WO2011022445A2 - Improved multiplexed, microparticle-based assay for antibodies to antigens - Google Patents

Abstract

The present invention provides improvements in assays for detection and identification of antibodies to viral antigens, such as Human Immunodeficiency Virus Type 1 (HIV-I), in a biological sample, such as human serum, blood, urine, plasma, cerebral spinal fluid and the like. The invention is advantageous for use to diagnose the presence of such antibodies in persons of unknown risk of infection, and to confirm the presence of such antibodies in samples from such persons, and is also intended to be utilized as a confirmatory assay for conventional enzyme immunoassay (EIA) and other rapid tests, to confirm the presence of antibodies against one or more viral antigens in a sample.

Description

Improved Multiplexed, Microparticle-Based Assay for

Antibodies to Antigens

Cross Reference to Related Application

This application is a non-provisional application, which is incorporated by reference herein and claims priority, in part, of US Provisional Application No. 61/235141, filed August 19, 2009.

Background

Field of the Invention

The present invention relates to diagnostic assays, and particularly to in vitro diagnostic assays and assay systems for detection and identification of antibodies to infectious disease antigens, such as Human Immunodeficiency Virus Type 1 (HIV-I), in biological samples from a human subject or patient. The present invention is therefore advantageous for use to diagnose the presence of such antibodies in persons of unknown risk of infection, and to confirm the presence of antibodies to infectious disease antigens, such as anti-HIV antibodies, in samples from such persons. The invention is also intended to be used as a confirmation immunoassay to confirm the results of other assays, such as conventional enzyme immunoassay (EIA) and other rapid tests to confirm the presence of antibodies to antigens, such as anti-HIV antibodies against one or more HlV-I antigens in a biological sample. The improvements provided by the invention enable previously known, labor-intensive and subjective confirmation methods to be advantageously improved to be more labor- friendly, objective, and automatable confirmation methods.

Background Art

For conventional antigen serology methodologies, such as in HIV serology, the process usually begins with a screening test that is relatively rapid to perform. If a positive result is obtained, the specimen is repeated. If a specimen is repeat reactive, they a confirmation assay is then performed on the specimen. In the case of HIV-I antigen, numerous conventional assays and procedures exist for confirming the presence of anti-HIV antibodies in a biological sample, such as blood, serum or cerebral spinal fluid from a human patient. For example, there are conventional assays such as the well-known Western Blot test, one specific example of which is the test commercially available from BioRad Genetic Systems, as the Human

Immunodeficiency Virus Type I Western Blot test kit.

A conventional Western Blot assay for HIV-I is generally produced by first separating the viral proteins via SDS PAGE and then transblotting those proteins on to a solid support (membrane) such as nitrocellulose. A strip containing these proteins is then incubated with the patient serum. If the patient possesses antibody to any of the viral proteins, that antibody will bind to that area of the nitrocellulose strip. The strip is then washed to remove any unbound serum antibody, and incubated with an enzyme conjugate, such as goat anti-human Ig labeled with horseradish peroxidase. Once again, the strip is washed to remove any unbound conjugate. Finally, the strip is soaked in a precipitating enzyme substrate. If antibody was bound to the nitrocellulose strip, a visible line will form. This line corresponds to a "positive" reaction to that particular viral protein. Depending on the intensity of the reaction to a particular protein, and the combination of viral proteins one possesses antibody to, a person is deemed to be confirmed positive for anti-HIV or not. These criteria have been clearly defined in the United States by the Centers for Disease Control. Conventional western blot assays, such as described above, are known to be extremely labor intensive, extremely subjective, difficult to manufacture, and require extremely skilled individuals to perform and interpret. Accordingly, there is a particular need for an improved assay system to diagnose the presence of such antibodies in persons of unknown risk of infection, and to confirm the presence of anti-HIV antibodies based on results from such conventional assays, and also to confirm results produced by other assays such as conventional enzyme immunoassay (EIA) and other rapid tests that are commonly utilized, to confirm the presence of anti-HIV antibodies against one or more HlV-I antigens in a patient sample.

