer margin
|
(+/-)
|
Intermediate
|
Definite large ring with a
rough multiform outer margin and peripheral agglutination
|
(+)
|
Reactive
|
Agglutinated particles spread
out covering the bottom of the well uniformly
|
(++)
|
Rheumatoid factor Test
Purpose: Detect rheumatoid factor in human serum as an aid in the
diagnosis of rheumatoid arthritis
Principle
The MBDr Rheumatoid factor latex
test provides a suspension of polystyrene latex particle which have been coated
with heat treated human IgG. The immunoglobulin present in most cases of
rheumatoid arthritis bind to the human IgG coating the latex particle. In
specimens having abnormally high level of RF, this binding is evident by rapid
agglutination of the latex
Method
- Allow kit reagent and patient serum to come to room temperature
- Transfer 50µl of patient serum to the test circle on the test card
- Shake the latent reagent, using the dropper provided, add one drop of suspension to the test card circle
- Mix drops using a disposable stirrer
- Gently and evenly, rock and rotate the test card for two minutes whilst examining the test slide for agglutination
Result And Interpretation
- Positive result is indicated by the obvious agglutination pattern in latex. in clear solution
- Negative result is indicated by no changes in the latex suspension on the test card
- Positive result will be obtaining at a RF serum concentration of 81U/ml and above
- Negative result will be obtaining at a RF serum concentration <81U/ml
Anti-Streptolysin O Test (ASOT)
Purpose: Detection of anti
Streptolysin antibody in human serum.
Principle: The ASO-Latex is a
rapid agglutination procedure for the direct detection and semi quantization on
slide of anti-streptolysin O (ASO). The antigen, a particulate latex suspension
coated with streptolysin O, agglutination in the presence of specific
antibodies in sera of presence with streptococcal Beta-hemolytic infection
(group A and C)
Method
- Allow kit reagent and patient serum to come to room temperature
- Transfer 50µl of patient serum to the test circle on the test card
- Shake the latent reagent, using the dropper provided, add one drop of suspension to the test card circle
- Mix drops using a disposable stirrer
- Gently and evenly, rock and rotate the test card for two minutes whilst examining the test slide for agglutination
Result And Interpretation
- Positive result is indicated by the obvious agglutination pattern in latex. In clear solution
- Negative result is indicated by no changes in the latex suspension on the test card
- Positive result will be obtain at a RF serum concentration of 200lµ/ml and above
- Negative result will be obtain at a RF serum concentration <200lµ/ml
Widal/Weil Felix Test
Purpose:
In this test, patient serum is by
2 methods which are using test card for screening test and by tube
agglutination test for titres the antibodies against H, O and VI. Suspension of
enteric bacteria that are commonly in an area such as Salmonella typhi and Salmonella
parathphi A
Principle
The test depends on the ability
of antibody in patient’s serum to agglutinate the stained bacterial antigens.
When this occurs the aggregates become clearly visible to the naked eye
Methodology
Test Card Test
- One drop of 50µl of patient serum was dropped onto 4 circle of test card.
- For each circle, one drop of reagent TO, TH and 2 type of TVI was used.
- Mix drops using a disposable stirrer
- Gently and evenly, rock and rotate the test card for two minutes whilst examining the test slide for agglutination
Test Tube Agglutination Test
Results
Test Card Test
Positive result is indicated by
the obvious agglutination pattern in any of each circle.
Negative result is indicated by
no changes in any circle
Tube Agglutination Test
Agglutination of the antigen indicates
the presence of antibody. Titres excess of 1/80 is probably significant. A
comparison between samples taken 10- 14 days apart may be value in acute
illness
Antigen
|
Dilution
|
Interpretation
|
Typhi O
|
Dil>1:80
|
Significant for Salmonella
infection
|
Typhi H
|
Dil<1:80
|
Not suggestive of enteric fever
infection. Suggest repeat within 10-14 day if clinically indicated
|
A(H) Paratyphi
|
Dil >1:80
|
Titres excess of 1:80 is
probably significant. Repeat test for comparison within 10-14 days apart
|
B(H) Paratyphi B-H
|
Dil < 1:80
|
Dengue Serology
Purpose: This analyzer is fully
automated to do the full serial testing of dengue serology by using Enzyme Immunoassay
method. This analysis is done by using a machine called Personal Lab machine
Method
- Start the personal Lab analyzer.
