CN112662610A - Process for removing newborn bovine serum endotoxin - Google Patents
Process for removing newborn bovine serum endotoxin Download PDFInfo
- Publication number
- CN112662610A CN112662610A CN202011466034.1A CN202011466034A CN112662610A CN 112662610 A CN112662610 A CN 112662610A CN 202011466034 A CN202011466034 A CN 202011466034A CN 112662610 A CN112662610 A CN 112662610A
- Authority
- CN
- China
- Prior art keywords
- stirring
- endotoxin
- treatment
- serum
- adsorption
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a process for removing newborn bovine serum endotoxin, which comprises the following steps: taking newborn calf serum which is fed by the small bull for 4-14 days and contains a large amount of lipids, endotoxin and hemoglobin exceeding standards; pre-filtering the newborn calf serum to obtain pre-filtered supernatant; adding active carbon into the pre-filtered supernatant for adsorption, wherein the mass fraction of the active carbon is 0.5-2% of the mass of the supernatant, and performing intermediate filtration after 50-90min of adsorption; and adding an endotoxin adsorption material for adsorption treatment after the middle filtration treatment, and performing post filtration treatment after the adsorption treatment to obtain the newborn calf serum without endotoxin after the post filtration treatment. The invention adopts prefiltering treatment, intermediate filtering treatment and postfiltering treatment to be matched with an endotoxin adsorption material, so that endotoxin in serum of newborn cattle can be better removed.
Description
Technical Field
The invention relates to the field of biology, in particular to a process for removing neonatal bovine serum endotoxin.
Background
Abundant nutrients necessary for cell growth are commonly used for in vitro culture of animal cells, and have extremely important functions. It provides hormones for maintaining the growth of the cell index, nutrients in the basal medium in no or low amounts, and major low molecular nutrients. It also provides binding proteins that recognize vitamins, lipids, metals and other hormones, and bind or modulate the activity of the substances to which they bind.
Bovine serum is the most abundant natural medium used in cell culture, contains endotoxin which is a general term for toxic substances existing in the cells of gram-negative bacteria, is a cell wall component of various gram-negative bacteria, is released after being lysed by the cells, and is also called as a pyrogen. The chemical component of the medicine is phospholipid polysaccharide-protein complex, and the toxic component of the medicine is mainly lipoid A. Endotoxin is located in the outermost layer of the cell wall, covering the mucin of the cell wall. Most of the endotoxins of various bacteria have certain toxicity, and can cause fever, microcirculation disturbance, endotoxin shock, disseminated intravascular coagulation and the like. Therefore, the serum containing endotoxin can not be used for vaccine production, and the serum can be used for vaccine production only by removing endotoxin and retaining the nutrient components in the serum, so that waste is changed into valuable, and the endotoxin is fully utilized.
Disclosure of Invention
The invention aims to provide a process for removing newborn bovine serum endotoxin, which aims to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a process for removing newborn bovine serum endotoxin comprises the following steps:
s1: taking newborn calf serum which is fed by the small bull for 4-14 days and contains a large amount of lipids, endotoxin and hemoglobin exceeding standards;
s2: pre-filtering the newborn calf serum to obtain pre-filtered supernatant;
s3: adding active carbon into the pre-filtered supernatant for adsorption, wherein the mass fraction of the active carbon is 0.5-2% of the mass of the supernatant, and performing intermediate filtration after 50-90min of adsorption;
s4: and adding an endotoxin adsorption material for adsorption treatment after the middle filtration treatment, and performing post filtration treatment after the adsorption treatment to obtain the newborn calf serum without endotoxin after the post filtration treatment.
Preferably, the pre-filtering process in S2 specifically includes the steps of: s21: filtering fibrin in the cattle serum to obtain a filtrate; s22: separating the filtrate into macromolecular compound and free molecules by an ultrafiltration column; s23: adding immunoglobulin and lipoprotein into the macromolecular compound and polymerizing into flocculent precipitate; s24: and centrifuging, clarifying and filtering the flocculent precipitate to obtain a pre-filtrate, and combining the pre-filtrate with free molecules to obtain a pre-filtered supernatant.
Preferably, in the filtration treatment in S3, the treatment solution is centrifuged to obtain a supernatant, and the supernatant is filtered through a rough filtration membrane.
Preferably, the activated carbon in S3 is activated carbon granules and/or powder, and the activated carbon granules and/or powder is added into serum and stirred gently until the mixture is uniform, and the activated carbon is removed through a middle filtration treatment to obtain a middle filtration treatment raw material.
