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WO2008133744A2 - Préparation d'échantillons pour l'essai de détection d'antigènes - Google Patents

Préparation d'échantillons pour l'essai de détection d'antigènes Download PDF

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Publication number
WO2008133744A2
WO2008133744A2 PCT/US2007/086894 US2007086894W WO2008133744A2 WO 2008133744 A2 WO2008133744 A2 WO 2008133744A2 US 2007086894 W US2007086894 W US 2007086894W WO 2008133744 A2 WO2008133744 A2 WO 2008133744A2
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saline solution
sample
physiological saline
samples
degrees celsius
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WO2008133744A3 (fr
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William E. Hessman
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Central States Testing LLC
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Central States Testing LLC
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding

Definitions

  • the present invention relates to tissue sample preparation methods for improving the ability to detect an analyte in a tissue using a test based on antigen capture technology.
  • a challenging problem is reliably detecting an analyte that is present, if at all, at a low concentration in a very small fraction, e.g., less than one percent, of a population of samples to be tested.
  • the various technologies that are available to detect an analyte present at a low concentration in a sample are generally based on specific high-affinity binding of a binding partner to the analyte. Examples include the specific binding of an antibody to an antigen or the binding of a probe, such as an oligonucleotide, to a nucleic acid molecule.
  • Much effort has been devoted to improving detection of an analyte by improving the specificity and/or affinity of the interaction between the analyte and the binding partner.
  • one or more amplification processes such as enzymatic production of the detected molecule or specific, template-dependent synthesis of a nucleic acid analyte using the polymerase chain reactive (PCR), are used to enhance the sensitivity of the test.
  • amplification processes such as enzymatic production of the detected molecule or specific, template-dependent synthesis of a nucleic acid analyte using the polymerase chain reactive (PCR)
  • PCR polymerase chain reactive
  • a known solution is to test first for the presence of the analyte in pooled aliquots of samples, then test separately the samples contributing to a pool that tests positive for the analyte.
  • the suitable pool size generally varies by analyte and detection technology, and would likely differ for a PCR-based test and an antigen capture ELISA (enzyme-linked immunosorbent assay) test.
  • Simple dilution of a single positive sample with several negative samples can reduce the concentration of the analyte below the detection threshold for the analyte and cause false negative errors.
  • a negative sample may also contain one or more factors that can suppress or inhibit the interactions required to detect the analyte, also causing false negative errors.
  • sample handling procedures have been found that reduce false negative errors and thereby improve the ability to detect an analyte that is present, if at all, at a low concentration in a only very small fraction, e.g., less than ten percent, and in preferred embodiments, less than one percent, of the population of samples to be tested.
  • the present invention provides methods for reducing false negative errors in antigen capture tests of pooled and single samples.
  • the methods are particularly useful for detecting BVDV (bovine viral diarrhea virus) antigen-positive samples when pooled with other samples that contain an inhibiting agent, such as an antibody to a BVDV antigen or other interfering substance.
  • BVDV bovine viral diarrhea virus
  • the invention provides a method of processing a tissue sample to provide a liquid sample to be tested for the presence of an analyte, comprising the steps of immersing a tissue sample in a buffered physiological saline solution; incubating the tissue sample in the buffered physiological saline solution at about 32-41 degrees Celsius for at least 1 hour; reducing the temperature of the tissue sample in buffered physiological saline solution by at least about fifteen degrees Celsius for at least fifteen minutes; and removing an aliquot of the buffered physiological saline solution thereby providing a liquid sample to be tested for the presence of an analyte.
  • Suitable buffered physiological saline solutions have a buffer, pH, inorganic components and organic components (if present) that are known in the art and are compatible with the tissue sample, the formation of the analyte/binding partner complex and the detection means.
  • a preferred buffered physiological saline solution is phosphate buffered saline solution.
  • the temperature of the tissue sample in buffered physiological saline solution is reduced to room temperature after incubation at about 32-41 degrees Celsius.
  • the temperature of the tissue sample in buffered physiological saline solution is reduced to about 2-7 degrees Celsius after being incubated at about 32-41 degrees Celsius.
  • the temperature of the tissue sample in buffered physiological saline solution is reduced to about —20 degrees Celsius after being incubated at about 32-41 degrees Celsius.