Summary of the Invention

The present invention provides improvements in diagnostic assays and assay systems for the detection and identification of antibodies to antigens, in particular viral antigens such as Human Immunodeficiency Virus Type 1 (HIV-I), in biological samples, especially in samples such as human blood, serum, or other body fluids. The present invention is advantageous for use to diagnose and to quantitatively determine the presence of such antibodies in persons of unknown risk of infection, and to confirm the presence of such antibodies in patient samples upon which another assay has been performed, and thus is also suitable to be utilized as a confirmatory assay for conventional enzyme immunoassays (EIA) and other tests, for example to confirm the presence of anti-HIV antibodies against one or more viral antigens, such as HlV-I antigens in a patient sample. The improvements provided by the invention thereby enable previously known, labor-intensive and subjective confirmation methods to be advantageously improved to be more labor-friendly, objective, and automatable confirmation methods.

An improved assay provided by the instant invention, exemplified in the practice of one preferred embodiment thereof for the detection and identification of antibodies to Human

Immunodeficiency Virus Type 1 (HIV-I), involves a procedure comprising the following steps:

1. incubating a biological sample to be tested from a subject suspected to have anti- HIV antibodies against one or more HlV-I antigens in a container containing a multiplexed mixture of a bead suspension comprising a mixture of distinguishable sets of microspheres made of a suitable material, for example polystyrene, each set being conjugated with a different HIV-I viral antigen;

2. rinsing the microspheres to remove any non-reactive proteins; 3. adding phycoerythrin-conjugated anti -human IgG, IgM or IgA, or a mixture thereof, to the container; and

4. incubating the container for a suitable period of time.

Detailed Description of the Invention

The present invention provides improvements in diagnostic assay methods, and assay systems, including an improved quantitative assay for detection and identification of antibodies to antigens, which can include but are not limited to Human Immunodeficiency Virus Type 1

(HIV-I) in biological samples, such as human serum, blood, urine, plasma, cerebral spinal fluid and the like. The present invention is advantageous for use to diagnose the presence of such antibodies in persons of unknown risk of infection, and also to confirm the presence of such antibodies in patient samples, and is also suitable to be utilized as a confirmatory assay for conventional enzyme immunoassay (EIA) and other rapid testing techniques, to confirm the presence of antibodies against one or more viral antigens, such as HlV-I antigens, in patient samples.

It is to be appreciated that the sample to be analyzed by the methods of the present invention can be any type of biological sample that can be taken from an organism, including without limitation a human subject or patient, and can include, without limitation, such samples as serum, blood, urine, plasma, cerebral spinal fluid and the like.

In the practice of the invention, if antibodies to a target antigen are present in the sample to be analyzed, specific antibodies will bind to the immobilized antigen on one or more of the sets of microspheres. In one example of a preferred embodiment of the invention, phycoerythrin- conjugated anti-human IgG, IgM, IgA, which for example can be a goat anti-human IgG, IgM, IgA, will react with human antibody immobilized on the solid phase in the first step of the assay procedure. The resulting bead suspension can then be analyzed by a conventional fluorescence analytical instrument, for example the AtheNA Multi-Lyte System®, including the Luminex® instrument, commercially available from Zeus Scientific, Inc., Branchburg, New Jersey, USA. The set(s) of microspheres can thereafter be sorted for identification purposes, and the amount of reporter molecule (phycoerythrin-conjugate) can be determined for each set. Using appropriate conventional means, well known to those in the art, to convert raw fluorescence produced by the instrument into outcome units, which can then be converted into a quantitive result, enables an indication of the presence and amount of anti— HIV antibodies present in the patient sample.