Preferably, the endotoxin adsorption material in S4 is prepared by coupling reaction of chitosan microspheres and glycine.
Preferably, the post-filtration treatment in S4 comprises sequential stepwise filtration using a 0.8 μm filter, a 0.65 μm filter, a 0.45 μm filter, and a 0.1 μm filter, thereby obtaining the endotoxin-depleted newborn bovine serum.
Preferably, the serum is added into the activated carbon particles and/or the activated carbon powder and the mixture is gently stirred to be uniform in a stirring device, the stirring device comprises a stirring box, a rack, a first stirring structure and a second stirring structure, the stirring box is fixed at the end part of the rack, and the first stirring structure and the second stirring structure are respectively arranged on the upper side and the lower side inside the stirring box; the first stirring structure comprises a sliding rail, a toothed plate, a gear and a first stirring rod, the sliding rail is fixed on the top wall of the stirring box, a spring is arranged on the inner side of the sliding rail, and the toothed plate is connected onto the sliding rail in a sliding manner; the gear is installed in the pinion rack downside and with the pinion rack meshing through the support, and gear end connection has first puddler, and first puddler can swing when gear, pinion rack intermeshing.
Preferably, the second stirring structure is a second stirring rod which is rotatably connected to the lower side in the stirring box, a driving structure for driving the second stirring rod and the first stirring rod to rotate is arranged on the outer side of the stirring box, the driving structure comprises a first rotating rod, a second rotating rod, a third rotating rod and a rotating column, the first rotating rod is arranged on the rack and is in transmission connection with the second stirring rod, a first helical gear is arranged on the first rotating rod, and a handle for driving the first rotating rod is arranged on the bottom side of the rack; the second dwang both ends are equipped with first band pulley respectively and with the second helical gear of first helical gear meshing, third dwang one end is equipped with the second band pulley that passes through the belt linkage with first band pulley, the end connection that the third dwang passed the agitator tank has the third bevel gear, and the meshing transmission of third bevel gear and rotation post, rotation post top is rotated and is connected in the agitator tank roof, the rotation post facial make-up is equipped with the cam of contradicting with the pinion rack.
Compared with the prior art, the invention has the beneficial effects that:
the invention can obviously reduce the content of endotoxin in the high-endotoxin bovine serum which is used as waste in the production process of the newborn bovine serum in the past, and increase the production efficiency and the added value of the newborn bovine serum; and the endotoxin in the serum of the newborn cattle can be better removed by adopting prefiltering treatment, intermediate filtration treatment and postfiltration treatment matched with an endotoxin adsorbing material.
Drawings
FIG. 1 is a process flow diagram of example 2 of the present invention;
FIG. 2 is a schematic structural view of a stirring apparatus in example 3 of the present invention.
In the figure: 1. a stirring box; 2. a frame; 3. a slide rail; 4. a toothed plate; 5. a gear; 6. a first stirring rod; 7. a second stirring rod; 8. a first rotating lever; 9. a second rotating lever; 10. a third rotating rod; 11. rotating the column; 12. a first pulley; 13. a second pulley; 14. a cam.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the description of the present invention, it should be noted that the terms "vertical", "upper", "lower", "horizontal", and the like indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of describing the present invention and simplifying the description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.
In the description of the present invention, it should also be noted that, unless otherwise explicitly specified or limited, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly and may, for example, be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
Example 1: the invention provides a technical scheme that: a process for removing newborn bovine serum endotoxin comprises the following steps:
s1: taking newborn calf serum which is fed by the small bull for 4-14 days and contains a large amount of lipids, endotoxin and hemoglobin exceeding standards;
s2: pre-filtering the newborn calf serum to obtain pre-filtered supernatant;
s3: adding active carbon into the pre-filtered supernatant for adsorption, wherein the mass fraction of the active carbon is 0.5-2% of the mass of the supernatant, and performing intermediate filtration after 50-90min of adsorption;
s4: and (4) after the middle filtration treatment, performing post filtration treatment to obtain the newborn calf serum with endotoxin removed.
In this embodiment, the pre-filtering process in S2 specifically includes the steps of: s21: filtering fibrin in the cattle serum to obtain a filtrate; s22: separating the filtrate into macromolecular compound and free molecules by an ultrafiltration column; s23: adding immunoglobulin and lipoprotein into the macromolecular compound and polymerizing into flocculent precipitate; s24: and centrifuging, clarifying and filtering the flocculent precipitate to obtain a pre-filtrate, and combining the pre-filtrate with free molecules to obtain a pre-filtered supernatant.
