Immunoblot
Immunoblot used at Rayan Specialised Laboratory EUROIMMUN immunoblots
Different technologies for comprehensive antibody testing.
- Extensive portfolio of products for autoimmune, infection and allergy diagnostics.
- Best test quality due to in-house antigen development and production.
- Multiplex tests with only one strip per patient serum.
- No additional reagents required, all necessary reagents included in the test kit.
- Ready-for-use reagents, exchangeable between products, with colour codes for safe assignment.
- Universal instead of individual incubation schemes for uncomplicated combined processing of different parameters.
- Optimised for automated processing with blot processors such EUROBlotOne and EUROBlotMaster.
- Automated evaluation, result reporting and archiving of blot strips with the EUROLineScan software.
Test principle
In the immunoblot, antigens coated on membranes are used as a solid phase in order to detect specific antibodies in patient samples. The test performance is either manual, semi- or fully automated. If a sample contains specific antibodies, these bind to the membrane-bound antigens. In the next step, an alkaline phosphatase (AP) labelled antibody (conjugate) is added, which binds to the specific antibodies. The alkaline phosphatase catalyses a colour reaction with the subsequently added nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP). If specific antibodies are present in the patient sample, a dark line appears at the respective antigen position. The intensity of the resulting staining is proportional to the antibody concentration in the sample.
EUROLINE
EUROLINE assays are multiparameter line blots which enable generation of comprehensive combined antibody profiles on one test strip. Membrane strips with multiple purified, biochemically characterised antigens or antigen extracts as thin parallel lines are used as the antigen-containing solid phase. The membranes are fixed as chips at exactly defined positions on plastic foils. The membrane strips do not contain superfluous proteins which may lead to unspecific positive results. On every EUROLINE test strip, there is a control band which indicates whether the individual incubation steps have been performed correctly. The evaluation is performed fully automatically with the EUROLineScan software.
Westernblot and EUROLINE-WB
The Westernblot is the method of choice when positive results from a screening test (indirect immunofluorescence or ELISA) are to be confirmed or further differentiated. Blot strips are used as antigen-containing solid phase. They are coated with antigen extracts electrophoretically separated according to molecular mass.
EUROLINE-WB assays are a combination of Westernblot and EUROLINE tests: The Westernblot strips are additionally coated with highly purified native or recombinant antigens in the form of membrane chips. The correct performance of the individual incubation steps is indicated by the staining of the control band at the lower end of each strip. The evaluation is performed fully automatically with the EUROLineScan software.
Different profiles for different Autoimmune diseases:
| ANA Profile 3 | S.Sclerosis Profile | Autoimmune Liver Profile 2 | Myositis Profile 3 | ANCA Profile | Neuronal Antigen Profile 2 | Gangloside Profile 2 IgM, IgG | S.Sclerosis Profile |
| nRNP/Sm | SCL-70 | AMA M2 | Mi-2 | Proteinase 3 | Amphiphysin | GM1 | SCL-70 |
| Sm | CENP A | M2-3E | Ku | MPO | CV2 | GM2 | CENP A |
| SS-A | CENP B | LKM-1 | PM-SCL100 | Elastase | PNMA2 | GM3 | CENP B |
| Ro-52 | RP11 | LC-1 | PM-SCL75 | Cathepsin G | Ma-2 \ Ta | GD1a | RP11 |
| SS-B | RP155 | SLA\LP | Jo-1 | BPI | Ri | GD1b | RP155 |
| Scl-70 | FIBRILLARIN | SRP | Lactoferrin | Yo | GT1b | FIBRILLARIN | |
| PM-Scl | NOR90 | PL-7 | Hu | GQ1b | NOR90 | ||
| Jo-1 | Th\To | PL-12 | Th\To | ||||
