ANALYTICAL AND PREPARATIVE METHODS BASED ON PRIMARY ANTIGEN-ANTIBODY BINDING

1. ANALYTICAL AND PREPARATIVE METHODS BASED ON PRIMARY ANTIGEN-ANTIBODY BINDING IMMUNOAFFINITY CHROMATOGRAPHY ELISA

2. SENSITIVITIES OF IMMUNOASSAYS

3. AFFINITY PURIFICATION OF ANTIBODIES USING AN ANTIGEN-SORBENT COLUMN column polymer beads affinity purified antibody : monoclonal antibodies which can be ordered from catalogues are also purified using this technique covalently bound antigen

4. STEPS OF PURIFICATION • Addition of antibodies to be purified • Binding • Washing • Elution

5. PURIFICATION OF ANTIGENS coloumn fixed antigen specific Abs on the surface of the bead • Loading the antigen mixture • Binding • Washing polimer bead • Elution Purified antigens

6. IMMUNOPRECIPITATION • isolation and concentration of a particular protein from a protein mixture • detection of protein associations (e.g. members of receptor signalization)

7. ELISAEnzyme Linked ImmuneSorbent Assay ELISA plate well

8. enzyme immune sorbent enzyme linked Antigen/antibody adsorbed to solid surface Antibody conjugated with enzyme

9. ENZYME ACTIVITY IN ELISA IS DIRECTLY PROPORTIONAL TO THE AMOUNT OF ANTIGEN PRESENT Enzyme activity is measured by the color reaction due to conversion of substrate Similar principle applies to many other antibody-based detection methods

10. Label Secondary antibodies Label Antigen BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Direct method Indirect method Primary antibodies

11. Enzyme Enzyme-specific antibody, same isotype as the primary antibody Secondary antibody Primary antibody Antigen BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Enzyme/anti-enzyme system PAP – peroxidase / anti-peroxidase APAAP – alkaline phosphatase / anti- alkaline phosphatase

12. Avidin-biotin enzyme complexes Avidin-enzymecomplexes Biotin-enzyme complex Avidin Biotinylated antibody Antigen BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Indirect systems combined with biotin-avidin signal amplification (Avidin binds biotin with very high affinity ) ABC Basic

13. SENSITIVITIES OF IMMUNOASSAYS

14. EXAMPLES FOR DIRECT, INDIRECT AND COMPETITIVE ELISAs

15. STEPS OF COMBINED SANDWICH ELISA For antigens present at low concentration in complex biological samples Removal of excess enzyme Coating with Ag-specific „capture” antibody Removal of unbound material Removal of unbound protein Blocking free plastic surface with inert protein Removal of unbound material Addition of antigen- containing solution Addition of biotinylated antibody specific to a different epitope on target protein Addition of avidin-conjugated enzyme Addition of substrate

16. PRACTICAL USE OF IMMUNOASSAYS hCG (human chorionic gonadotropin) – pregnancy test Detection of tumor antigens, cytokines, hormones doping/drug assay: EPO (erythropoietin), steroids sandwich assays or competitive tests

17. Tumor Ag specific detecting/reporter antibody (suplemented) Tumor antigen (patient serum) Tumor Ag specific capture antibody precoated plate (as a part of the kit) TUMORDIAGNOSTICS • Tumor specific (TSA) and tumor associated (TAA) antigenrecognizing antibodies can be used in diagnostics • The antigens can be detected by sandwich techniques • PROSTATE CANCER: • Prostate-Specific Antigen (PSA) derives its name from its first known site of origin, the prostate gland. Serum concentrations of PSA are elevated in patients with prostate cancer, benign prostatic hypertrophy (BPH) and prostatitis. In addition, PSA serum levels appear to correlate with the volume and clinical stage of prostate cancer. • Human prostatic acid phosphatase (PAP) in human serum can be usedsimilarlyinquantitative measurement.

18. Bladder cancer: NMP22 is a Nuclear Matrix Protein found in human epithelial cells. In the urine of healthy individuals, the protein is present at low levels. The majority of patients with bladder cancer release large quantities of NMP22 into their urine, that can be detected by immunoassay Thyroid Cancer: Increased Serum Thyroglobulin (sTG) can be detected by immunoassay Alpha Fetoprotein (AFP)is a 68 kDa protein, which is produced primarily during fetal life by the fetal liver and yolk sac. AFP also appears in the maternal serum, presumably by transplacental transfer . After birth, serum AFP levels decline rapidly during the first year of life and low basal levels are then apparently maintained throughout childhood and adult life. Its normal concentration in serum is below 9 ng/mL; higher concentrations are associated with hepatoma and ovarian, testicular and presacral teratocarcinomas, and other cancers. Carcinoembryonic Antigen (CEA)is a glycoprotein involved in cell adhesion. It is normally produced during fetal development. serum from individuals with colorectal and other carcinomas had higher levels of CEA than healthy individuals and can be used to monitor the response to colon cancer treatment. Her-2/neu protein is a 185 kD trans-membrane glycoprotein associated with tyrosine kinase activity. Approximately 20-30% cases of breast cancer show an amplification and/or over-expression of Her-2/neu in tumor cells. Since the introduction of Herceptin as a targeted therapy for breast cancer, the clinical testing of Her-2/neu in breast carcinoma has become very important in patient care. It can be shown by immunohistochemistric or immunofluorescent methods from biopsy.