In this example, the activated carbon in S3 is activated carbon particles and/or powder, and the activated carbon is removed by a middle filtration treatment and a middle filtration treatment raw material is obtained by adding the activated carbon particles and/or powder to serum and gently stirring until uniform.
In this example, the filtration treatment in S3 is performed by centrifuging the treated liquid to obtain a supernatant, and filtering the supernatant through a rough filtration membrane.
In this example, the post-filtration treatment in S4 comprises sequential stepwise filtration using a 0.8 μm filter, a 0.65 μm filter, a 0.45 μm filter, and a 0.1 μm filter, thereby obtaining a newborn bovine serum with endotoxin removed.
Example 2: referring to fig. 1, the present invention provides a technical solution: a process for removing newborn bovine serum endotoxin comprises the following steps:
s1: taking newborn calf serum which is fed by the small bull for 4-14 days and contains a large amount of lipids, endotoxin and hemoglobin exceeding standards;
s2: pre-filtering the newborn calf serum to obtain pre-filtered supernatant;
s3: adding active carbon into the pre-filtered supernatant for adsorption, wherein the mass fraction of the active carbon is 0.5-2% of the mass of the supernatant, and performing intermediate filtration after 50-90min of adsorption;
s4: and adding an endotoxin adsorption material for adsorption treatment after the middle filtration treatment, and performing post filtration treatment after the adsorption treatment to obtain the newborn calf serum without endotoxin after the post filtration treatment.
In this embodiment, the pre-filtering process in S2 specifically includes the steps of: s21: filtering fibrin in the cattle serum to obtain a filtrate; s22: separating the filtrate into macromolecular compound and free molecules by an ultrafiltration column; s23: adding immunoglobulin and lipoprotein into the macromolecular compound and polymerizing into flocculent precipitate; s24: and centrifuging, clarifying and filtering the flocculent precipitate to obtain a pre-filtrate, and combining the pre-filtrate with free molecules to obtain a pre-filtered supernatant.
In this example, the activated carbon in S3 is activated carbon particles and/or powder, and the activated carbon is removed by a middle filtration treatment and a middle filtration treatment raw material is obtained by adding the activated carbon particles and/or powder to serum and gently stirring until uniform.
In this example, the filtration treatment in S3 is performed by centrifuging the treated liquid to obtain a supernatant, and filtering the supernatant through a rough filtration membrane.
In this embodiment, the endotoxin-adsorbing material in S4 is chitosan microspheres and glycine, which are prepared by coupling reaction.
In this example, the post-filtration treatment in S4 comprises sequential stepwise filtration using a 0.8 μm filter, a 0.65 μm filter, a 0.45 μm filter, and a 0.1 μm filter, thereby obtaining a newborn bovine serum with endotoxin removed.
Example 3: referring to fig. 2, in this embodiment, the adding of the serum into the activated carbon particles and/or the activated carbon powder and the gentle stirring until the serum is uniformly mixed are performed in a stirring device, the stirring device includes a stirring box 1, a frame 2, a first stirring structure and a second stirring structure, the stirring box 1 is fixed at an end of the frame 2, and the first stirring structure and the second stirring structure are respectively installed at an upper side and a lower side inside the stirring box 1; the first stirring structure comprises a sliding rail 3, a toothed plate 4, a gear 5 and a first stirring rod 6, the sliding rail 3 is fixed on the top wall of the stirring box 1, a spring is arranged on the inner side of the sliding rail 3, and the toothed plate 4 is connected onto the sliding rail 3 in a sliding manner; gear 5 installs in pinion rack 4 downside and with the meshing of pinion rack 4 through the support, 5 end connection of gear has first puddler 6, and first puddler 6 can swing when gear 5, pinion rack 4 intermeshing.
In this embodiment, the second stirring structure is a second stirring rod 7 rotatably connected to the lower side inside the stirring box 1, a driving structure for driving the second stirring rod 7 and the first stirring rod 6 to operate is arranged outside the stirring box 1, the driving structure comprises a first rotating rod 8, a second rotating rod 9, a third rotating rod 10 and a rotating column 11, the first rotating rod 8 is installed on the frame 2 and is in transmission connection with the second stirring rod 7, a first helical gear is installed on the first rotating rod 8, and a handle for driving the first rotating rod 8 is arranged at the bottom side of the frame 2; the both ends of second dwang 9 are equipped with first band pulley 12 respectively and with the second helical gear of first helical gear meshing, third dwang 10 one end is equipped with the second band pulley 13 that passes through the belt linkage with first band pulley 12, the end connection that third dwang 10 passed agitator tank 1 has the third helical gear, and the transmission of third helical gear and 11 meshing of rotation post, 11 tops of rotation post rotate and connect in 1 roof of agitator tank, 11 facial make-ups of rotation post are equipped with the cam 14 of contradicting with pinion rack 4.
