# Presentation: 329406

## HIV TESTING TECHNOLOGIES 

**Dr. V. RAMESH BABU, MD (Micro)**
**GOVT. HOSPITAL OF THORACIC MEDICINE**
**TAMBARAM SANATORIUM**
**CHENNAI-47**

**HIV TESTING TECHNOLOGIES **
**ELISA / WESTERN BLOT**

## Introduction

**GENERAL TREND OF OUR COUNTRY                            IN DIAGNOSIS**
**VARIOUS ISSUES IN HIV TESTING**
**TESTING GUIDELINES**

**Introduction**

## Objectives for HIV testing

** ****Surveillance**
** ****Blood transfusion safety**

**Objectives for HIV testing**

## Kinectics of Antibody

**  ****STRUCTURAL PROTEIN OF HIV1 AND HIV 2 **
**     ****HIV 1 ENV – gp41, 120, 160 core – p55, 18, 24**
**     ****pol – p31, 51, 65**
**  ****HIV 2 ENV – gp36, 140, core – p56, 26, 16 **
**     ****pol – 68, 53, 34**
**  ****Viral entry, Target cell (CD4) **
**  ****Window period **
**  ****IgM. IgG**

**Kinectics of Antibody**
**Response to HIV**

## HIV STRUCTURE

## Evolution of Immune response 

**Evolution of Immune response **

## Different Test for HIV

**DIRECT **
**INDIRECT**
**PRE-TEST COUNSELING, **
**INFORMED CONSENT,**
**CONFIDENTIALITY.**

**Different Test for HIV**

## Challenges of HIV Testing

**Sensitivity- Early diagnostic ****( window period)**
**Specificity- Cross reactivity**
**Easy to perform, low cost **
**Detection of HIV-1 and HIV-2 and discrimination between the two viruses**
**One test can not fulfill these requirements **
**Need to perform a combination of HIV tests for screening and confirmation **

##     Current HIV technologies 

-  **Screening tests**
** ****Enzyme immunosorbent assays (EIAs)**
** ****Simple/rapid immuno-diagnostics  assays**
-  **Confirmatory or supplemental tests**
-  **Western blot (WB) **
-  **Alternatives to confirmatory tests**
** ****Repetitive EIA or rapid assays**

**    ****Current HIV technologies **

## EIAs (Enzyme Immunosorbent  Assays)

**This enzyme acts on its substrate to produce a  colour change.Test results are measured by measuring this colour. **
**Four immunologic principles**
**Indirect**
**Competition**
**Sandwich**
**Immuno-capture****  **

**EIAs (Enzyme Immunosorbent  Assays)**

## Indirect EIA

**Indirect EIA**

## Competitive EIA

**A measured amount of known enzyme-labeled component (being measured) is added to the reaction at the same time patient sample is added.**
**The labeled component therefore **_**competes**_** ****against the unlabeled component in the patient sample for binding sites.  **
**Results**
**Negative Reaction has color change**
**Positive Reaction no color change**

## Sandwich EIA (Ab-Ag-Ab)

**Sandwich EIA (Ab-Ag-Ab)**

## IgG-Capture EIA

**Serum (1/100)**

**Biotin**

- -

**Labeled Ag**

**Strepavidin**

- -

**Peroxidase**

**TMB**

**Substrate**

**Goat**

- -

**anti**

- -

**Human**

- -

**IgG**

**Capture Antibody**

## Reason for EIA 

**Easy to Perform**
**Use to screen large number of sample**
**Sensitive**
**Specific**
**Cost effective**

**Reason for EIA **

## Evolution of HIV ELlSAs

-  **First generation**
** ****Ag : Purified lysates of HIV**
** ****Poor sensitivity and specificity**
** ****Second generation**
** ****Ag : HIV-recombinant proteins and/or peptides**
** ****Detection of HIV-1 and HIV-2**
** ****Poor sensitivity, improved specificity**
** ****Third generation**
** ****Ag : HIV-recombinant proteins and/ or peptides**
** ****Detection of IgM and IgG, improved sensitivity **
** ****Detection of HIV group O **
** ****Fourth generation**
** ****Capacity to detect Ag (P24) and antibodies **

## Components of Commercially Available EIA Kits

** ****Solid–Phase Support**
**  ****Antigens bound to polystyrene microtiter plates                                          ****	****(passive absorption)**
**  ****Blocking is necessary to reduce nonspecific ****	****binding (test accuracy)**
**  ****96 microwell format**
** ****Antigens**** **
**  ****The use of cloned antigens has reduced non-****	****specific binding**
** ****Antibodies**** **
**  ****Monoclonals of high titer, affinity and avidity**

## Components of Commercially Available EIA Kits

**  ****Conjugates**
**  ****Ab conjugated with enzyme                                                   ****	****(without effecting binding site)**
**   ****Enzyme**
**  ****HRP (horseradish peroxidase)**
**   ****Substrate (chromogenic)**
**  ****Colorless chromogen reacts with enzyme     color**
**   ****Stop Solution**
**  ****Typically an acid, stabilizes the color for a limited ****	****time**