Example 1 : In one specific example of a preferred embodiment of the present invention, an assay kit which can be used to perform an improved assay in accordance with the present invention includes the components described below:

Reactive Components (All reactive components contain sodium azide as a preservative at a concentration of 0.1 % w/v:

1. Multiplexed bead suspension, ready-to-use, 5.5 mL bottle. The suspension contains separate distinguishable 5.6 micron polystyrene beads that are conjugated with the following

recombinant HIV-I proteins: gpl60, gpl20, p66, p55, gp41, p31, p24 and pl7. The bead mix also contains one bead set designed to detect non-specific antibodies in the patient sample (if present) and four separate bead sets used for assay calibration. It is to be appreciated, however, that in the practice of the invention any type of known HIV-I viral protein can be used, whether recombinant or non-recombinant, and such antigens can be varied, and other suitable antigens utilized, rather than those specifically listed above, as is well-known to those skilled in the art. 2. Phycoerythrin conjugated goat anti-human IgG (y chain specific), IgM (p chain specific) and IgA (oi chain specific), ready to use, 15 mL amber bottle. It is to be appreciated, however, that in the practice of the invention any type of anti-human IgG or other suitable immunoglobulin moieties as are well known to those in the art may be used, rather than the goat anti-human IgG described above.

3. Human positive serum control. One, 0.2mL vial. (PC-I)

4. Human low positive serum control. One, 0.2 mL vial. (PC— 2)

5. Human negative serum control. One, 0.2mL vial.

6. SAVe Diluent® (a readily available commercial sample diluent). One 50 mL bottle containing phosphate-buffered— saline, ready to use. NOTE: The sample diluent will change color in the presence of serum.

7. Wash Buffer Concentrate (10X): Dilute 1 part concentrate + 9 parts deionized or distilled water. One 5OmL bottle containing

1OX concentrate of phosphate buffered saline. Non-reactive Components:

1. One 96-well dilution plate containing micro wells.

2. One 96 well filtration plate containing micro wells.

3. Calibration CD. A compact disc is provided that includes all lot- specific kit calibration values required for sample analysis and assay quality control. To optimize read times, it is especially preferred that the bead suspension be thoroughly mixed just prior to use. It has been found that one effective means to accomplish the foregoing is to resuspend the beads is to first vortex the bead suspension for approximately 30 seconds, followed by sonication of the bead suspension for approximately 30 seconds in a small commercially-available bath sonicator. In addition, it has been found that the time and temperature of incubations is important to obtain suitable results. All reagents should be allowed to reach room temperature (20-25⁰C) before starting the assay procedure. Assay Procedure

Materials required to perform the assay procedure, but generally not provided in the assay kit of the invention:

1. Pipettes capable of accurately delivering 10 to 20OuL.

2. Multichannel pipette capable of accurately delivering (50-20OuL)

3. Reagent reservoirs for multichannel pipettes.

4. Serological pipettes.

5. Disposable pipette tips.

6. Laboratory timer to monitor incubation steps.

7. AtheNA Multi-Lyte System (Luminex instrument).

8. Distilled or deionized water.

Performance of the Assay Remove the individual components from storage and allow them to warm to room temperature (20-25⁰C). Determine the total number of controls and samples to be tested. It is necessary to include the Negative Control, the Positive Control and the Low Positive Control with each run. The Negative Control should be tested in well Al of the reagent reservoir, the Positive Control should be tested in well Bl and the Low Positive Control should be tested in well Cl. Each control and sample requires one microwell for processing.

To optimize read times, the bead suspension must be thoroughly mixed just prior to use. The most effective means to re-suspend the beads is to first vortex the bead suspension for approximately 30 seconds and then sonicate for approximately 30 seconds in a small bath sonicator.

For proper performance of the assay, it is important that the contents of the assay are thoroughly mixed. Suitable means of mixing include mixing the plate on a plate shaker for approximately 30 seconds at approximately 800 RPM or to set a pipettor to approximate the volume in the plate and repeatedly aspirate and expel (pump up and down) the contents of the well for a minimum of 5 cycles.