In this embodiment, drive first dwang 8 through the handle, make first dwang 8 drive second puddler 7 and second dwang 9, through second dwang 9 linkage third dwang 10, thereby make the cam 14 of third dwang 10 tip contradict in pinion rack 4, make pinion rack 4 remove and with the meshing of gear 5, and then make gear 5 drive the swing of first puddler 6, realize better active carbon granule and/or powder stirring effect through wobbling first puddler 6 and pivoted second puddler 7.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A process for removing newborn bovine serum endotoxin is characterized by comprising the following steps:
s1: taking newborn calf serum which is fed by the small bull for 4-14 days and contains a large amount of lipids, endotoxin and hemoglobin exceeding standards;
s2: pre-filtering the newborn calf serum to obtain pre-filtered supernatant;
s3: adding active carbon into the pre-filtered supernatant for adsorption, wherein the mass fraction of the active carbon is 0.5-2% of the mass of the supernatant, and performing intermediate filtration after 50-90min of adsorption;
s4: and adding an endotoxin adsorption material for adsorption treatment after the middle filtration treatment, and performing post filtration treatment after the adsorption treatment to obtain the newborn calf serum without endotoxin after the post filtration treatment.
2. The process according to claim 1, wherein the pre-filtering treatment in S2 comprises the following steps: s21: filtering fibrin in the cattle serum to obtain a filtrate; s22: separating the filtrate into macromolecular compound and free molecules by an ultrafiltration column; s23: adding immunoglobulin and lipoprotein into the macromolecular compound and polymerizing into flocculent precipitate; s24: and centrifuging, clarifying and filtering the flocculent precipitate to obtain a pre-filtrate, and combining the pre-filtrate with free molecules to obtain a pre-filtered supernatant.
3. The process according to claim 1, wherein the activated carbon in S3 is activated carbon particles and/or powder, the activated carbon particles and/or powder is added into serum and stirred gently until the mixture is uniform, and the activated carbon is removed through a middle filtration treatment to obtain a middle filtration treatment raw material.
4. The process according to claim 1, wherein the filtering treatment in S3 is performed by centrifuging the treated solution to obtain a supernatant, and filtering the supernatant through a coarse filter membrane.
5. The process according to claim 1, wherein the endotoxin-adsorbing material of S4 is chitosan microsphere and glycine prepared by coupling reaction.
6. The process according to claim 1, wherein the post-filtration treatment in S4 comprises sequential step-by-step filtration with 0.8 μm filter, 0.65 μm filter, 0.45 μm filter and 0.1 μm filter, thereby obtaining the de-endotoxin newborn bovine serum.
7. The process for removing the endotoxin in the newborn bovine serum according to claim 3, wherein the adding of the serum into the activated carbon particles and/or the activated carbon powder and the gentle stirring until the serum is uniformly mixed are carried out in a stirring device, the stirring device comprises a stirring box (1), a rack (2), a first stirring structure and a second stirring structure, the stirring box (1) is fixed at the end part of the rack (2), and the first stirring structure and the second stirring structure are respectively arranged on the upper side and the lower side inside the stirring box (1); the first stirring structure comprises a sliding rail (3), a toothed plate (4), a gear (5) and a first stirring rod (6), the sliding rail (3) is fixed on the top wall of the stirring box (1), a spring is arranged on the inner side of the sliding rail (3), and the toothed plate (4) is connected onto the sliding rail (3) in a sliding manner; gear (5) are installed in pinion rack (4) downside and with pinion rack (4) meshing through the support, gear (5) end connection has first puddler (6), and first puddler (6) can swing when gear (5), pinion rack (4) intermeshing.