- Preparing conjugate. Dengue antigen will be diluted with antigen diluents before mixed with HRP (Mab tracer) to set amount of solution that will be needed. (Dilution 1/250)
Prepare positive control,
negative control and calibrator.
- 10µl negative control + 1000µl serum diluents (pink color)- vortex
- 10µl positive control + 1000µl serum diluents (pink color)- vortex
- 10µl calibrator + 1000µl serum diluents (pink color)- vortex
Prepare patient sample. Patient
sample diluted with serum diluents solution
- 10µl patient sample + 1000µl serum diluents (pink color) - vortex.
- Put diluted patient sample serum test tube into plab instrument (black rack) from right to left start at no 1 in the Personal Lab analyzer.
- Calibrator, Positive control, negative control and conjugate were inserted into the Personal Lab analyzer.
- Switch on the Personal Lab analyzer.
- Let the Personal Lab analyzer work and result will be automatically display on the computer.
Anti Nuclear Antibody Screening
Intended Use
The Bio-Rad Autoimmune EIA ANA
Screening Test is a qualitative enzyme immunoassay (EIA) intended to screen for
the presence of antinuclear antibodies (ANAs) in human serum as an aid in the
diagnosis of certain systemic rheumatic diseases. This assay collectively
detects, in one well, total ANAs against double stranded DNA (dsDNA, nDNA),
histones, SS-A/Ro, SS-B/La, Sm, Sm/RNP, Scl-70, Jo-1 and centrometic antigens,
along with sera positive for immunofluorecent (IFA) Hep-2 ANAs.
Explanation Of The Test
Antinuclear antibodies (ANAs)
directed against a variety of macromolecule occur in extraordinarily high
frequency in systemic rheumatic diseases. Although these antibodies were first
associated with systemic lupus eryhematosus (SLE), the list of implicated
diseases has expanded and many rheumatic diseases are characterized by the
presence of one or more of these Sjogren’s Syndrome (SS), anti-dsDNA and
anti-Sm antibodies with SLE, anti-histone antibodies with SLE, anti-Scl-70
antibodies with scleroderma (progressive systemic sclerosis [PSS]), anti-Jo-1
antibodies with polio myositis and dermatomyositis and anti-centromere
antibodies with CREST syndrome.
The immunofluorescent assay (IFA)
has been used as the standard of method in the detection of ANAs. Although the
IFA is a sensitive test, it is laborious when testing large numbers of patient
samples and is subject to errors from human interpretation and from variability
in fluorescent microscopes. The IFA Hep-2 ANA test is also subject top the
following concerns : it is sometimes insensitive to certain sera containing
antibodies to SS-A, SS-B, Sm, or dsDNA and it tends to find sera positive in a
large number of patients who do not develop systemic rheumatic diseases within
a follow-up two year period. The Enzyme Immunoassay (EIA) test system is an
excellent alternative to the IFA test system for screening patient’s serum for
the presence of ANAs of clinical significance. The EIA test system efficiently
screens large numbers of patient samples and reduces human error.
The Bio-Rad Autoimmune EIA ANA
Screening Test collectively detects, in one well, total ANAs against double
stranded DNA (dsDNA,nDNA), histones, SS-A/Ro, SS-B/La, Sm, Sm/RNP, Scl-70, Jo-1
and centrometic antigens, along with sera positive for immunofluorecent (IFA)
Hep-2 ANAs. Sera positive on the EIA ANA Screening Test should be tested for
specific autoantibodies indicative of various systemic rheumatic diseases.
Principle
Purified antigens (dsDNA,
histones, SS-A/Ro, SS-B/La, Sm, Sm/RNP, Scl-70, Jo-1, centromere and other
antigens extracted from the Hep-2 nucleus) are bound to microwells. Antibodies
to these antigens, if present in diluted serum, bind in the microwells. Washing
of the microwells removes unbound serum antibodies.
Horseradish peroxidase (HRP)
conjugated anti-human IgG immunologically binds to the bound patient antibodies
forming “conjugate-antibody-antigen” sandwich. Washing of the microwells
removes unbound conjugate. An enzyme substrate in the presence of bound
conjugate hydrolyzes to form blue color. The addition of an acid stops the
reaction, forming the yellow end product. The intensity of the color is measured
photometrically at 450 nm.