## Sources of Error for HIV EIA TestsDocumented Sources of False Negative Results

**AIDS Patients**
**Early Seroconverters**
**Operator Error**
**Equipment Error**

## Operator Error

**FAIL TO ADD SERUM OR REAGENT TO THE CORRECT WELL **
**REAGENT DILUTED IN WRONG DILUVENT                  IN WRONG DILUTION**

**Operator Error**

## Source of False Positive Results

**MULTIPLE TRANSFUSION**
**AUTO IMMUNE DISORDER**
**CHRONIC HEPATITIS,**
**CHRONIC ALCOHOLIC**
**HBV VACCINATION**
**ANTIBODY TO POLYSTERENE**

**Source of False Positive Results**

## Trouble shooting EIA

**Kit integrity**
**Controls (kit and in-house) **
**Equipment**
**Cross contamination**
**Sample quality**
**Personnel training**
**Correct validation and interpretation of results**

## Kit integrity

- Was cold chain maintained during kit transport?
- Has the kit expired?
- Has the kit been stored properly in your lab?

## Controls

**Kit Controls should be run on each EIA **_**plate**_**.**
**Indicates whether the kit components are functioning.**
**Used to validate EIA run, calculate cut off**
**In-house controls**
**Kit controls are designed to be quite robust and          do not reflect subtle changes in testing.**
**In-house controls should be calibrated to test as        a low positive (above cut-off, below maximum)**

## Equipment : Pipettes

**Single and Multi channel Pipettes should be                   calibrated on a monthly basis.  **
**This can be done using a balance. **
**Inaccurate pipetting**

## Equipment : Microplate Washers 

**Daily**
- Prime the washer with wash solution before running sample plates
- Set the washer to wash the recommended number of times               (with correct volume)
- Check for accurate dispensing and complete aspiration in each plate well, if not clean the washer head
- Listen for changes in the sound the washer makes, this can  indicate a vacuum leak
- At the end of the day prime the washer with DI water

## Equipment : Micro-plate Washers

**Weekly**
**If a washer is not used during the week rinse it out with DI water to reduce microbial growth.**
**Monthly**
**Run a 10% solution of ethanol through the washer to disinfect. This can also be done if          the washer exhibits signs of contamination (high background).**
**Thoroughly rinse the washer after alcohol is used.  **

## Equipment : Micro-plate Reader

**Daily**
**Each time a reader is turned on it runs a              self test, it will then report any errors****.**
**Weekly**
**Run a control plate weekly. Variations in positive or negative specimens could be                 a  sign of a bad diode or a spill on a diode****.**

## Cross contamination

**Can be caused by:**
**Reusing pipette tips (contaminated                 with + plasma)**
**Splashes from one well to another**
**During removal of plate covers**

## Sample Quality

**Properly collected (no haemolysis)**
**Transport conditions**
**Storage conditions**
**Number of freeze/thaw cycles**
**Age of sample**

## Validation and Interpretation of Results

**Product inserts provide guidelines**
**Positive and Negative controls must fall within a certain range.**
**Controls are used to calculate a cut-off.**
**Samples below cut-off are negative, those above are positive**

## Western Blot (Immunoblotting)

**Solid-phase EIA with immobilized viral antigens to detect antibodies to specific HIV proteins.**

## Creating Western Blot Strips

***Diagram reproduced from ***_***Commercial Methods in Clinical Microbiology***_***, ASM Press***

**HIV lysate proteins are separated by size using gel electrophoresis**

**Proteins are transferred (blotted) onto the surface of a membrane**

**Strips are incubated with patient serum and antihuman IgG conjugated with an enzyme (and chromagen)**

**The membrane is**
**cut into strips**

## HIV Western Blot Banding Pattern

***Image reproduced from ***_***Commercial Methods in Clinical Microbiology***_***. 2000. ASM Press.***

**env****	****gp160**
**	****gp120**
**	****gp 41**
**gag****	****p55**
**	****p18**
**	****p24**
**pol****	****p65**
**	****p51**
**	****p31**
**	**

## Interpretation of Results(General Consensus)

**Negative:****	****               No bands present**
**Positive:****	****               2 ENV band present                                                  ****	****                        (WHO Guidelines) **
**	****	****	****	**
**Indeterminate:****	****      Any bands present but           ****	****	****	****      do not meet criteria for ****	****	****	****	****      positive**

## Western Blot Banding

***Image reproduced from ***_***Commercial Methods in Clinical Microbiology***_***, 2000. ASM Press.***

- *

- *

- *

## HIV-1 Seroconversion – Western blotBBI - Panel C

**gp160/120**
**p66**
**p55/51**
**gp41**
**p32**
**p24**
**p17**

**HIV-1 Seroconversion – Western blot*****BBI - Panel C***

## When should WB be used? 

- Western Blot assay should not be used as a screening test.
- WB should be viewed as a supplemental test which can be used to confirm positive results obtained from EIA.
- HOWEVER:
-  Specificity is less than that of EIA
-  A significant number of indeterminate blots are seen    	in low risk populations

## Advantages

**Specific interaction of antibody and antigen         can be directly visualized.**

**Disadvantages**

**  ****Technically demanding**
**  ****Expensive**
**  ****Subject to interpretation**
**	****Presence or absence of bands**
**	****Intensity of those bands**

**Western**** Blot**

## Thank You