8. The process for removing the endotoxin in the newborn bovine serum according to claim 7, wherein the second stirring structure is a second stirring rod (7) rotatably connected to the lower side in the stirring tank (1), a driving structure for driving the second stirring rod (7) and the first stirring rod (6) to operate is arranged at the outer side of the stirring tank (1), the driving structure comprises a first rotating rod (8), a second rotating rod (9), a third rotating rod (10) and a rotating column (11), the first rotating rod (8) is arranged on the rack (2) and is in transmission connection with the second stirring rod (7), a first helical gear is arranged on the first rotating rod (8), and a handle for driving the first rotating rod (8) is arranged at the bottom side of the rack (2); second dwang (9) both ends are equipped with first band pulley (12) respectively and with the second helical gear of first helical gear meshing, third dwang (10) one end is equipped with second band pulley (13) through the belt linkage with first band pulley (12), the end connection that agitator tank (1) was passed in third dwang (10) has third helical gear, and third helical gear and rotation post (11) meshing transmission, rotation post (11) top is rotated and is connected in agitator tank (1) roof, rotation post (11) facial make-up is equipped with cam (14) of contradicting with pinion rack (4).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011466034.1A CN112662610A (en) | 2020-12-14 | 2020-12-14 | Process for removing newborn bovine serum endotoxin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011466034.1A CN112662610A (en) | 2020-12-14 | 2020-12-14 | Process for removing newborn bovine serum endotoxin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112662610A true CN112662610A (en) | 2021-04-16 |
Family
ID=75405605
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011466034.1A Pending CN112662610A (en) | 2020-12-14 | 2020-12-14 | Process for removing newborn bovine serum endotoxin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112662610A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114225542A (en) * | 2021-12-17 | 2022-03-25 | 呼和浩特市草原绿野生物工程材料有限公司 | Method for efficiently removing toxin in newborn bovine serum |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103725645A (en) * | 2014-01-07 | 2014-04-16 | 济南劲牛生物科技有限公司 | Process for removing newborn bovine serum endotoxin |
| CN105664868A (en) * | 2016-01-22 | 2016-06-15 | 广州康盛生物科技有限公司 | Endotoxin adsorption material for blood purification and preparation method and application of endotoxin adsorption material for blood purification |
| CN110066762A (en) * | 2018-01-22 | 2019-07-30 | 范清春 | The method of industrialized production new born bovine purified blood serum |
-
2020
- 2020-12-14 CN CN202011466034.1A patent/CN112662610A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103725645A (en) * | 2014-01-07 | 2014-04-16 | 济南劲牛生物科技有限公司 | Process for removing newborn bovine serum endotoxin |
| CN105664868A (en) * | 2016-01-22 | 2016-06-15 | 广州康盛生物科技有限公司 | Endotoxin adsorption material for blood purification and preparation method and application of endotoxin adsorption material for blood purification |
| CN110066762A (en) * | 2018-01-22 | 2019-07-30 | 范清春 | The method of industrialized production new born bovine purified blood serum |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114225542A (en) * | 2021-12-17 | 2022-03-25 | 呼和浩特市草原绿野生物工程材料有限公司 | Method for efficiently removing toxin in newborn bovine serum |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112662610A (en) | Process for removing newborn bovine serum endotoxin | |
| CN101948494B (en) | Method for extracting cobamamide | |
| CN101690555B (en) | High-pressure homogeneous honey and preparation method for product thereof | |
| Ju et al. | Improvement of bilirubin adsorption capacity of cellulose acetate/polyethyleneimine membrane using sodium deoxycholate | |
| JP7008540B2 (en) | Toxin separator | |
| CN101311191A (en) | Method for preparing immune globulin against lymphocyte of human | |
| CN108504695B (en) | Extracellular polymer and preparation method thereof, heavy metal treatment agent and heavy metal wastewater treatment method | |
| CN102660504A (en) | Preparation method of lymphocyte separating medium | |
| CN111921565B (en) | Method for reducing aluminum content in amino acid injection | |
| CN1703972A (en) | Method for making crystal honey | |
| CN1849903A (en) | Method for inductrialized separation of purified lactoferrins from cattle colostrum | |
| CN113318125B (en) | Preparation method of echinacea polysaccharide nanoparticles | |
| CN209198173U (en) | A kind of membrane type pelleter | |
| CN107446905B (en) | Method for purifying recombinant human lysozyme | |
| CN110272486B (en) | Method for extracting phycocyanin from blue algae | |
| CN111087443A (en) | Method for extracting multiple urine proteins from urine | |
| CN110804578B (en) | Production method for removing IgG from bovine serum | |
| CN108192799B (en) | Composite clarifying agent and preparation method and clarifying method thereof | |
| CN110777107B (en) | Production method for removing lipoprotein from horse serum | |
| CN103159643A (en) | Technology for whole membrane extraction of L-glutamine fermentation broth | |
| CN105906738A (en) | Method for extracting and separating polysaccharides from Celosia cristata | |
| CN1164600C (en) | Process for preparing bifidobacterium factor oligose from gestumor tuber | |
| CN1431044A (en) | Adsorbent based on montmorillonite for purifying blood and its preparing method | |
| CN106166311B (en) | A kind of plasma purification system and its application | |
| CN110665467A (en) | Preparation method of straw-based adsorbent for adsorbing anions/cations |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |