Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/16—Blood plasma; Blood serum
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
  • C07K16/1036—Retroviridae, e.g. leukemia viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria

Definitions

• the present invention relates to the use of porcine seroconversion panels for determining the sensitivity and/or specificity of assay systems for detecting the presence or absence of antibodies to viral and/or other microbial infective agents, and to a method of producing the porcine seroconversion panels.
• the invention pertains to the use of porcine immune IgG, porcine immune IgA, and porcine immune IgM seroconversion panels.
• Assay systems capable of detecting the presence or absence of antibodies generated in response to the presence of antigens are well known. Such assay systems have proved useful in, inter alia, the diagnosis of various diseases. For example, viral infections, such as AIDS (acguired immune deficiency syndrome) and CMV (cytomegalovirus) may be diagnosed with assays which detect the presence of viral antibodies including immunoglobulins IgG, igA, and IgM, in patients suspected of having these diseases. Examples of such assay systems which employ antigen-antibody binding include ELISA, Western Blot, Quick Western Blots (U.S. Patent Nos. 4,816,387 and 4,855,235) and RIA. Such diagnostics uniformly include controls to insure the integrity of the test system.
• AIDS abbrevired immune deficiency syndrome
• CMV cytomegalovirus
• the diagnostics have both positive and negative controls.
• the positive control provides pertinent information concerning the activity of the test system, i.e., that reactive antibodies specific to the antigens used in an antibody test system are bound to the antigens (indicating that the antigens used in the test system are working properly), an that the anti-immunoglobulin used to detect the bound immunoglobulin is working.
• the anti-immunoglobulin may be labeled with an enzyme (conjugate) which activates a substrate added to the system to give a chromogen reaction; in this case the positive control indicate whether the conjugate has reacted, and whether the substrate h worked properly as an activated chromogen.
• a negative control provides information about the absence of reactive antibodies specific to the particular antigens used in a test system. It also provides information as to the reaction level, determined by the signal used in a particular test, at which a specimen m be considered negative. The cut-off point in a particular test is often based upon the relative valve obtained by a positive control and/or b the negative control. An acceptable detection range obtained b the controls utilized with a particular type of test kit is specifically designed and titrated for that type of kit.
• the positive control "value" obtained in a particular test system affects the sensitivity of that test system; the negative control "value" affects the specificity of the test system.
• Seroconversion panels are conventionally used to estimate the sensitivity and specificity of diagnostic tests or assay systems.
• a seroconversion panel is made by drawing consecutive blood samples over time from a donor infected with known microbial organism. The day that the blood sample is taken is the time point for that sample.
• the blood serum contains antibodies to the microorganism such as igG, IgA, and IgM.
• Seroconversion panels containing antibodies to retroviruses such as HIV-1, HIV-2, HTLV-1, and HTLV-II, and to other microorganisms such as toxoplasma, cytomegalovirus, Borellia b.
• LYME " LYME "
• Rubella have been obtained by followin certain plasmaphoresis donors or by repeatedly drawing blood samples from high risk individuals. The collected blood from each drawing is then tested for the presence of antibodies to the microbial organism and aliquotted in minute volumes, typically 100 to 250 microliters (ul) and stored for future use. The consecutive time point samples as a group constitute seroconversion panel. Notably, the supply of antibodies is scarce and uncertain and the quality and characteristics of the antibody varies from donor to donor. Further, as more successful therapies become known and used, fewer seropositive donors will be available, and thus the required antibody even more difficul to obtain.
• Seroconversion panels disclose how early an infection can be detected.
• IgG immunoglobulin G
• assays detect the presence or absence of IgG (immunoglobulin G) .
• Such assays only allow "controlled" detection (measurement defined by use of anti-IgG conjugate an of antibody positive control) of the presence of IgG in blood and body fluids directed to antigens used in the test systems.
• the appearance of detectable IgG directed to antigens in an infected/immunized individual does not occur until 30-40 days after initial infection in many instances.
• the IgG class antibodies are often present for months or years after infection/immunization.
• IgG The presence of circulating IgG directed to immunizing antigens during the course of an infection (or after immunization) is preceded by the presence of circulating IgM and/or IgA antibodies directed towards the antigens/immunogens.
• IgM and/or IgA antibodies directed to antigens in an infected/immunized individual are often present in detectable quantities as early as 14 days (or earlier) after the infection/immunization.