| CENP B | PM-Scl100 | EJ | PM-Scl100 | ||||
| PCNA | PM-Scl75 | OJ | PM-Scl75 | ||||
| dsDNA-NcX | Ku | Ro-52 | Ku | ||||
| nucleosome | PDGFR | PDGFR | |||||
| Histones | Ro-52 | Ro-52 | |||||
| Rib.P-prot | |||||||
| AMA M2 |
ANA Profile (Anti nuclear autoantibodies): The most important associated diseases
| Antigen | Disease | Prevalence |
| Double stranded DNA | Systemic lupus erythematosus (SLE) | 60 – 90 % |
| Single stranded DNA | Systemic lupus erythematosus Drug induced SLE MCTD (Sharp syndrome) Polymyositis / Dermatomyositis Progressive systemic sclerosis (PSS), Sjogern’s syndrome, rheumatoid arthritis | 70 – 95 % 60 % 20 – 50 % 40 – 50 % 8 – 14 % |
| RNA | Systemic lupus erythematosus PSS, Sjogern’s syndrome | 50 % 65 % |
| Histones | Drug induced SLE Systemic lupus erythematosus Rheumatoid arthritis | 95 % 95 % 15 – 50 % |
| U1-nRNP | MCTD (Sharp syndrome) Systemic lupus erythematosus Rheumatoid arthritis | 95 – 100 % 15 – 40 % 3 % |
| Sm | Systemic lupus erythematosus | 5 – 40 % |
| SS-A (Ro) | Sjogern’s syndrome Systemic lupus erythematosus Neonatal Lupus syndrome | 40 – 95 % 20 60 % 100 % |
| SS-B (La) | Sjogern’s syndrome Systemic lupus erythematosus | 40 – 95 % 10 – 20 % |
| Fibrillarin | PSS, diffuse form | 5 – 10 % |
| RNA polymerase I RNA helicase A | PSS, diffuse form Systemic lupus erythematosus | 4 % 6 % |
| PM-Scl (PM-1) | Poly-/ dermatomyositis / overlap syndrome PSS, diffuse form | 50 – 70 % 5 – 10 % |
| Centromeres | PSS, limited form | 80 – 95 % |
| Scl -70 | PSS, diffuse form | 25 – 75 % |
| Cyclin (PCNA) | Systemic lupus erythematosus | 3 % |
| Ku | Systemic lupus erythematosus Poly-/ dermatomyositis,PSS | 10 % 30 – 55 % |
| Mi-1, Mi-2 | Dermatomyositis | 5- 10 % |
- Autoimmune diseases can be treated.
- A positive ANA test means autoantibodies are present. By itself, A positive ANA test does not indicate the presence of an autoimmune disease or need for therapy.
- Some medications cause A positive ANA test. Tell your doctor all prescription, over the counter, street drugs and supplements you take.
- ANA testing can produce a positive result without any actual disease process. This typically signals the presence of antinuclear antibodies in a healthy individual.
- Talk to your doctor about a positive ANA and the best next steps for further evaluation.
Profiles for Food & Inhalation Allergy:
| INHALATION PROFILE | FOOD PROFILE | ||
| (g6, g12, t2, t3, t7, t9, w1, w6, d1, d2, i6, e1, e2, e3, m1, m2, m3, m5, m6, CCD) | (f1, f75, f2, f4, f14, f45, fs36, f13, f92, f33, f44, f93, f25, f31, f48, f83, f88, f3, f23, CCD) | ||
| g6 | Timothy grass (عشب تموثي) | f1 | Hens egg white (بياض البيض) |
| g12 | Rye (عشب) | f75 | Hens egg yolk (صفار البيض) |
| t2 | Alder (عشب ألدر) | f2 | Cow’s milk proteinالبقر) بروتين لبن) |
| t3 | Birch (شجرة البتولا) | f4 | Wheat flour (دقيق القمح) |
| t7 | Oak (شجرة البلوط) | f14 | Soybean (فول الصويا) |
| t9 | Olive (زيتون) | f45 | Bakerꞌs yeast S. cerevisiae (خيره الخبز) |
| w1 | Ragweed (عشب) | fs36 | Nut Mix2 (Pea nut,Hazel nut, almond) (مكسرات البندق واللوز والفول السوداني) |
| w6 | Mugwort (عشب الميرميه) | f13 | Peanut (الفول السوداني) |
| d1 | Dermatophagoides pteronyssinus (عت غبار المنزل الاوربيه) | f92 | Nutmeg (جوز الطيب) |
| d2 | Dermatophagoides farina (عت غبار المنزل الامريكيه) | f33 | Orange (برتقال) |
| i6 | German cockroach (صرصور) | f44 | Strawberry (فراوله) |
| e1 | Cat epithelium (القط) | f93 | Mango (مانجو) |
| e2 | Dog hair (الكلب) | f25 | Tomato (طماطم) |
| e3 | Horse epithelium (حصان) | f31 | Carrot (الجزر) |
| m1 | Penicilium notatum (فطر) | f48 | Apple (تفاح) |
| m2 | Cladosporium herbarum (فطر) | f83 | Chicken (الدجاج) |
| m3, | Aspergillus fumigates (فطر) | f88 | Mutton /Lamb (لحم الضأن) |
| m5 | Candida albicans (فطر) | f3 | Codfish (سمك البحر) |
| m6 | Alternaria tenuis (فطر) | f23 | Crab (سرطان البحر) |
| CCD | CCD marker | CCD | CCD marker |