  • the method further includes the steps of performing the test for the analyte on the liquid sample, and evaluating the test measurements using a standard that has been empirically determined using known positive and negative samples to minimize false negative errors, hi preferred embodiments, the liquid sample is a pooled sample that includes aliquots of 2 to 20 samples.
  • the present invention provides a method of increasing the sensitivity of a test that measures the presence or amount of an analyte located in a tissue based on the specific high-affinity binding of a binding partner to the analyte, comprising the steps of immersing a tissue sample in a buffered physiological saline solution; incubating the tissue sample in the buffered physiological saline solution at about 32-41 degrees Celsius for at least 1 hour; reducing the temperature of the tissue sample in buffered physiological saline solution by at least about fifteen degrees Celsius for at least fifteen minutes; removing an aliquot of the buffered physiological saline solution thereby providing a liquid sample to be tested for the presence of an analyte; testing the liquid sample using the test based on the specific high-affinity binding of a binding partner to the analyte; detecting the presence or amount of the analyte by measuring the formation of an analyte/binding partner complex; and evaluating the test measurements using a standard
  • Suitable buffered physiological saline solutions have a buffer, pH, inorganic components and organic components (if present) that are known in the art and that are compatible with the tissue sample, the formation of the analyte/binding partner complex and the detection means.
  • a preferred buffered physiological saline solution is phosphate buffered saline solution.
  • the temperature of the tissue sample in buffered physiological saline solution is reduced to room temperature after incubation at about 32-41 degrees Celsius.
  • the temperature of the tissue sample in buffered physiological saline solution is reduced to about 2-7 degrees Celsius after being incubated at about 32-41 degrees Celsius.
  • the temperature of the tissue sample in buffered physiological saline solution is reduced to about -20 degrees Celsius after being incubated at about 32-41 degrees Celsius.
  • the present invention provides a method of increasing the sensitivity of a test that measures the presence or amount of an antigen located in a tissue, where the antigen is likely to be detected in less that 10% of the samples to be tested, comprising the steps of immersing a tissue sample in a buffered physiological saline solution; incubating the tissue sample in the buffered physiological saline solution at about 32-41 degrees Celsius for at least 1 hour; reducing the temperature of the tissue sample in buffered physiological saline solution by at least about fifteen degrees Celsius for at least fifteen minutes; removing an aliquot of the buffered physiological saline solution thereby providing a liquid sample to be tested for the presence of an antigen; pooling an aliquot of b liquid samples to form a pooled sample, where b is an integer from 2 to 20 inclusive; testing the pooled sample for the presence and amount of the antigen; evaluating the test measurements for each pooled sample using a standard that has been determined empirically using known positive and negative samples to minimize
  • Suitable buffered physiological saline solutions have a buffer, pH, inorganic components and organic components (if present) that are known in the art and that are compatible with the tissue sample, the formation of the analyte/binding partner complex and the detection means.
  • a preferred buffered physiological saline solution is phosphate buffered saline solution.
  • the temperature of the tissue sample in buffered physiological saline solution is reduced to room temperature after incubation at about 32-41 degrees Celsius.
  • the temperature of the tissue sample in buffered physiological saline solution is reduced to about 2-7 degrees Celsius after being incubated at about 32-41 degrees Celsius.
  • the temperature of the tissue sample in buffered physiological saline solution is reduced to about -20 degrees Celsius after being incubated at about 32-41 degrees Celsius.
  • test is a BVDV antigen capture ELISA test
  • S/P ratio of the sample absorbance to the positive control absorbance
  • the buffered physiological saline solution contains a non-interfering colorant.
  • a preferred colorant is a mixture of FD&C Yellow 5 and FD&C Blue.
  • Bovine viral diarrhea virus is a positive stranded RNA virus of the Pestivirus genus of the Flaviviridae family. This virus is found worldwide in ruminant animals both domestic and wild, including sheep, goats and deer as well as cattle. In addition to having several genetic subtypes (BVDV Ia, BVDV Ib and BVDV 2), the virus can exist in cytopathic and noncytopathic forms, producing a wide range of clinical disease.
  • Persistently infected animals are persistently viremic animals. Cattle persistently infected with BVDV act as the major reservoir of BVDV and are the primary source of infection, with transiently infected cattle considered a less important source. See generally, Larson, R.L., et al., Bovine Viral Diarrhea (BVD): Review for Beef Cattle Veterinarians, Bov Pract 38:93-102, 2004. Persistently infected animals are much more efficient transmitters of BVDV than transiently infected animals because they secrete much higher levels of virus for a much longer period of time.