• the IgM class antibodies gradually los titer 30-35 days after initial infection/immunization.
• IgM immunoglobulin G
• assays capable of detecting IgM and/or IgA will facilitate earl detection of numerous diseases.
• IgM is, however, a relatively short-lived antibody. While it may be produced shortly after infection, IgM levels fall, eventually below detectable levels, as IgG is produced in increasing amounts. Because IgM has a short life span, IgM levels are typically below detectable levels before many diseases are even diagnosed.
• Seroconversion panels can be used to test each manufactured batch of assay systems or diagnostic test kits to ensure that the assay system performs with the same high sensitivity and specificity each time it is to be released for distribution. Test laboratories which use such assay systems o test kits can use seroconversion panels to ensure that the laboratories are performing high quality testing.
• the present invention overcomes the previously mentioned disadvantages because it provides the ability to produce the desired seroconversion panels, i.e., porcine IgG, IgA, and IgM antibody seroconversion panels.
• the porcine seroconversion panels to determine the sensitivity and/or specificity of assay systems detecting antibodies to viral and/or microbial infective agents.
• the present invention provides a use of porcine seroconversion panels for determining the sensitivity and/or specificity of assays that detect the presence or absence of antibodies which bind to viral and/or other microbial antigens and production of porcine seroconversion panels.
• the invention is useful in connection with assays in which predetermined antigens are sequentially contacted with a biological fluid and positive control comprising antibodies to the antigens for times and under conditions sufficient for the antigens and any antibodies in the biological fluid, and the antigens and antibodies in the positive control, to form antigen-antibody complexes, and the formation of the complexes is therewith detected.
• the sensitivity and/or specificity of the assay is determined by using seroconversion panels containing human antibodies to the antigens instead of the biological fluid, the improvement comprising the use of seroconversion panels containing porcine antibodies which bind to the antigens and react to anti-human antibody.
• the present invention can be used to provide seroconversion panels containing known titers of antibodies to predetermined antigens.
• the sensitivity and/or specificity of different assays for detecting antibodies to the predetermined antigens can be compared by using each assay to measure the presence of the antibodies in the seroconversion panels and comparing the measurements. The comparison of the measurements obtained by the different assays will show which assay is more sensitive and/or more specific.
• porcine immune IgG, IgM, and/or IgA seroconversion panels It is presently preferred to use porcine immune IgG, IgM, and/or IgA seroconversion panels.
• porcine seroconversion panels can be produced with antibodies reactive to predetermined antigens of only one viral or other microbial infective agent or of a plurality of viral or other microbial infective agents. No such manipulation is possible with human donor derived seroconversion panels.
• a further advantage of the present invention is that a porcine seroconversion panel containing a plurality of antibodies to a group of microorganisms, such as human retroviruses HIV-1, HTLV-1, HIV-2 and HTLV-II can be used by makers of assay systems to develop and control the sensitivity and specificity of their assay systems to detect any one of the plurality of antibodies in the same assay.
• Such an assay would enable one test to be performed on a sample instead of a series of individual assays to detect one antibody at a time. If a positive reaction is obtained, then the sample can be tested further, if necessary, to determine the exact antibody that is reacting. For example, blood banks which screen donated blood for the presence of AIDS antibodies, could ' use an assay containing antigens to HIV 1, HTLV-1, HTLV-II and HIV-2, inste of four individual assays using only one antigen. If the test sample is not reactive, then no further testing is required. the test sample is reactive, then the sample could be further tested, if necessary, in assays using one of the four antigens to identify the viral antigen to which the test sample is reacting.
• FIG. 1 presents the results of a Western Blot assay measuring the presence of HTLV-1 IgG in a porcine seroconversi panel and the corresponding results of the ELISA assay on the same panel.
• Samples are numbered 1 to 15, the negative contro is identified as "NC", and the positive control is identified a "PC”. The time point is listed below the sample.
• the ELISA optical density (O.D.) reading of the sample is presented below the time point.
• the Western Blot result for a sample is shown above the sample number with protein identifying band numbers 15, 19, 24 , 26, 28, 32, 36 and 53 and glycoprotein identifying band number 46 presented in numerical order to the right of the PC sample.
• the band numbers are used to identify the band in the sample that aligns with the corresponding band number in th right hand column.
