E2.L4 - Molecular Diagnostics - P1

Molecular diagnostics

identification of organisms as causative agents of disease.

Antibody Structure

2 identical heavy chains and 2 identical light chains. One variable chain on each heavy chain, the rest are constant domains. The heavy chains are stabilized and attached with disulfide bonds. The 2 light chains also attached by disulfide bonds to the heavy chains. Each light chains has one variable domain and one constant domain.

Polyclonal Response

Activation of multiple B-cells

Monoclonal response

Antibodies produced by a single B-cell clone.

Making a monoclonal antibody (Fusion)

Animal immunized with antigen. Spleen harvested and the leukocytes are isolated. Need to induce fusion or myeloma(cancer cells) and B-cells in order to grow immortal cells. Long polymers of ethylene glycol (~4,000 daltons) added to cause crowding conditions. This creates random collisions between myeloma and Bcells. May have myeloma-myeloma or Bcell-Bcell interactions due to the random aspect of the conditions. The myeloma-Bcell is called a Hybridoma.

Selecting for a Hybridoma Fusion event: Denova Pathway

No nucleotides used to start. Precursors are used which eventually lead to nucleotides. Aminopterin used to block denova pathway.

Selecting for a Hybridoma Fusion event: Salvage Pathway

Uses preexisting nucleotides. Hypoxanthine and Thymadine used to create nucleotides. 

This media is called HAT (Hypoxanthine Aminopterin Thymadine). Only hybridoma can survive using this media. Bcell-Bcells die on their own. Myeloma can not use salvage pathway.

Cultivating Hybridoma

Dilution that isolates single hybridoma in well. They are allowed to proliferate. Then a small volume of the antibodies are pulled and tested to see if they bind to the gene product of interest. If they cannot, they are discarded. The antibodies that can bind, were produced from cell producing the desired antibody.

Uses of Antibodies

A blood type has universal H antigen and N-Acetylgalactosamine. B has H and Galactose. AB has both N-Galactosamine and Galactose added to the H antigen. Type O express only H antigen.

Enzyme Linked Immunosorbent Assay (ELISA)

Capable of reacting with colorless substrate to produce a colored product. Enzyme linked to a monoclonal antibody.

Direct ELISA: Used in wastewater treatment to detect contaminated treated water. Contaminanted and uncontaminated samples put in wells. The contaminant sticks nonspecifically to the well walls. Enzyme conjugated antibody added to both wells. It binds to the contaminants. The wells are washed and the Ab that did not bind are removed. Colorless substrate added to both wells. Colorless in uncontaminated well, but purple in contaminated well.

Indirect ELISA:

for detection of Rheumatoid factor. IgG antigens on microscope slides. Serum of Rh+ and Rh- individuals gathered and added to the slides. Rh factor antibodies binds to IgG. Conjugated alphaRh secondary antibodies bound to primary antibodies. Colorless substrate added and Rh+ serum will have color while Rh- will not.

Sandwich (capture) ELISA

Antibody attached to well, sample added to well, antigen captured by Ab. 1st Zone captures Conjugated antibody, 2nd zone captures polyclonal antibody, 3rd zone contains alpha IgG. 2nd and 3rd zones produce color if positive and only 3rd for negative. If third zone does not produce color, then the test is invalid and the antibody has expired.

Sandwich (capture) ELISA: ovarian cancer screening

Normal serum does not have human epididymis protein 4, while afflicted serum does. Biotin binds to HE4 in afflicted serum. Samples passed over wells containing streptavidin which binds biotin. Conjugated antibody added and following wash, substrate added. Color development = positive test

Immunofluorescent Assays

Antibodies attached to fluorophores. Antibody glows when exposed to UV light. Infected individual sample smeared on slide. Fluorophore conjugated antibody added to slide, Ab attach to infectious organisms, and they will fluoresce when illuminated by UV light. Glowing = positive test.

Immunofluorescent Assays for Allergies

Protein microarrays used with probe cells. Serum containing IgE added to allergens/antigens in probe cell. Fluorophore Conjugated anti IgE Ab added. Wash, then UV light to excite fluorophore. Allergens that glow indicate positive test for allergy.