Serum incubation Prepare a 1 :21 dilution of the Negative Control, the Positive Control, and the Low Positive Control and each of patient sera.

(Example: Combine lOuL of serum with 20OuL of sample diluent). The sample diluent will undergo a color change confirming that the specimen has been combined with the diluent. For proper performance, it is important that the sample dilutions be thoroughly mixed.

After determining the total number of microwells to process, use a multichannel or a repeating pipette to dispense 50 uL of the bead suspension into each of the microwells of the filtration plate.

Thereafter, transfer lOuL of each diluted sample (1 :21) and control from the dilution plate to the filtration plate. For proper performance, it

is important that the sample dilution and bead suspension are thoroughly mixed.

The plate is then incubated at room temperature (20-25⁰C) for about 30 +/— 10 minutes. After the incubation, rinse the beads by vacuum filtration using the supplied Wash Buffer diluted to the IX concentration, and perform the following steps:

a. Place the filtration plate on the vacuum manifold and remove the solution, leaving the beads behind.

b. Turn off the vacuum and add 200 uL of IX Wash Buffer.

c. Apply the vacuum and remove the solution.

d. Repeat steps b and c for a total of three rinses.

e. Following the final wash, gently blot the bottom of the filter plate and allow the plate to air dry for 3-5 minutes before proceeding

to the next step. Conjugate Incubation

15OuL of the conjugate solution is added to each well at the same rate and in the same order as the specimens were added. For proper performance, it is important that the conjugate solution and bead suspension are thoroughly mixed. Incubate the plate at room temperature (20— 25°C) for about 30 +/— 10 minutes.

Specimen Analysis

The instrument is set-up, calibrated and maintained according to the manufacturer's instructions prior to reading the assay results, and then set to analyze the reactions by selecting the appropriate template in accordance with the operations manual for the operation of the instrument. Thereafter, the plate should be read within approximately 60 minutes after the completion of the conjugate incubation. One may decide to shake the plate for approximately 15 seconds prior to reading. This optional step may reduce the amount of time required to read the plate.

Example 2:

In another preferred embodiment of the invention, a shortened version of an assay procedure as described above can be conducted as follows:

1. Dilute specimens 1 :21 in diluents; mix well.

2. Combine 5OuL of bead suspension and lOuL of diluted specimen in a microwell; mix well.

3. Incubate at room temperature for about 30 +/— 10 minutes.

4. Rinse the microspheres three times with IX Wash Buffer.

5. Gently blot the bottom of the microwell plate and air dry for from 3— 5 minutes.

6. Add 15OuL of conjugate to each well; mix well and transfer to a reaction plate.

7. Incubate at room temperature for about 30 +/— 10 minutes.

8. (optional)Shake plate.

9. Read results within about 60 minutes.

Conversion of Fluorescence to Unit Values

A. Calculations: 1. Assay Calibration

The improved diagnostic assays provided by the present invention can be calibrated, as will be appreciated by those skilled in the art, by utilizing a technique called "Intra- Well Calibration Technology®", commercially available from Zeus Scientific, Inc., Branchburg, NJ, USA. Intra- WeIl Calibration Technology includes a multi-point standard curve within the bead suspension. With Intra-Well Calibration Technology, each well of the assay plate is calibrated internally without any user intervention. The standard curve is designed to self-adjust based upon the unique characteristics, for example, of the patient or control serum. Calibrator values are then assigned to the internal standards. These values are lot specific and are encoded within the lot specific calibration CD included in the kit box.

2. Analyte Cut Off Values

Each analyte of the improved assay system provided by the invention has an assigned cutoff value. Cutoff values are determined for each kit lot, and are encoded within the lot specific Calibration CD included in the kit box. 3. Calculations

Through Intra- Well Calibration Technology, all calculations are performed automatically when using the improved assay system of the invention. Intra-Well Calibration Technology performs a regression analysis of the internal standards and then adjusts the calculated unit values based upon an additional standard and the characteristics of the sample.