• FIG. 2 presents the results of a Western Blot assay meas ⁇ ring the presence of HTLV-1 IgM in a seroconversion panel and the corresponding results of the ELISA assay on the same panel illustrated in the same manner as FIG. 1.
• porcine seroconversion panels are obtained by immunizing a pig with viral or other microbial material against which it is desired t raise specific antibodies and drawing sequential time point samples of antibody-containing blood from the immunized pig, with aliquots of these time point samples being used to make seroconversion panels.
• suitable immunizing agents include HIV-1, HIV-2, HTLV-1, HTLV-II, CMV, LYME, toxoplasma. Rubella, and Epstein-Barr virus.
• immunizing agents as may be known to those skilled in the art are also useful.
• the immunization begins with a first vaccination of th animal with a preparation comprising 10-500 micrograms (ug) of viral or microbial lysate or selected portions of the viral cor or envelope proteins or of the microbial core or membrane proteins.
• Viral material is generally solubized in Triton X-100, SDS (sodium dodecyl sulfate) , mercaptoethanol, and/or NP 40 (Novidet P40 detergent, nonionic detergent), and suspended i phosphate buffered saline (PBS), pH 7.2-7.4.
• the first injection contains an adjuvant; Freund's complete adjuvant (FCA) is preferred for this purpose.
• FCA Freund's complete adjuvant
• the first vaccination typically comprises a tot volume of 2 ml, one ml of viral material in buffer plus one ml of adjuvant. The viral material and adjuvant should be thoroughly mixed immediately prior to injection. In a preferr embodiment, the first vaccination contains 50-100 ug of viral material in 1.0 ml of 1% SDS mixed with 1.0 ml of FCA.
• Booster immunizations are generally prepared with adjuv-it and typically contain 10-500 ug of viral proteins, preferably from about 50-100 ug per injection in PBS, pH 7.2-7.4. It is preferred that booster immunizations are prepared with Freund's incomplete adjuvant. It is further preferred that the booster immunization contains 50-100 ug of viral material solubilized in 0.1% NP 40, heated for up to 30 minutes at 50-60°C, and diluted with 1% SDS. Booster injectio begin from about 7 to 30 days after the initial vaccination a every 7 to 30 days thereafter until the desired number of samples needed for the seroconversion panel is obtained. It i preferred that the first booster injection be given twenty-fo hours after the first vaccination.
• booster immunizations up to four booster immunizations are given. It is further preferred that the fourth or final booster immunization be prepared with Freund's incomplete adjuvant and with viral lysate solubilized in 1% SDS and heated for up to 40 minutes at 65-75°C to elicit unfolding of the outer shell or envelope proteins and to expos the interior or core proteins, thereby allowing an immunoresponse to these interior proteins.
• a first blood sample is generally drawn and tested prior to the initial vaccination to establish a baseline antibody level. It is presently preferred to vaccinate by intracutaneous or subcutis injection, but other modes of administration, such as intramuscular injection may be used.
• the vaccination site is preferably the side of the neck, typically injecting three to four different locations. Vaccination on the neck is considered to yield higher antibody i ⁇ r ⁇ f but other vaccination sites as are known to those ⁇ killed in the f frt are suitable for raising the desired immune
• Blood is collected from the immunized animal by consecutive bleedings after the first immunization, preferably every 2 to 3 days, until a sufficient number of consecutive blood.samples, preferably 8 to 16 time points, are obtained fo the desired seroconversion panel.
• the titer of the desired antibody of each blood sample or serum extracted therefrom is measured.
• ti points Once the timing and frequency of blood drawings (ti points) is established for the particular immunogen being used a multiple number of animals can be immunized.
• the animal is sacrificed and the blood collected at only one time point, ea animal's time point being different and including an animal fo a baseline time point.
• the total collected blood of one anima would represent one time point sample.
• the advantage is that large amounts of antibody-containing blood for a particular ti point in a seroconversion panel can be readily obtained.
• serum from the immunized anim in a seroconversion panel.
• whole blood, plasma or a other form of antibody as may be known to those skilled in th art may be used.
• the serum may be freeze-dried or dried or fractionated to enrich the IgG, IgM or IgA fractions.
• the previously described method can be used to produce seroconversion panels containing antibodies against several viruses including HIV-1, HIV-2, HTLV-1, HTLV-II, cytomegalovirus, Epstein-Barr virus, toxoplasma, and hepatitis virus.