19. THE ROLE OF THE HER-2/NEU PROTEIN IN THE PATHOGENESIS OF BREAST CANCER AND THE HERCEPTIN THERAPY

20. STEPS OF BASIC INDIRECT ELISA Detection of antigen or specific antibody Removal of excess antibody Adsorption of antigen (coating) Removal of excess antigen Saturation of uncovered surface area with proteins Removal of excess protein Addition of Ag-specific antibodies Addition of Secondary Ab conjugated with enzyme Addition of chromogenic substrate

21. Human Ig specific labeled antibodies indicate the presence of the virus specific antibodies. The serum of the infected person with virus specific antibodies. The antibodies could bind the virus antigens. viral antigen precoated test plate TESTING VIRAL INFECTION Viral antigens cannot be detected efficiently in lot of case (latency), but you can efficiently detect the antibodies that were produced by the body in response to the viral infection. These antibodies can serve as diagnostic markers. EXAMPLES: • Epstein-Barr Virus (EBV) test kit →ELISA method for the qualitative detection of IgG antibody to Epstein-Barr Virus nuclear antigen-1 (EBNA-1) in human serum • HIV assay kit →enzyme-linked immunosorbent assay for the detection of antibody to HIV-1 in serum, plasma or dried blood spots • Toxoplasmosis (Toxoplazma gondii – parasitic protozoa) → chromatographic immunoassay for the qualitative detection of human IgM antibodies against Toxoplasma in serum or plasma

22. AUTOIMMUNITY Autoantibodies from different autoimmune diseases can be detected similarly. • In 60% of SLE patients autoantibodies can be detected against double strand DNA. Anti-ENA antibodies can be seen in many systemic AIDs. (See Ouchterlony method in the previous practice) • Type I (autoimmune) diabetes can be detected by the presence of antibodies against islet-cell specific antigens. • Glutamic acid decarboxylase (GAD65), specific antibodies have been found in 70-90% of prediabetic and Type 1 diabetic patients (including approximately 7-10% of adult onset diabetics with Type 1 diabetes) • IA-2 (a tyrosine phosphatase-like protein) specific Ab. are found in 50-75% of Type 1 diabetic patients at and prior to disease onset, are generally more prevalent in younger patients, and are associated with rapid progression to overt disease. These autoantibodies have been detected in some ICA positive/GAD Ab negative patients, and therefore can be considered independent markers of disease. • Insulin: anti-insulin ABs are found predominantly, though not exclusively, in young children (<5 years) developing Type 1 diabetes. In insulin-naive (untreated) patients, the prevalence of autoantibodies to insulin is almost 100% in very young individuals and almost absent in patients with adult onset of Type 1 diabetes. (It should be noted that insulin autoantibodies are indistinguishable from insulin antibodies that commonly develop with insulin therapy).

23. isotype specific antibody antibody from the serum antibody capture antibody SERUM ANTIBODY ISOTYPE DETERMINATION Sometimes the antigen specific antibodies could refer the presence of the antigen in the body (see the”viral infection testing” part ) The isotypes of these antibodies additionally could refer the fresh/persistent (IgM dominance) or repeated/memory immuneresponse (IgG dominance) against the parasite. The possibilities can be discriminated by the use of isotype specific secondary antibodies. α-IgM α-IgG Memory response IgM IgG Antigen • Increased IgE level can be seen in atopic allergy cases, and some autoimmune process, and in the case of some parasite infection • Abnormal levels of serum immunoglobulin isotypes can be seen in the case of some immunodeficiencyies (e.g. hyper IgM syndrome, multiplex myeloma (case study!))

24. COMPETITIVE ELISA Highly sensitive method used to detect and quantitate small amounts of antigen in complex biological samples. Antigen in solution and on the solid surface compete for the binding site of labeled specific antibody. - Coating with antigen, blocking - Addition of experimental sample that contains or lacks antigen - Addition of labeled antibody  binding - Washing - Addition of enzyme substrate

25. Maybe you have already met such kind of diagnostic tools or you are going to meet them during your career e.g. detection of human chorionic gonadotropin in serum or urine (pregnancy test) The principles of these tools are similar to the ELISA assay you have met before.

26. hCG Rapid One-Step Immunochromatographic Assay strip side view front view absorbtion pad (cellulose) control antibody lane (detection antibody capture) nitrocellulose membrane (signal detection pad) hCG capture antibody lane glass fiber membrane with visually labeled detection antibodies sample application pad urine

27. detection antibody capture antibodies hCG positive hCG negative control lane (C) test lane (T) control antibody lane hCG capture antibody lane detection antibodies hCG

28. Competitive system hCG positive hCG negative control lane test lane control lane detection antibody capture antibody hCG lane ( bound hCG)

29. You don’t need to use additional enzymatic incubation steps, because the labels on the detection antibodies can be observed by naked eye: • colloidal gold („surface plasmon” resonance) • paint-filled latex beads The assay can be used in semi-quantitative manner Other uses of immunechromatographic test strips: detection of toxins in food e.g.: aflatoxins (mycotoxins of Aspergillus spp.)

30. ELISA PLATES - RESULTS ELISA plate with different concentration samples after the addition of the chromogen substrate ELISA plate with different concentration samples after stopping the enzyme reaction

31. DETERMINATION OF THE CONCENTRATION A quantitative property of an indicator refers to the concentration: • color (absorbance, optical density) • fluorescence • cell number (e.g. in determination of growth factor concentration) Quantified concentration can be obtained by comparison with known concentration sample (standard) The principle of comparison: equal absorbances  equal concentrations PARTIAL TRUTH !!!

32. The serial dilution of the standard OD The sample with unknown concentration You should also dilute the unknown sample This region could indicate the concentration According to OD: it could be anyone ? 62 31 16 7.8 3.9 1.9 500 250 125 0 0.97 0.49 0.24 0.12 1000 0.061 0.030 0.015 0.007 0.004 concentration