7th week practice

1. Immunological techniques based on primary antigen-antibody reactions 7th weekpractice ELISA

2. May be you have already met such kind diagnostic tools, or you are going to meet them during your career eg. detection of human chorionic gonadotropin in serum or urine (pregnancy test) The principles of these tools are similar as the ELISA assay you have met before.

3. capture/sandwich simple Ag specific detection/reporter antibody detection/reporter antibody Ag specific antibody antigen Ag specific capture antibody antigen competitive Ag specific antibody antigen ELISA types (review) eg. for detecting Ag specific antibodies antigen detecting purpose the Ag is present in the solution Ag is not present You can detect the Ag with a single antibody, or you can detect Ag specific antibodies with higher sensitivity

4. Bioactive material assays hCG (human chorionic gonadotropin) – pregnancy test doping/drug assay: EPO(erithropoetin), steroids sandwich assays or competitive tests

5. 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 antybody lane glass fiber membrane with visually labeled detection antibodies sample application pad urine

6. detection antibody capture antibodies hCG + hCG negative control lane (C) test lane (T) control antibody lane hCG capture antibody lane detection antibodies hCG

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

8. 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 colors”) • colored latex beads The assay can be used in semi-quantitative manner competitive immunochromatographic test strips for detecting aflatoxin

9. 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 Viral infection testing 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

10. HIV supplemental confirmatory tests: Western-blot: The solid phase antigens are derived from LAV strain of HIV-1 grown in the CEM cell line. You can obtain pre-wet blot membrane strips and incubate with the examined serum. HIV specific serum antibodies result specific bands on the Western blot. At least two envelop protein specific band or one envelop specific and the p24 antigen specific band can be considered as positive test. Immunofluorescent assay (IFA): The assay uses immortalized human T-cells which express HIV-1 antigens on their surface. The cells are fixed to the surface of an IFA glass slide. Fixed, uninfected T-cells are provided as a control. The serum or plasma sample HIV- I antibodies comes in contact with the HIV-1 antigens on the slide. Fluorescent secondary antibodies detects them. The interpretation of the degree and pattern of fluorescence of the infected cells of the IFA slide compared to the uninfected cells determines the confirmed HIV-1 status of the sample.

12. Autoimmunity Autoantibodies from different autoimmune diseases can be detected similarly. • eg. • 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/GADAb negative patients, and therefore can be considered independent markers of disease. • insulin: spec. Ab. 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).

13. Antinuclear (ANA) autoantiboies from the serum of a SLE patient can be visualized in cell culture (Hep-2) by indirect fluorescent labeling (immunofluorescence)

14. Tumordiagnostics Tumor Ag specific detecting/reporter antibody (suplemented) Tumor antigen (patient serum) Tumor Ag specific capture antibody precoated plate (as a part of the kit) • Tumor specific (TSA) and tumor associated (TAA) antigen recognizing 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 used similarly in quantitative measurement.

15. 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 (HTG) 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. The 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.

16. 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 immunresponse (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 autoimmun process, and in the case of some parasite infection • Abnormal levels of serum immunoglobulin isotypes can be seen in the case of some immunodeficiency (eg. hyper IgM syndrome) • (See the past case study about myeloma multiplex)

17. Practice:

18. 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 !!!

19. 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

20. Estimating the concentration with a „ruler” The OD are proportional with the concentrations in this range points with identical OD 62 31 16 7.8 3.9 1.9 500 250 125 0.97 0.49 0.24 0.12 1000 0.061 0.030 0.015 0.007 0.004 0.002 2X 4X 8X 16X 32X 64X 128X 256X Dilutions of the unknown sample So, the concentration of the (undiluted) unknown sample: 1.9x128 = 243.2μg/ml OD The concentrations are equal in the tubes The 1.9μg/ml diluted standard corresponds to the… conc. of the standard (µg/ml) … 128-fold diluted unknown sample

21. You can use linear regression (Least-squares analysis), and calculate the concentrations with the equations (formula) of the lines fitted on the linear parts of the dilution curves 62 31 16 7.8 3.9 1.9 500 250 125 0.97 0.49 0.24 0.12 1000 0.061 0.030 0.015 0.007 0.004 0.002 2X 4X 8X 16X 32X 64X 128X 256X Dilutions of the unknown sample OD Ysample=mx+b ystd=mx+b conc. of the standard (µg/ml) (two-fold dilution (log scale!))

22. This OD range results false concentrations also The range of suitable OD values The OD of any highly diluted solutions will be located on this range of the dilution curve.If you insert this OD value into the formula and calculate the concentration by multiplying it with the dilution, then you get enormous high FALSE concentration. Serious errors You must know the optical density range that you should use to calculate the concentration with the equation(formula) of the dilution line! OD The fitted line with its equation(formula) y=mx+b  OD=m(concentration)+b Dilution curve concentration

23. Presentation ELISA plate with serially diluted IFNγ standard and Tcell culture supernatants Which is the concentrated sample? • Try to calculate the concentration of the given ELISA data at home! • calculate the mean of the 3 parallel data • use the logarithm of the dilution to draw the dilution curves • try to use a computer with spreadsheet program