Diluent Buffer and Method for Diluting an Assay Component

This invention relates generally to solutions useful as diluents in the biological sciences which are commonly known as "buffers". Such solutions are capable of neutralizing both acids and bases and thereby maintain the original acidity or basicity of the solution. More particularly, the invention relates to an improved buffer useful for the dilution of immunohematological, immunological and immunochemical assay components, such as antibodies and antigens.

Immunohematological analysis of the blood is performed routinely on the red blood cells of donors and recipients in order to determine whether the red blood cells of a possible blood donor is compatible with with red blood cells an intended recipient. The standard analysis performed in these laboratories is an ABO-Rh blood typing, with the determination of other blood groups, such as Kell and Duffy, sometimes required in order to complete the analysis or so-called "match" of the red blood cells. The ABO blood group is comprised of antigens A and B, with the determination of the blood groups A, B, AB and O based on an agglutination reaction of the test sample with the anti-A, anti-B and anti-A,B antisera. The Rh, or Rhesus blood group comprises several antigens, including the antigens C, D, E, c, and e. Test samples which do not react with anti-D (anti-Rh.sub.o) are preliminarily classified as "Rh negative". These presumptive Rh negative test samples are retested with a manual antiglobulin test in which elevated incubation temperature, washing, and the use of a secondary antibody is required to confirm the "negative" result.

No direct agglutination technique, manual or automated, has demonstrated equivalent or greater sensitivity than the manual antiglobulin test with respect to detection of the D antigen. Several attempts have been made to improve the sensitivity of the anti-D reagent in order to eliminate the necessity for performing confirmatory testing. Blood grouping reagent manufacturers have added various components to their reagents to enhance the quality of the agglutination of tests performed in tubes. Thus, for example, a monoclonal IgM anti-D antibody has been included to supplement the human plasma IgG source, with the aim being to provide improved sensitivity for the determination of agglutination of the test sample with anti-D antisera. See, for example, Product insert, Ortho Diagnostics Systems, Blood Grouping Serum Anti-D (Anti-Rh.sub.o) (polyclonal and monoclonal blend). However, even the use of this reagent to improve agglutination does not eliminate the need for the use of the confirmatory testing techniques required for anti-D antisera negative determinations, or to dilute the antibody used in the determinations.

Traditional methods for immunohematological analysis which utilize microtiter methods, both manual and automated, require the use of undiluted or low dilutions of the antibody components in order to perform the immunohematological blood group analysis with specificity and sensitivity. No diluent buffer is known to be available commercially for blood grouping reagents. Products are available to add directly to the microplate to increase sensitivity, but these products only are added in conjunction with washing steps and a secondary antibody. See, for example, Product Insert, Gamma Biologicals, Micro-U Enhancement Reagent (Low Ionic) for Antibody Detection tests in Microplates, February (1986). Thus, these methods typically do not offer much of a cost savings to the user, since the user is not able to dilute the reagents required to perform the test.

Other blood group reagent manufacturers have incorporated high molecular weight additives, high protein and surfactants into their reagents to enhance aggultination reactions. Applying these reagents to microplate methodologies, however, also requires no or a very low dilution of antibody reagent in isotonic NaCl or Bovine Serum Albumin (BSA). See, for example, Product Insert, Ortho Diagnostics Systems, Blood Grouping Reagent Anti-D for Slide and Modified Tube Tests, and F. K. Widman, American Association of Blood Bank Techniques, "Serologic Techniques and Alloantibodies," pages 435-451 at 440 (1985). Further, pretreatment of the microplate, such as treatment with an anti-static gun or prewetting, previously has been required with some systems. F. K. Widman, American Association of Blood Bank Techniques, "Serologic Techniques and Alloantibodies," pages 435-451 at 439 (1985).

The present invention overcomes the above-identified problems of the prior art by providing an improved buffer useful as a diluent which eliminates the need to perform required confirmatory anti-D testing procedures in cases of a negative or non-reactive result when using a commercially available anti-D antisera or human source antibody. It also provides such a buffer useful for diluting assay components such as antibodies, without sacrificing the sensitivity or specificity of the assay. The buffer of the invention also can be used to dilute out other assay components such as a test sample or an antigen preparation for use in an assay. The buffer provided allows the user the ability to dilute the antibody, for example, to a level not achievable in previous microtiter methods. This dilution of antibody results in both a cost savings to the user, and also serves to preserve the human source antibody which must be used in the majority of blood group antigen testing, where monoclonal antisera are not available. Also, the buffer may be used without pretreatment f the microtiter plate. Thus, treatment with an anti-static gun or prewetting is not required when using the buffer of the present invention.

