7th weekpractice

ELISA

Immunological techniques based on primary antigen-antibody reactions

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.

ELISA types (review)

simple capture/sandwich

antigen

Ag specificantibody

detection/reporterantibody

competitive

eg. for detecting Ag specific antibodies

antigen

Ag specificcapture antibody

Ag specificdetection/reporter

antibody

antigen detecting purpose

the Ag is present in the solution

Ag specificantibody

You can detect the Ag with a single antibody, or you can detect Ag specific antibodies with higher sensitivity

antigen

Ag is not present

Bioactive material assays

hCG (human chorionic gonadotropin) – pregnancy test

doping/drug assay: EPO (erithropoetin), steroids

sandwich assays or competitive tests

hCG Rapid One-Step Immunochromatographic Assay strip

nitrocellulose membrane(signal detection pad)

glass fiber membrane with visually labeled detection antibodies

front view side view

hCG capture antybody lane

control antibody lane(detection antibody capture)

absorbtion pad (cellulose)

sample application pad

urine

hCG capture antibody lane

control antibody lane

detection antibodies

hCG

control lane (C)

test lane (T)

hCG + hCG negative

detection antibody capture antibodies

hCG lane( bound hCG)

control lanedetection antibody

capture antibodycontrol lane

test lane

hCG positive hCG negative

Competitive system

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

competitive immunochromatographic test strips for detecting aflatoxin

The assay can be used in semi-quantitative manner

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.

viral antigen precoated test plate

The serum of the infected person with virus specific antibodies. The antibodies could bind the virus antigens.

Human Ig specific labeled antibodies indicate the presence of the virus specific antibodies.

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

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.

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/GAD Ab 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).

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

Tumordiagnostics Tumor specific (TSA) and tumor associated (TAA) antigen

recognizing antibodies can be used in diagnostics The antigens can be detected by sandwich techniques

Tumor antigen (patient serum)

Tumor Ag specific detecting/reporter

antibody (suplemented)

Tumor Ag specificcapture antibody precoated plate

(as a part of the kit)

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.

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.

Serum antibody isotype determinationSometimes 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.

Antigen

IgM IgG

α-IgM α-IgG

Memory response

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)antibody capture antibody

antibody from the serum

isotype specific antibody

Practice:

Determination of the concentrationA 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 !!!PARTIAL TRUTH !!!

concentration

10

00

50

0

25

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12

5

62

31

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7.8

3.9

1.9

0.9

7

0.4

9

0.2

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0.1

2

0.0

30

0.0

61

0.0

07

0.0

15

0.0

04

0

The sample with unknown concentration

ODThe serial dilution of the standard

According to OD: it could be anyone

?

You should also dilute the unknown sample

This region could indicate the concentration

This region could indicate the concentration

ODEstimating the concentration with a „ruler”

conc. of the standard (µg/ml)

The

OD

are

pro

port

iona

l with

the

co

ncen

trat

ions

in t

his

ran

ge

2X 4X 8X 16X 32X 64X 128X 256XDilutions of the unknown sample

10

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50

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25

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12

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62

31

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7.8

3.9

1.9

0.9

7

0.4

9

0.2

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0.1

2

0.0

30

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0.0

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points with identical OD

The concentrations are equal in the tubes

The 1.9μg/ml diluted standard corresponds to the…

… 128-fold diluted unknown sample

So, the concentration of the (undiluted) unknown sample: 1.9x128 = 243.2μg/ml

OD

conc. of the standard (µg/ml)(two-fold dilution (log scale!))

2X 4X 8X 16X 32X 64X 128X 256XDilutions of the unknown sample

10

00

50

0

25

0

12

5

62

31

16

7.8

3.9

1.9

0.9

7

0.4

9

0.2

4

0.1

2

0.0

30

0.0

61

0.0

07

0.0

15

0.0

04

0.0

02

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

ystd=mx+b

Ysample=mx+b

Serious errorsYou must know the optical density range that you should use to calculate the

concentration with the equation(formula) of the dilution line!

Dilution curve

The fitted line with its equation(formula)y=mx+b OD=m(concentration)+b

OD

concentration

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.

This OD range results false concentrations also

The range of suitable OD values

PresentationELISA 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