  • BVDV is shed by a PI animal throughout life from virtually all secretions and excretions including nasal discharge, saliva, semen, urine, tears, milk, and to a lesser extent, feces.
  • Fetuses, placentae and fetal fluids, from BVDV-induced abortions can also contain BVDV.
  • Horizontal transmission of BVDV to seronegative cattle has been shown to occur after only one hour of direct contact with a single PI animal.
  • room temperature is a temperature of 18-25 degrees Celsius.
  • the temperature of a refrigerator when not stated explicitly, is 2-7 degrees Celsius.
  • ALL sample handling procedures whether involving intermediate steps of freezing or heating, the samples were allowed to come to room temperature and mixed gently before a liquid sample aliquot was removed for testing.
  • the manufacturer's product insert describes the test kit (HerdchekTM, EDEXX, Westbrook, Maine) as a simple, fast and inexpensive means of detecting BVDV antigen in either serum samples or ear notch biopsy samples that is based on ELISA methodology, using a BVDV antigen-specific monoclonal antibody as the capture antibody, a goat polyclonal anti-BVDV antibody as the detector, and a horseradish peroxidase-anti-goat conjugate.
  • the ELlSA procedure is carried out at room temperature (18-25 degrees Celsius).
  • the test kit insert instructions were followed with the following modifications to the sample preparation instructions and the guidelines for interpretation of results.
  • an ear-notch biopsy sample approximately 1 cm x 1 cm should be suspended in phosphate buffered saline (PBS, 0.1 M phosphate buffer (pH 7.4) with 0.9% NaCl) to prepare a liquid sample extract for use in the assay.
  • PBS phosphate buffered saline
  • pH 7.4 0.1 M phosphate buffer
  • the processed ear-notch sample can be either refrigerated at 2°-7°C for short-term storage (1-2 days) or frozen (-20 0 C or colder) for long-term storage.
  • the ELISA procedure yields a colored reaction product in the presence of BVDV antigen, the test result for each sample is calculated as the sample to positive control (S/P) ratio, which is calculated as (sample A 450 - negative control mean A 45 o)/(positive control mean A 450 - negative control mean A 450 ).
  • a first stage of testing pooled samples is followed by testing of individual samples that contributed to a positive pool.
  • aliquots of several samples are tested as a pooled sample.
  • the pooled sample approach also has the advantage of increasing the speed at which samples can be tested, since at a PI prevalence rate of 0.3 - 0.4 percent or less, more than 99% of the samples are expected to be BVDV negative.
  • aliquots of the samples contributing to the few positive pooled samples can be retested separately to identify the individual positive samples.
  • aliquots of 100/6 ⁇ l are taken from each sample vial, where b is the number of samples to be tested in each pooled sample.
  • Low values of b provide minimal cost and time advantage, but high values of b may reduce the sensitivity of the test due to increased dilution of antigen in the pooled samples.
  • b is suitably in the range of 2 to about 20. In certain preferred embodiments, b is 6-12.
  • the first adjustment to the standard kit protocol was the introduction of an additional category for pooled samples.
  • S/P sample to positive control
  • the standard IDEXX kit protocol for single samples provides the following categories for S/P values:
  • the manufacturer's instructions provide that samples with S/P values falling within the suspect range should be assayed again, using a different "modified" working detector reagent containing no detector antibody.
  • the suspect sample and the kit controls should be included in the same run using the standard working reagents.
  • breakpoints defined in the manufacturer's instructions are referred to herein as "IDEXX" breakpoints.
  • IDEXX IDEXX breakpoints.
  • S/P values ⁇ 0.20 were considered negative and those with values > 0.20 were considered positive.
  • the raw OD (optical density) values for negative and positive controls in the kit must have been ⁇ 0.5 and > 0.8, respectively.
  • CST CST breakpoints
  • Routine pooling of up to ten non-fresh samples including a known positive sample resulted in S/P values that while reduced, still fell in the "positive” or “suspect” ranges.
  • Initial studies using more than seventy-five known positive samples in pools of various sizes showed that false negative errors can occur in tests of pools of eighteen and more samples.
  • studies of sample pools of ⁇ 100 samples reported false negative in a few samples that were negative by antigen capture ELISA for both pooled and individual samples but positive by PCR tests. See Kennedy, J. A., Diagnostic efficacy of a reverse transcriptase-polymerase chain reaction assay to screen cattle for persistent bovine viral diarrhea virus infection, J. Amer. Vet. Med. Assoc, November 1 , 2006, 229(9): 1472-1474.
  • sample pooling was discovered while testing a series of samples from a group of cattle in a feedlot that were primarily (>80%) from the same herd.
  • two of the six negative samples were found to interfere with the measurement of the antigen level in the known positive samples. In general, this effect was more prominent in fresh ( ⁇ 3 hours old) samples.
  • Table 1 summarizes a study of this effect using nineteen known positive samples pooled with three different groups of samples from BVDV antigen negative animals (Group I, animals 1641, 1642, 1643, 1644, 1645, 1646; Group II, animals 4711, 4712, 4713, 4714, 4715, 4716; Group m, animals 4676, 4677, 4678, 4679, 4680, 4681) showing the effect of pooling a known positive sample with samples containing an interfering agent from negative animals. In all cases, the fresh samples were incubated in PBS for less than an hour before aliquots of the PBS were taken for testing.
  • the groups of six negative samples are denoted by the number of the first of the six samples; Groups I, II and III of Examples 2 and 3 correspond to 1641, 471 1 and 4676.
  • Group I had two animals with a moderate inhibitory effect, Groups II and III each had one animal with a substantial inhibitory effect, 4715 and 4676, respectively.
  • the fresh samples were incubated in PBS for less than an hour before aliquots of the PBS were taken for testing.
  • Table 4 summarizes the results of a study using the same samples, but using a different sample handling procedure.
  • the samples were incubated in PBS for 2 days, frozen overnight at -20° Celsius (-5° Fahrenheit), thawed to room temperature, aliquoted & pooled and tested.
  • Example 4 The negative samples from the same set of cattle used in Example 4 were used in this study, including Groups I, II and III, that included samples having interfering agents. Samples from a known PI animal (4718) were used as known positive samples. The results are summarized in Table 6, with the data presented in Tables 7 and 8. Eight sample handing procedures were studied.
  • Table 7 presents the data from three sample handling procedures in which a fresh sample was subjected to three hours incubation in PBS at room temperature (A), refrigeration in PBS for three hours (B) or freezing - storage at -20° C for three hours followed by thawing to room temperature (C) before testing.
  • A room temperature
  • B refrigeration in PBS for three hours
  • C freezing - storage at -20° C for three hours followed by thawing to room temperature (C) before testing.
  • Using the manufacturer's breakpoints produced 10-11 false negative errors out of 12 samples, while the adjusted breakpoints produced three false negative errors for the three pooled samples (Groups I, II, and EI) having interfering agents.
  • Table 7 also provides the results for tissue handling procedure D, which was 48 hours incubation in PBS at room temperature, frozen overnight at -20° C, and allowed to thaw at room temperature before testing.
  • Tissue handling procedure D using the IDEXX breakpoints produced 3 false negative errors out of 12 samples, while the adjusted breakpoints produced zero false negative errors.
  • Table 8 presents the data from four tissue sample handling procedures in which a fresh sample was subjected to various treatments that lasted about two days or longer before testing.
  • Treatments E and F involved a long period of refrigeration in PBS (overnight or two days) either preceded by 12 hours in PBS at room temperature or followed by heating for one hour at 35° C.
  • Treatments G and H involved two overnight freeze-thaw cycles; in H, the overnight freeze-thaw cycles were preceded by 2 days in PBS at room temperature.
  • Treatment H produced the greatest reduction in false negative errors, but at a cost of four days added to the testing process.
  • sample handling procedures such as holding a sample overnight at -20° C, could further reduce false negative errors.
  • the tissue sample was incubated in PBS at 4-5° C for 24 hours in a refrigerator, and then stored overnight in a -20° C freezer.
  • Table 1 1 compares the degree of "inhibition” according to results in Table 10 and the serum neutralizing antibody titers to each BVDV subtype.
  • Example 8 Effect Of Tissue Handling Procedures Table 12 summarizes the tissue handling procedures used in the following studies. Table 13 shows the outcomes of using different tissue handling procedures with inhibitors at 2:1 and 7:1 dilutions and compares the percentage improvement in the S/P ratio by a tissue handling procedure.