• the previously described method can be used to produce antibodies for seroconversion panels in animals other than pigs, including rabbits, goats, mice, rats, sheep, cows, horses, and monkeys.
• An immunogen for injection was prepared with inactivated HTLV-1 lysate, containing proteins (p) 19, 24, 26, 38, and glycoprotein (gp) 46.
• the HTLV-1 lysate present in a concentration of 1 mg/ml in 0.1% NP 40 (a nonionic detergent), 0.5m NaCl and TNE buffer (29.2 g/1 NaCL, 1.2 g/1 Tris, and 0.37g/l EDTA) , was heated for 30 minutes at 56°C.
• One hundred ul of the HTLV-1 viral lysate were diluted with 900 ul of 1% SDS. Shortly before injection, this diluted viral lysate was mixed with 1.0 ml Freund's cornple adjuvant (FCA) to make the immunogen.
• FCA 1.0 ml Freund's cornple adjuvant
• Booster injections contained 100 ul of HTLV-1 viral lysate in 900 ul of 1% SDS mixed with 1 ml Freund's incomplete adjuvant (FIA) .
• the first booster was administered twenty-four hours later (day 1), a second booster 16 days later (day 17), and a third booster 14 days later (day 31).
• a fourth booster was prepared where 100 ul of HTLV-1 viral lysate diluted in 900 ul 1% SDS was first heat treated for 40 minutes at a temperature from 65 to 70 ⁇ C and th mixed with 1 ml FIA.
• the pig was vaccinated with this fourth booster on day 48.
• the pig received a total of five injection
• Example 1 The method of Example 1 was used except that inactivated HIV-1 viral lysate was used instead of inactivated
• the HIV-1 lysate contained pl8, p2 ' 4, p31, gp41, gp48, p53, p56, ⁇ 64, gpllO, gpl20, and gpl60.
• Example 1 The aliquots of the seroconversion panel were diluted 1 to 30 with PBS Tween. To a single Western Blot strip,
• NBT nitrobluetetrazolium salt
• Example 3 The method of Example 3 was repeated except that the conjugate used was 2 ml of goat anti-human IgM, mu-chain, conjugated with alkaline phosphatase (Calbiochem, Lot # 041890) diluted 1:500 in PBS Tween. The resulting strips are shown in FIG. 2.
• FIG. 2 shows the presence of
• IgM by day 9 with the appearance of three protein bands, ⁇ l9, ⁇ 26, and p28.
• a high titer of IgM is indicated by the appearance of protein bands at ⁇ l5, pl9, p24, p26, p28, p32 and ⁇ 36. This remains fairly constant until day 48 where, in contrast to the IgG seroconversion panel of Example 3, the IgM titer declines as shown by the decreasing number of protein bands.
• the ELISA assay was carried out accordance with the manufacturer's instructions with two modifications.
• the seroconversion panel Prior to starting the ELISA assay, the seroconversion panel was pretreated to remove IgG, which interferes with the ELISA measurement of IgM. Each aliquot of serum from the seroconversion panel was diluted 1:10 in PBS Tween. The amoun of IgG present in the sample was estimated and an equivalent amount of Protein G (Pierce, Immuno Pure Immobilized Protein G was mixed in with the diluted serum and incubated at 37°C for minutes. The diluted sample was then centrifuged for 5 minute at 10,000 rpm. The supernatant was removed and then used in t ELISA assay.
• the second modification was the replacement of the Ig conjugate of the test kit with an IgM conjugate, using goat anti-human IgM, mu-chain, conjugated with alkaline phosphatase (Calbiochem, Lot # 041890) which was diluted 1:4000 in PBS Twe
• Example 5 In contrast to Example 5, on day 9, on O.D. reading o 1.22 was obtained, indicating a high titer of IgM. On day 23, the titer peaked, giving an O.D. reading of 1.75. On day 38, O.D. reading of 1.17 indicated the titer was decreasing. The titer continued to decrease as shown by the decreasing O.D. values.

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• 1991
  • 1991-11-06 JP JP4501275A patent/JPH06502723A/ja active Pending
  • 1991-11-06 KR KR1019930701388A patent/KR930701975A/ko not_active Ceased
  • 1991-11-06 AU AU90942/91A patent/AU650707B2/en not_active Ceased
  • 1991-11-06 WO PCT/US1991/008296 patent/WO1992008471A1/en not_active Application Discontinuation
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