Patent Application: US-21661894-A

Abstract:
a factor that indicates fertilization of an ovum before implantation , preimplantation factor , has been found . pif also provides an early indication of pregnancy , malignancy , and autoimmune diseases . assays for pif are described . detection of pif in mammalian blood and other fluids is utilized in various diagnostic tests .

Description:
in a retrospective study , serum samples from 60 human individuals ( including 10 males , 20 non - pregnant females and 30 pregnant females ) were analyzed in the lymphocyte / platelet binding assay . blood was drawn at various times throughout the menstrual cycle , and at different times after transfer of in vitro embryos . three non - pregnant women received progesterone replacement therapy , and one was taking oral contraceptive pills containing estrogen and progesterone . guinea pig complement ( gpc ), histopaque and dulbecco &# 39 ; s phosphate buffered saline ( pbs ) were obtained from sigma ( st . louis , mo .). t - 11 monoclonal antibody which recognizes the cd2 receptor on the lymphocytes was obtained from ortho pharmaceuticals ( raritan , n . j .). one can advantageously use other reagents , for example , rabbit complement ( sigma ), t - 11 antibody ( dakko , denmark ). eight ml of blood from a healthy o + donor was diluted 1 : 1 with pbs and was layered over 12 ml of histopaque column in a 50 ml test tube and centrifuged at 2400 rpm for 20 minutes to isolate lymphocytes plus platelets by density gradient . following centrifugation the buffy coat was removed , diluted 1 : 1 with pbs and washed twice with pbs at 1100 rpm for 10 minutes each time removing the supernatant . during each washing process 2 ml of double distilled water was added to lyse the residual red blood cells . the lymphocytes and platelets recovered were diluted in pbs to 10 12 cells per ml . the lymphocyte / platelet binding assay was carried out in microcentrifuge tubes in a total volume of 60 μl . assay mixture consisted of 15 μl of o + lymphocytes plus platelets , 15 μl heat inactivated serum of the test subject , 15 μl activated guinea pig complement , and 15 μl t11 antibody ( anti cd2 ). following gentle mixing for 5 minutes 10 μl of the sample was withdrawn and placed on a slide with a cover slip . the number of lymphocytes with or without bound platelets were counted . a total of 200 cells in three different fields were counted . samples then were recounted by placing another 10 μl of mixture on a slide and the average of the duplicate measurements was reported . all lymphocytes were counted at × 400 magnification using a nikon inverted microscope and were categorized as bound ( even if one platelet was bound to the surface ) or free if no platelet binding was observed . data are expressed as % platelets - bound lymphocytes per total bound and non - bound lymphocytes . statistical analysis was carried out by using chi - square analysis with fischers exact test and anova one way analysis of variance . a difference of p & lt ; 0 . 05 was considered significant . fig1 is a scattergram of pif activity (% platelet binding to lymphocytes ) in pregnant women with intact ovaries and subsequent viable pregnancy and in non - pregnant subjects . the percentage of lymphocytes bound by platelets is 63 %± 10 % ( mean ± sd ) among pregnant women and 23 %± 6 % in non - pregnant individuals . the difference between serum samples taken from pregnant and non - pregnant women is significant ( p & lt ; 0 . 001 ). pooled pregnancy sera ( n = 10 ) had 59 % lymphocytes bound to platelets ( data not shown ). intra and interassay variabilities were each less than 3 %. table i compares the percentage of lymphocyte binding in the pif assay with the clinical characteristics of the 60 subjects studied . no differences in percentage binding were observed when nonpregnant females in follicular phase of the menstrual cycle were compared with the luteal phase . all male and non - pregnant females had less than 33 % binding . using these values as the non - pregnant controls , 2 standard deviations from the mean is less than 35 % binding . therefore , a test was considered positive when the percentage of lymphocytes bound to platelets was greater than 35 %. five women who underwent in vitro fertilization / embryo transplants and had successful pregnancies had blood drawn serially : the day of embryo transfer , 4 days after embryo transfer when hcg concentrations were negative , and 11 days after embryo transfer when hcg was positive . pif activity became detectable in the sera in all women by 4 days after embryo transfer . table i______________________________________percentage of lymphocyte binding in 30 pregnantand 30 nonpregnant individuals % binding n mean sd sem 95 % ci______________________________________nonpregnant 30 21 . 