Immunoassays for Conformation Specific Disorders

Alzheimer’s caused by Amyloid Beta plaques at end of inflamed nerves. Synthetic DNA expression allows for production of different sequences of the Abeta42 plaques. Motifs bound to Ab. mAb allows for detection of the Abeta plaques to diagnose Alzheimer’s

Nucleic Acid Hybridization Probes

Often times specific gene only found in pathogenic organism. Frequently a toxin or other virulence determinant. Stool sample applied to Nitrocellulose in dysentery testing. Positive dot blot test will have the nucleic acid of interest. Nucleic acid hybridization probe allows for detection of the positive test. Horse raddish peroxidase allows for luminescence on positive test. Luminol is a substrate that is hydrolyzed by HRP for emission of photon. Exposure to X-ray film will show a black spot on positive test.

Allele Specific Hybridization

Detects specific gene mutation that causes disease. Cystic fibrosis transmembrane conductance regulator (CFTR) gene defect causes CF. Common mutation is delta F508 3 nucleotide single codon deletion. Homoz Wt individual, Carrier, and Homoz deltaF508 individual. DNA PCR amplification for each individual. Wt has 2 F508, Carrier - 1F508 and 1 deltaF508, Infected has 2 delta508. Dot blot done with fluorophore conjugated WT sequence probe and fluorophore conjugated deltaF508 probe. Wt will fluoresce one color, infected another color, and the carrier will fluoresce a mix of the 2 colors.

Oligonucleotide Ligation Assay

Homz WT, carrier, and Homz Mutant. Single nucleotide polymorphism associated with mutant. Hmz WT has to copies of WT gene, carrier has 1 and 1. Hmz Mutant has 2 mutant copies. Common oligonucleotide probe will bind to Wt and is also labeled with green Fluorophore. Common Allele specific oligonucleotide probe binds to mutant gene and labeled with orange fluorophore. Both common probes added. DNA ligase added, then polyacrylamide gel ran. Hmz Wt will have green at higher point indicating higher molecular mass = this indicates complete base pairing from the ligation. Orange signal will be lower due to failure to ligate. Carrier will be mix of the 2 colors. Hmz Mutant results will be opposite of Wt.

Padlock Probes

Hmz Wt, carrier, and Hmz mutant. Single nucleotide polymorphism associated with disease. The probe detects SNP’s with a single oligonucleotide labeled with a fluorophore. DNA extracted and PCR. Dot blot for each individual DNA. DNA denatured to ssDNA. Padlock probes added. Wt has complete basepairing with Wt probe, but incomplete base pairing for Mut probe. Complete pairing of each probe for carrier. Complete pairing of mut and incomplete for wt probe in mutant DNA. DNA ligase catalyzes formation of phosphodiester bond at complete base pairing interactions. Wash removes unligated padlocks. Exposure to UV light shows one color for WT, another for mutant, and mixed for carrier .

More Efficient Nucleic Acid Hybridization Diagnostics

Makes use of molecular beacon. Specialized hybridization probe that uses fluorescence for diagnosis. Complementary sequence on ends of oligonucleotides. They form a hairpin, non-complementary sequence used for GoI. Quencher on 5’ and 3’ ends to prevent fluorescence. When complementary sequence binds to GoI, the ends separate. Exposure to UV light will now allow for fluorescence. No need for substrate or xray film.

Using a Molecular Beacon

Detecting different alleles of a gene that are associated with disease. HzWT, carrier, and HzMut. PCR. Dot blot. Denature, then Wt and mut beacons added. Basepairing to complementary sequences. Following wash and UV, HzWT fluoresces one color, Mutant fluoresces another, and carrier fluoresces a mix. Not commonly used for SNPs.

Allele Specific PCR

Used to detect SNP. 3 primers and 2 PCR needed. HmWT, carrier, and HzMut. PCR. One of the forward primers terminates with WT SNP at 3’ position and the other terminates with Mut SNP at 3’ position. Common reverse primer used in all PCR. Gel electrophoresis run. 2 lanes for each, WT and Mut. Product at WT lane for WT, Product at both lanes for carrier, and product at Mut lane for mutant.