The present invention accordingly provides an improved buffer solution, comprising a conventional, biological buffer such as a phosphate buffer, a high molecular weight polymer such as polyvinylpyrrolidone (PVP), a biological detergent such as Triton X-100.RTM., bovine serum albumin and sodium chloride all at effective concentrations, and wherein the buffer has a pH in the range of from about pH 5 to about pH 9. Cholic acid is also present in the solution at an effective concentration for visual readout up to four hours after completion of the assay. The buffer also can include sodium azide as a preservative at an effective concentration. The preferred phosphate buffer which is used can comprise sodium phosphate, monobasic and further comprise sodium phosphate, dibasic.

The pH of the buffer of the present invention can range from a pH of about 5.0 to a pH of about 9.0, preferably from about 5.6 to about about 7.2, more preferably the pH is in the range of from about pH 6.0 to about pH 6.8, and most desirably the pH of the buffer is about pH 6.4.

A method of using the buffer provided by the invention comprises diluting the selected assay component(s) in the buffer. A kit containing the buffer as a constituent also is provided.

As previously stated, this invention provides an improved buffer which is useful for diluting assay components without sacrificing either the sensitivity or specificity of the assay. The buffer is provided in two compositions. One composition of the buffer is preferred when visual readout up to four hours after completion of the assay is desired. The second composition of the buffer can be used and is preferred when automated assays are to be performed and visual readout is not desired, or when visual readout will be immediate. The latter buffer composition comprises a biological buffer, a high molecular weight polymer, a biological detergent, and bovine serum albumin, all provided at effective concentrations. If visual readout at up to four hours after assay completion is desired, an anionic surfactant is added to the aforedescribed buffer composition in an effective amount. A buffer composition according to the invention has a pH in the range of from about 5 to about 9. Sodium azide may be added to either of the foregoing preferred compositions at an effective concentration to be used as a preservative. If necessary, the overall pH of the buffer of the invention can be adjusted with the addition of an alkaline buffer, such as one containing sodium hydroxide.

The improved biological buffer composition of the invention can include a conventional buffer such as a phosphate buffer, MES (morpholino-ethanesulfonic acid) buffers, BIS-TRIS buffers, citrate buffers, TRIS-HCl buffers and borate buffers, at an effective concentration which can range from about 10 to 100 mM, preferably in the range of from about 10 to 30 mM, and most preferably about 20 mM. The preferred buffer is a phosphate buffer, preferably comprising sodium phosphate, monobasic and sodium phosphate, dibasic, at concentrations such that the effective concentration of buffer is achieved.

Effective concentrations of PVP in the buffer of the invention range from about 0.05 to about 0.25%, (w/v) preferably range from about 0.05 to about 0.15%, and most preferably the concentration is about 0.085%. Effective concentrations of the biological detergent range from about 0.005 to about 0.06%, (w/v) preferably range from about 0.01 to about 0.025%, and most preferably the concentration is about 0.017%. The preferred detergent is Triton X-100.RTM.. Effective concentrations of cholic acid range from about 0.01 to about 0.3%, (w/v) preferably range from about 0.13 to about 0.2%, and most preferably the concentration of cholic acid is about 0.15%. Bovine Serum Albumin is provided in effective concentrations which range from about 1.5 to about 3.5%, (w/v) preferably in the range of about 1.75 to about 2.75%, and most preferably the concentration of BSA is about 2.25%. Effective concentrations of sodium chloride range from about 0 to about 300 mM, preferably range from about 100 to about 200 mM, and most preferably the concentration of sodium chloride is about 154 mM.

The high molecular weight polymer selected for use in the invention can include polyvinylpyrrolidone (PVP) having molecular weights of from about 10 kd to about 1500 kd, dextrans with molecular weights ranging from about 10 kd to about 2000 kd, polyethylene glycols (PEG) having molecular weights in the range of from about 200 d to about 10,000 d, polyvinyl alcohol having a molecular weight of about 10,000 d to about 100,000 d, polybrene (hexadimethrine bromide), methylcellulose, gum acacia, protamine sulfate, merquat, celquat and magnafloc, provided at an effective concentration. The preferred high molecular weight polymer is polyvinylpyrrolidone (PVP); most preferably, PVP-360 is provided at an effective concentration. The high molecular weight polymers serve as agglutination "enhancers"; the majority of these high molecular weight polymers which can be utilized in the invention have a positive charge, although a positive charge is not necessarily required.