B. Interpretation of Antibody Reactivity to Each Viral Protein:

The improvements to an assay system provided by the invention enables the determination of the antibody results of the patient for each of the viral proteins. Individual results

for each anti— viral antibody are assigned a numerical outcome or arbitrary unit value.

Specimen unit values for antibody to each

of the viral proteins are interpreted as follows:

Anti— Viral Reactivity per Bead Unit Value:

Negative or non— reactive: < 100 AU/mL, patient sample does not contain antibody to that particular viral protein.

Positive or reactive: > 120 AU/mL, patient sample contains antibody to that particular viral protein.

Borderline or Equivocal: 100— 120 AU/mL, patient sample reacts too strongly with the protein to be scored as negative or non— reactive but not enough to be considered positive or reactive.

C. Interpretation of results: It is to be appreciated that in a preferred embodiment, the improved assay system of the invention is capable of determining if the patient is anti-HIV-1 negative, indeterminate, or positive based upon the following criteria:

Patient Outcome: Reactivity to each viral protein:

POSITIVE: The patient sample must be positive for antibody to at least two of the major viral proteins: gpl60/120, gp41 or p24. Results must be greater than 120 AU/mL for any one viral protein to be considered positive.

INDETERMINATE: The patient is positive for one or more of the viral proteins (results are greater than 120 AU/mL), or equivocal for one or more of the viral proteins, but the patient does not meet the criteria above for a positive outcome.

NEGATIVE: The patient is negative for antibody to all of the viral proteins. While certain aspects of preferred embodiments of the present invention have been described, it is not intended that the invention be limited to such embodiments. For example, it will be apparent to those skilled in the art that the improvements provided by the invention in the procedures and techniques described herein for the detection and identification of antibodies to Human Immunodeficiency Virus Type 1 (HIV-I) can also be applied to the improvement of any traditional assay, such as a western blot serology assay, into a multiplex bead serology assay for various infectious disease antigens and that such improvements enable a simpler, more objective, more automatable assay system to be available to the user, than has been previously known. Examples of antigens capable of detection and having detectable antibodies thereto in accordance with the present invention can include, but are not limited to, such pathogens as Toxoplasmosis, Borrellia, Epstein Barr Virus, Cytomegalovirus, Rubella Virus and Herpes Simplex Viruses, and can include as well other bacterial, viral or other infectious and disease causing agents. Thus, the aforedescribed procedures and techniques performed in accordance with a preferred embodiment of the present invention should not be considered as limitations thereon, and it is apparent that various modifications may be made thereto without departing from the spirit of the invention, the full scope of which is delineated in the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for performing an assay for detection of antibodies against one or more antigens in a biological sample comprising the steps of:

a. obtaining a biological sample to be tested from a subject suspected of having said antibodies;

b. incubating said sample in a container containing a multiplexed mixture of a bead suspension comprising a mixture of distinguishable sets of microspheres, each set being conjugated with a different antigen;

c. rinsing the microspheres to remove any non-reactive proteins;

d. adding phycoerythrin-conjugated anti-human IgG, IgM, IgA, or a mixture thereof, to the container;

e. incubating the container for a suitable period of time; and

f. reading an assay result from the performance of steps a through e.

2. The method of claim 1 , wherein the antigen is a viral antigen.

3. The method of claim 1 wherein the antigen is selected from the group consisting of

Toxoplasmosis, Borrellia, Epstein Barr Virus, Cytomegalovirus, Rubella Virus and Herpes Simplex Viruses.

4. The method of claim 1 wherein the antigen is abacterial antigen or another infectious agent.

5. The method of claim 4 wherein the bacterial or other infectious agent is a cellular

component, or a biological specimen capable of being analyzed via traditional serological methods such as western blot.

6. An assay kit for performing a method in accordance with any of claims 1 through 5.