  • a sample from animal No. 4715 which was shown to have a large "inhibition" effect above was used as a negative sample in the 2: 1 and the 7: 1 pools.
  • the negative samples used for the pools were the same for all tissue handling procedures.
  • the charts to the right of the outcomes illustrates the increase in values obtained when comparing the means for each tissue handling procedure to incubation for 10 minutes in PBS at room temperature procedure (standard test kit protocol) which was used as control group.
  • the tissue handling procedure highlighted showed the greatest increase in S/P.
  • Table 16 summarizes the results of studies in the effects of higher temperatures followings by freezing for various times were examined. All samples (including the controls) were incubated in PBS for one hour at 105 F° (41° C), and then frozen for the indicated time.
  • Table 17, above summarizes the results of studies in the effects of room temperature incubations of various durations with or without freezing for various times, or higher temperatures with or without freezing for various times were examined. Samples processed according to the manufacturer's recommended test kit protocol (room temperature for 10 minutes) were used as the control. As in Table 13, those positive results that would have been found using the standard test kit protocol breakpoints are indicated by (b), positive results that would have been found using the CST breakpoints but not by the standard test kit protocol breakpoints are indicated by (a) and false negative errors by both are without an indicator letter.
  • the chart below the data outcomes illustrates the increase in S/P values obtained when comparing the means for each tissue handling procedure to control. The tissue handling procedure highlighted showed the greatest increase in S/P value.
  • Table 19 compares the results obtained after testing 190 known positive samples after six different sample handling procedures (described in Table 12, above) using both the IDEXX and the CST breakpoints. Pooled samples consisted of an aliquot of one known positive sample and aliquots of six known negative samples. Note that the manufacturer's protocol was particularly likely to produce false negative errors when known positive samples were pooled with negative samples that produced "inhibition," i.e., samples 3, 18, 30, 32, 65, 120, 125, 150, and 173. As above, positive results that would have been found using the CST breakpoints but not by the standard test kit protocol breakpoints are indicated by "(a)”. Here false negative errors produced by both sets of criteria are indicated by "(n)”.
  • Table 20 summarizes the effectiveness of different sample handing procedures and the adjusted breakpoints in reducing the numbers of false negative errors
  • a dilute solution of a visible colorant in phosphate buffered saline is useful in positioning a sample in the wells on a microtiter plate.
  • the colorant does not interfere with the accuracy of the testing procedure since the colored PBS is replaced during the testing protocol before an optical measurement is made.
  • Table 21 provides the S/P results for known positive samples in PBS with colorant ("+ Dye") and without a colorant ("No Dye”).
  • the colorant is a green mixture of FD&C Yellow 5 and FD&C Blue at a final concentration of 0.005% v/v.
  • tissue samples were cut from the animals' ears using a round biopsy punch having a 5 mm diameter that produced a tissue plug about 15.7 mm in circumference and having a height of the thickness of the ear tissue.
  • Samples were prepared from the plugs and from the corresponding adjacent plugs after being bisected in a plane perpendicular to the ear surface and containing the long axis of the plug. Average S/P values obtained from 36 samples prepared from biopsy plugs before and after bisection showed an increase in S/P from 0.17 to 0.31 for a increase in cut surface edge length from 15.7 mm to 25.7 mm. Similarly, ear notch tissue was used to prepare fifty samples from tissue biopsies having a cut edge length of about 26 mm and after a cut that split the corresponding adjacent biopsies to produce a cut edge length of about 52 mm, resulting in measured S/P levels of 1.32 and 1.53, respectively.

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Abstract

La présente invention concerne des procédés permettant de réduire des erreurs de faux négatif dans des essais de capture d'antigènes d'échantillons regroupés ou uniques. Les procédés sont particulièrement utiles pour détecter des échantillons contenant des antigènes lorsqu'ils sont regroupés avec d'autres échantillons contenant un agent inhibiteur.
PCT/US2007/086894 2006-12-08 2007-12-08 Préparation d'échantillons pour l'essai de détection d'antigènes Ceased WO2008133744A2 (fr)

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US5415996A (en) * 1993-10-20 1995-05-16 Olsson; Lennart Prognostic markers in human breast cancer
WO2002090572A2 (fr) * 2001-05-09 2002-11-14 Third Wave Technologies, Inc. Detection d'acides nucleiques dans des echantillons groupes

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