2 5 . 9 1 . 0 19 . 2 - 23 . 3females 20 23 . 6 5 . 5 1 . 2 21 . 0 - 26 . 1follicular 10 22 4 . 6 1 . 5 18 . 7 - 25 . 3luteal 10 25 . 5 6 . 1 1 . 9 20 . 7 - 29 . 5males 10 17 . 7 4 . 7 1 . 3 14 . 8 - 20 . 5pregnant 30 56 . 1 15 . 9 2 . 9 50 . 2 - 62 . 0______________________________________ fol vs lut ns . 2 ♀ vs ♂ p = 0 . 007 fol vs ♂ p = 0 . 05 lut vs ♂ p = 0 . 007 preg vs np = p & lt ; 0 . 0001 preg vs ♀ p & lt ; 0 . 0001 fig2 is microphotograph showing results of platelet binding to lymphocytes using sera from non - pregnant and pregnant women . low percentage of binding of serum lymphocytes to platelets is seen when serum from a non - pregnant subject is used ( panel a ) and high percentage of binding of lymphocytes to platelets is observed when serum from a pregnant woman is used ( panel b ). in addition to lymphocyte binding of platelets , a difference in the appearance of background is observed . increased platelet binding to lymphocytes is associated with a clearing up of the background , thus making it appear empty . table ii shows results of percentage of lymphocyte binding to platelets using sera from non - pregnant women receiving progesterone replacement therapy during the luteal phase or oral contraceptives ( estrogen and progesterone ). all of the values are within the non - pregnant range demonstrating no effect of these steroids on the results of the pif assay . table ii______________________________________percentage of lymphocytes binding to platelets inthe lymphocyte / platelet binding assay using sera fromnon - pregnant women , receiving estrogen and / orprogesterone therapy . subject treatment % binding______________________________________1 progesterone 172 progesterone 223 progesterone 324 estrogen and 30 progesterone______________________________________ table iii shows the effect of hcg on the results of the lymphocyte / platelet binding assay . no effects on the binding of platelets were found whether hcg was added to the assay mixture alone or premixed with non - pregnant serum . table iii______________________________________the effect of human chorionic gonadotropin on theresults of the lymphocyte / platelet binding assay . amount of hcgadded to assay % bindingunits exp 1 exp 2 exp 3 exp 4______________________________________0 25 19 28 185 25 21 11 2010 23 23 9 2315 (+ 15 μl serum ) 28 20 15 16______________________________________ forty - five women undergoing assisted reproductive technology were prospectively followed . blood was collected either 4 days after embryo transfer following ivf ( in vitro fertilization ) or 6 days after insemination , and subjected to the pif assay . it was later determined whether each patient did or did not become pregnant . table iv shows the predictive value with respect to pregnancy in these patients , who were tested blindly . obtain 5 ml of blood in blue top tube ( sodium citrate ) from a healthy o (+) donor . dilute 1 : 2 with pbs and isolate lymphocytes by density - gradient technique : histopaque - 1077 ( sigma ). check the number of spontaneous bindings ( background ). count % of 1 lymphocyte attached to 3 or more platelets . a good donor has no more than 3 - 4 %. after 5 - 10 minutes assess percentage of platelet - bound lymphocytes . double blinded and averaged from the two scorers . monoclonal antibodies to pif are obtained by isolation of proteins constituting pif by known techniques of chromatography and sds - page electrophoresis , using the lymphocyte / platelet binding bioassay for identification of fractions , followed by known methods for production and isolation of monoclonal antibodies from hybridomas . 1 . rolfe b e . detection of fetal wastage . fertil steril . 1982 ; 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