The biological detergents (surfactants) used in the present invention can include non-ionic surfactants, anionic surfactants, zwitterionic surfactants and cationic surfactants. Non-ionic detergents useful in the invention include polyoxyethylene sorbitan monolaurate (Tween.RTM. 20), polyoxyethylene sorbitan monooleate (Tween.RTM. 80), polyoxyethylene ethers(Triton.RTM., Brij.RTM.)and octylphenel ethylene oxide (Nonidet.RTM.). Anionic surfactants include caprylic acid, cholic acid, dexocholic acid, glycocholic acid and sodium dodecyl sulfate. Zwitterionic surfactants include CHAPS .RTM. (3-[3-Cholamidopropyl)-dimethyl;ammonio]-1-propanesulfonate). Useful cationic detergents include cetylpyridinium chloride. Preferably, non-ionic detergents are used. The most preferred non-ionic detergent is Triton.RTM.X-100. When visual readout is desired for either manual or automatic procedures, the addition of an anionic surfactant to the composition of the buffer is preferred. The most preferred anionic surfactant is cholic acid.

The buffer of the invention is particularly useful as a diluent of assay components. The term "assay components", as used herein, means whole blood or whole blood components including serum, plasma, red blood cells, white blood cells, and platelets. Thus, human and animal test samples or commercial preparations of these components may be diluted with the present buffer. In addition, assay components include commercial assay reagents such as polyclonal and/or monoclonal antibodies, particularly of IgG or IgM class, and fragments thereof; antigens and fragments thereof; antigenic lysates, recombinant proteins, synthetic peptides, and the like. It also is contemplated that inert assay materials, such as latex particles, magnetic beads, microparticle and the like, if provided in a suspension, may be suspended in the buffer of the invention, or if dilution of these materials in a suspension is desired, the dilution may be performed with the buffer of the invention. The buffer also can be used as a substitute for BSA or isotonic NaCl for use in "U"-, "V"-, or flat-bottom microplate systems, whenever the assay procedure calls for dilution of an assay component with these constituents.

The assay component, especially a cell, may be treated prior to dilution with the buffer of the invention with an enzyme such as bromelin, papain, trypsin, chymotrypsin, ficin, neuraminidase, urokinase and the like, or, the assay component may be treated (without enzyme treatment). Preferably, the assay component is treated with an enzyme in order to optimally use the buffer of the invention to its maximum capability, especially when performing agglutination assays. Preferably, the enzyme used is bromelin.

The buffer provided by the invention may be used to dilute assay components prior to performing the assay itself, or, it can be used to dilute assay components when performing the assay, such as when serial dilutions of test sample or other assay components are performed according to a particular assay method. The buffer provided is especially useful for diluting antibodies which are used in determining the presence or absence of specific antigens on red blood cells (erythrocytes). When antibody is diluted with the buffer of the invention, it allows the red blood cells possessing the specific antigen and present in the test sample to agglutinate with the antibody. The agglutination (clumping) of the red blood cells indicates a positive reaction, while no agglutination, indicated by a uniform cell suspension, indicates a negative reaction. Non-specific reactivity has not been found while employing the buffer as a diluent. In the past, antibody testing such as described above, and performed against human source polyclonal plasma, has required that an additional control test be performed in which the test sample is tested against the supplementary components present in commercial antibody reagents in order to determine whether the test sample reacts with the commercial antibody component itself or with the supplementary components. Preliminary test results obtained with two rare known samples which react with supplementary components indicate, however, that when the present buffer is used, this additional testing need not be performed, since the test samples diluted with the buffer of the invention do not react with the supplementary components.

Other advantages of the buffer provided by the present invention have been disclosed. For Example, the present buffer can be used when testing for the presence of the D antigen on red blood cells. It has been determined that the sensitivity of the results is increased such that no additional testing is required to confirm the result, thus eliminating the standard procedure of confirmatory testing which is the conventional procedure used when employing microplate testing methodologies. The buffer also can be used in other antigen-antibody interactions to enhance the reactions, such as for Hepatitis Surface Antigen and Syphillis testing.

It has been found that the reagents comprising the buffer of the invention can vary significantly in concentration without detracting from the performance of the buffer in assays. Table 1 lists the reagent components of the buffer and their range of effective concentrations.

These concentrations are based on a 60 .mu.l diluted antibody per test, and 30 .mu.l red cell suspension (3 to 5%). It is understood that the component cholic acid is not required for performance of the buffer as a diluent when visual readout for up to four hours is not desired. Thus, cholic acid may be removed from the buffer composition in this situation. Sodium chloride may be used to adjust the pH. Therefore, sodium chloride may be provided as follows:

In its most preferred composition for visual readout, the buffer is provided at the following concentration.

To approximately 8 liters of Di water, add the Sodium Phosphate, Monobasic and the Sodium Phosphate, Dibasic, and mix until dissolved. Then add the Sodium Chloride to the mixture and mix again until dissolved. Add the PVP-360 next and vigorously mix. Following that addition, add the Cholic Acid and mix unit it is dissolved. Then, add the BSA and mix until it is dissolved. Finally, add the Triton-X100.RTM. and mix until it is dissolved, which mixing is accomplished in approximately 15 minutes. Adjust the mixture to pH 6.4 with Sodium Hydroxide. Q.S. to 10 L. If visual readout is not required, the cholic acid may be deleted from the formula, and the concentration of Triton may be increased within the range provided herein. If the presence of sodium azide is desired, then 10.0 grams (0.1% w/v) of sodium azide is added to the buffer when adding sodium chloride. The pH of the buffer may be adjusted with sodium hydroxide.

It has been found, for example, that the effective antibody dilution range varies from 1:2 to about 1:100,000. The effective dilution range will vary depending upon the assay component being diluted; it is contemplated, however, that dilutions greater than 1:100,000, and up to approximately 1:500,000, will be able to be achieved with the present buffer using different assay components. This dilution may be performed prior to performing the assay itself. It also is contemplated that the buffer can be used to dilute components such as antibodies, test samples, or antigens, when performing the assay itself, such as when serial dilutions of test sample or other possible assay components are performed according to assay method.

The present invention now will be described by way of examples, which are meant to illustrate preferred embodiments of the invention, but not to limit the spirit and scope thereof.

An evaluation of commercially available antisera diluted in the buffer of the invention (provided in the preferred format, with cholic acid and sodium azide) was performed using a prototype automated blood bank analyzer (prototype of Abbott Laboratories, Abbott Park. Ill. 60064). Five commercially available antisera (anti-D, anti-C, anti-E, anti-c and anti-e), were serially diluted. Each dilution was tested against four homozygous, 4 heterozygous and one antigen negative donor samples. The materials used in the experiment were as follows: The buffer of the invention, provided as described herein; anti-D (Ortho Diagnostics Bioclone.phi., Lot No. DB113A), anti-C (Ortho Diagnostics, Lot. No. CS163B), anti-E (Ortho Diagnostics, Lot No. ES124B), anti-c (Ortho Diagnostics, Lot No. SC826A), anti-e (Ortho Diagnostics, Lot No. SE133A). The assays sere performed in microtiter trays. The phenotypes of the test cells were unknown at the time of performing the experiment. The test cells provided were analyzed at the Blood Center of Southeaster Wisconsin (Milwaukee, Wis.) for complete Rh-hr phenotype prior to testing at Abbott Laboratories; however, the test cells were coded prior to testing at Abbott Laboratories in order to avoid any subjectivity when determining titers. Each diluted antisera was tested against the following coded test cells using the instruction sheet send from the Blood Center.

Anti-D Plate: Cells labelled as A, B, D, E, F, H, I, J, M

Anti-C Plate: Cells labelled as A, C, D, E, H, I, J, L, O

Anti-E Plate: Cells labelled as C, D, F, J, M, N, O, Q, R

Anti-c Plate: Cells labelled as A, B, D, E, G, I, K, O, P

Anti-E Plate: Cells labelled as C, D, F, H, I, M, N, O, P

The test cell diluent comprised the following:

b 7.5 mls of 2% Bromelin (obtained from Sigma Chemical Co., St. Louis, Mo., lot No. 37033-15),

1.0 mls of 1% Tween.RTM. 20 (obtained from Sigma Chemical Co., St. Louis, Mo., lot No. 21197-12), and

91.5 mls of 0.9% NaCl, 0.1% Sodium Azide

Serial two-fold dilutions were prepared individually using each of the antisera described herein and following the procedure outlined in the AABB Technical Manual, 9th Edition, page 239. The dilutions prepared ranged from 1:2 to 1:131072. The dilutions were utilized in performing red cell phenotyping with a prototype automated blood bank analyzer (prototype of Abbott Laboratories, Abbott Park, Ill. 60064).

The automated blood bank analyzer format comprised a track which functioned as a "conveyor-type" mechanism, in which microtiter trays were placed on a track and advanced from station to station at 4.8 minute intervals.

Station 1 was a reagent station. 60 .mu.l of antisera reagent was dispensed, in its diluted form, to the designated locations on the microtiter tray. In addition, 230 .mu.l bromelin reagent was dispensed to three cell dilution wells. One well was designated as a primary dilution well wherein packed red cells from a "pilot" tube that contained whole blood which had been centrifuged to pellet the red cells were placed.

Station 2 was the sample handling station. 70 .mu.l of packed red cells were pipetted from the pilot tube to the primary cell dilution well which contained 230 .mu.l of bromelin solution. The cell suspension was mixed by aspirating and dispensing within the pipet itself.

Station 3 was the cell diluter/distributor. 70 .mu.l of the primary cell dilution (approximately a 15.4% cell concentration) was transferred to the two secondary dilution wells for each test sample. The secondary dilution wells were mixed, and 30 .mu.l of these suspensions (approximately 4-5% cell concentration) were distributed to the designated test wells.

The tray was then transported along the following track:

Station 4--Horizontal

Station 5--50.degree. tilt in direction A

Station 6--50.degree. tilt in direction B

Station 7--65.degree. tilt in direction A

Station 8--Horizontal

Station 9--65.degree. tilt in direction B

Station 10--Horizontal

Station 11--50.degree. tilt in direction A

Station 12--Horizontal

Station 13--The tray was read for the presence or absence of cells in the light path.

A positive result was determined to be high light counts, in which crescent-shaped agglutinates formed, and a negative result was determined to be low light counts, in which uniform cell suspension was present.

Eight cells were tested per tray. Two separate trays were required for the number of antisera dilutions tested. Tray #1 contained dilutions 1:2 through 1:4096. Tray #2 contained dilutions 1:8192 through 1:131,072.

Tables 2-6 show data which demonstrate that the commercially available antisera can be diluted to a great extent when utilizing the buffer of the invention.

Assays were manually performed at the Southeastern Blood Center of Wisconsin following the procedures detailed in the AABB Technical Manual, 9th Edition, page 239. The test cells were treated with Ficin at 0.5% prior to assay. The assays were performed in U-bottom microtiter plates on donor plasma. Visual interpretation of results was performed. The antibodies tested were the same as described in Example 1. Each diluted antisera was tested against the same panel as that described in Example 1. In addition to testing with the buffer of the invention, a 3% BSA buffer also was tested. The results are detailed below in Tables 7-11.

This experiment was performed to compare and contrast the performance of 3% BSA and the buffer diluent when utilized in Du phenotyping on an automated bidirectional tilt system as described in Example 1. The more sensitive buffer, in this context, was defined as the buffer which allowed the utilization of the lower amount of antibody to detect the presence of the Du phenotype by agglutination technology. To those skilled in the art, Du is classified as a red blood cell possessing the D antigen in which the presence of the D antigen cannot be demonstrated by using initial testing methods and, therefore, it requires more detailed testing to demonstrate its presence, such as by Indirect Antiglobulin Methodology. This definition of Du can depend both on the methodology used, (manual or automated), and also on the antisera source used. Du is defined in this experiment as red cell samples initially screened D negative on Dynatech Microbank Systems (Badger Region Red Cross, Madision, Wis.), and retested to be D positive by Indirect Antiglobulin Test.

The experiment followed the procedure of Example 1 with the following changes. Only a single antisera was tested, Ortho Diagnostics' Slide Anti-D, lot no. D5670D. The method of preparing a two-fold serial dilution was performed as in Example 1, with the exception that the initial dilution was 1:6.25. This dilution method is described in the AABB Technical Manual, 9th Edition, p. 239. (Technique for Master Dilution [1985]).

In the past, there have been inherent problems with utilizing commercially available antisera in microplate methodology, manual or automated. The antisera has needed to be diluted such that the additives which are incorporated into the antisera reagent are diluted out to a satisfactory level to eliminate false positive reactions (agglutination in the absence of specific antibody). Thus, controls were necessary to prove that, at low dilutions, there was specific agglutination occurring. As the additives were diluted out further, and the amount of diluent present overcame those additives, no non-specific agglutination occurred at higher dilutions. The level of non-specific agglutination which would occur with both the diluent buffer and 3% BSA when the commercial antisera were diluted in them was initially determined. The levels of dilution at which non-specific agglutination occurred were 1:12.5 with 3% BSA and 1:25 with the diluent buffer. All positive reactions above these dilutions were considered true positive results, and all reactions at or below are to be considered false positive results.

The results of the comparison are shown in Table 12.

It will be apparent to one of ordinary skill in the art to which this invention pertains that various changes and modifications can be made in the specifically-described embodiments of the invention disclosed herein, without departing from the spirit and scope of the invention, which is defined solely by the following claims: