Test Methodologies

ELISA (Enzyme-linked Immunosorbent Assay)

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying pathogen specific antibodies. Cell lysate or antigens (including peptides) are immobilized to a solid surface and then complexed with patient’s anti-pathogen antibody (primary antibody). The pathogen bound human antibody is captured by anti-human antibody, linked to an enzyme (Secondary antibody). Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measureable product.

image02

IFA (Indirect Immunofluorescent Assay)

This immunofluorescent assay indirectly detects pathogen-specific IgG or IgM antibodies in patient serum.

image01

Step 1: Patient serum is added on to the slide with fixed pathogens. If pathogen-specific IgG or IgM antibodies are present in the human serum they will bind to the fixed pathogen on the slide.

Step 2: Unbound serum is removed by washing the slide with a wash buffer, and a fluorescent labelled anti-human antibody is added.

Step 3: After washing of the excess secondary antibody, the slide is viewed under a fluorescent microscope. If pathogen-specific antibodies were present in the human serum, fixed pathogen on the slide will fluoresce bright green when viewed with a fluorescent microscope.

PCR

Currently IGeneX offers in-house developed PCR tests (Traditional and real-time PCR tests) for detection of tick-borne pathogens directly from clinical samples using their proprietary hybrid-select method.

The standard polymerase chain reaction (PCR) test is not always sensitive enough when very few organisms are present in a sample. Furthermore, PCR sensitivity is well-known to be reduced in the presence of inhibitors. Therefore, IGeneX, Inc. has developed nucleic acid-based PCR diagnostic assays that are highly sensitive and specific. Theoretically, these PCR tests can be performed on any type of sample. These PCR tests offer enhanced performance compared to microbiological, immunological, and amplified tests currently available for the detection of microorganisms in test samples. The PCR is a three-step amplified nucleic acid assay that detects pathogen specific DNA sequences in a clinical sample.

Principle
The multiplex PCR-based diagnostic test is performed directly on the clinical specimen in a three-step assay. The steps are:

  • Selection Hybridization Step
  • PCR Amplification
  • Detection of Amplified Products

image04

Selection Hybridization Step: Specifically removes the “common PCR inhibitors” from the clinical sample while simultaneously selects and purifies the DNA fragment of interest. This procedure also concentrates the fragment of interest, and therefore, improves sensitivity.

PCR Amplification: The purified pathogen DNA fragment of interest is PCR amplified with–pathogen specificprimers. This sequence “hybridizes” or binds specifically to pathogen DNA of interest and under predetermined PCR conditions. Therefore, only pathogen specific DNA is amplified.

Detection of Amplified Products: In the third step, the PCR amplified products are transferred and bound to a nitrocellulose membrane. The membrane bound PCR-amplified products are hybridized with pathogen specific probes. Only samples that have pathogen specific DNA hybridize to the probes and give a blue-purple color dot on the membrane.

The combination of these three steps imparts very high specificity and sensitivity to the test.

PCR tests are useful for the following reasons.
Accurate identification of biochemically unusual strains of pathogen is possible.
The assay is independent of the host’s immune response schedule. Therefore, much earlier detection of the microorganism is possible. Direct testing allows the monitoring of the efficacy of an antibiotic regime. The assay can be performed on any type of sample: EDTA whole blood, serum, cerebral spinal fluid, synovial fluid, urine, breast milk, tissue biopsy, and ticks.

FISH (Fluorescent In Situ Hybridization)

The Fluorescent in situ Hybridization Test (FISH) provides a significant increase in sensitivity and specificity over standard Geimsa- stained smears for the presence of bacteria, fungi and intraerythrocytic parasites (piroplasts) in RBCs.

Step 1: Methanol fixed blood smear from patient’s blood.

Step 2: After addition of hybridization buffer with pathogen specific probe labeled with fluorescent dye the slide incubated for a short time at 37*C. Pathogen rRNA hybridizes with the pathogen if present in the blood smear.

Step 3: After hybridization is complete, the excess probe is removed by washing with a special a wash solution.

Step 4: Smear is dried, counter stain is added and viewed with fluorescent microscope. Pathogens if present in the blood smear will fluoresce.

image05

Western Blots

The IgG and IgM Western Blot are qualitative immunoassay in which antibodies are visualized. The IgG and IgM Western blot are a qualitative immunoblot assays that determines whether the pathogen specific antibodies are present in patient serum or plasma. These tests are generally more sensitive and specific than the ELISA and IFA tests

Principle:
WB Strip Preparation: WB strips are prepared from a cell lysate of the pathogen of interest. Briefly, pathogen cell lysate is fractionated on acrylamide gel by electrophoresis. The separated proteins are transferred from the gel to a membrane. Membranes are washed, dried and sliced into 5 mm strips.

Immunoblotting: Patient serum or plasma is incubated with WB strip. If specific antibodies to pathogen antigens are present they bind to the corresponding antigen bands. After washing off the unbound serum the strip is incubated with alkaline phosphate-conjugated goat anti-human antibody. Bound antibodies react with BCIP/NBT, a chromogenic substrate. A dark purple colored precipitate develops on the antigen-antibody complexes. Bands are visualized, scored for intensities relative to the positive and negative controls.

Patients Serum tested by B. burgdorferi Western Blot

image07

LDA Lyme Dot Blot Assay

Lyme Dot-blot Assay (LDA) is a qualitative immunoassay for the direct detection of Borrelia burgdorferi specific antigens in urine using anti-B. burgdorferi antibodies.

image06

Purpose
The antigens present in urine are immobilized onto a membrane in a Dot-blot format and incubated with anti-B. burgdorferi specific antibodies. After washing the bound anti-B. burgdorferi specific antibodies are reacted with anti-rabbit IgG, which is visualized by an enzyme/substrate reaction.

If the initial Lyme panel tests on blood samples are negative, including PCR, but symptoms for Lyme disease are present, the Lyme Dot-blot assay on urine, especially test panel #875 consisting of Lyme Dot-blot assay and PCR, can be helpful in making the diagnosis.