Anti Double Stranded DNA
Intended Use
The Bio-Rad Autoimmune EIA
Anti-dsDNA Screening Test is a quantitative enzyme immunoassay (EIA) intended
to screen for the presence of antinuclear antibodies (ANAs) in human serum as
an aid in the diagnosis of certain systemic lupus erythematosus.
Explanation Of The Test
Antinuclear antibodies (ANAs)
directed against a variety of macromolecule occur in extraordinarily high
frequency in systemic rheumatic diseases. Many rheumatic diseases are
characterized by the presence of one or more of these ANAs. Therefore, the
identification of the specific antibody is useful in the detection and
diagnosis of the disease.
Anti-dsDNA is present in 50-70%
of patients with SLE. Circulating DNA /anti-DNA immune complexes are considered
to play a part in the pathogenesis of SLE. The presence of anti-dsDNA is one of
the diagnostic criteria for SLE. IgG antibodies to dsDNA are considered
clinically most useful for the diagnosis and management of SLE. Antibodies to
single stranded DNA (ssDNa) and IgM antibodies to dsDNA are found in a number
of other connective diseases, liver diseases, as well as in some normal
individual. Accurate detection of anti-dsDNA is important in the diagnosis and
management of SLE. EIA tests for anti-dsDNA have demonstrated greater
sensitivity than standard IFA and RIA tests allowing for improved detection of
low titer antibodies to dsDNA.
Principle
Purified dsDNA is bound to
microwells. The DNA retain its antigenicity and remain double stranded.
Antibodies to dsDNA, if present in diluted serum, bind in the microwells.
Washing of the microwells remove unbound serum antibodies. Horseradish
peroxidase (HRP) conjugated anti-human IgG immunologically binds to the bound
patient antibodies forming “conjugate-antibody-antigen” sandwich. Washing of
the microwells removes unbound conjugate. An enzyme substrate in the presence
of bound conjugate hydrolyzes to form blue color. The addition of an acid stops
the reaction, forming the yellow end product. The intensity of the color is
measured photometrically at 450 nm.
Extractable Nuclear Antigens Plus Screening
Intended Use
The Bio-Rad Autoimmune EIA ENA
Screening Test is an enzyme immunoassay (EIA) intended for the detection and
quantitative measurement of antibodies against Extractable Nuclear Antigens
(SS-A/Ro, SS-B/La, Sm, RNP, Jo-1, Scl-70) in human serum as an aid in the
diagnosis of certain systemic rheumatic diseases..
Explanation Of The Test
Antinuclear antibodies (ANAs)
directed against a variety of macromolecule occur in extraordinarily high
frequency in systemic rheumatic diseases. Many rheumatic diseases are
characterized by the presence of one or more of these ANAs. Therefore, the
identification of the specific antibody is useful in the detection and
diagnosis of the disease.
Extractable Nuclear Antigens
(ENAs) are acidic (non-histone) macromolecules extracted from the saline soluble fraction of cell
nuclei;hence the term ENA. There are now 20 different saline-extractable
antigens identified. Antibodiies to ENAs are seen in several clinical syndromes
including Systemic Lupus Erythematosus (SLE), Mixed Connective Tissue Diseases
(MCTD), Sjogren’s Syndrome (SS), Myositis and Progressive Systemic Sclerosis (PSS)
and are considered to be markers for these diseases.
The Bio-Rad Autoimmune EIA ENA
Plus Screening Test simultaneously
screens in a single well for SS-A/Ro, SS-B/La, Sm, RNP, Jo-1, Scl-70
autoantibodies. Sample found to be negative can be considered not positive for SS-A/Ro,
SS-B/La, Sm, RNP, Jo-1, Scl-70.
Principle
Purified ENAs (SS-A/Ro, SS-B/La,
Sm, RNP, Jo-1, Scl-70) are bound to microwells. Antibodies to these antigens, if
present in diluted serum, bindto the ENAs in the microwells. Washing of the
microwells remove unbound serum antibodies. Horseradish peroxidase (HRP)
conjugated anti-human IgG immunologically binds to the bound patient antibodies
forming “conjugate-antibody-antigen” sandwich. Washing of the microwells
removes unbound conjugate. An enzyme substrate in the presence of bound
conjugate hydrolyzes to form blue color. The addition of an acid stops the
reaction, forming the yellow end product. The intensity of the color is
measured photometrically at 450 nm.
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